Ammonium (NH4 +) and nitrate (NO3 -) are the two main forms of inorganic nitrogen (N) that exist in soil and both can be absorbed and utilized by plants. As a vast and crucial biome, soil microorganisms are responsible for mediating the inorganic N assimilation process and enhancing nitrogen use efficiency. Understanding how these microorganisms assimilate different forms of inorganic nitrogen is crucial. There are a handful of microorganisms that play a dominant role in the process of soil inorganic nitrogen assimilation and have a significant advantage in abundance. However, microbial preferences for ammonium or nitrate, as well as differences in their metabolic pathways under co-existing ammonium and nitrate conditions, remain unclear.

In this study, two microbial strains with nitrogen assimilation advantages, Burkholderia sp. M6-3 and Arthrobacter sp. M7-15 were isolated from an acidic Chinese soil and then incubated by different sources of inorganic N to investigate their N preferences. Furthermore, RNA sequencing-based transcriptome analysis was used to map the metabolic pathways of the two strains and explore their explanatory potential for N preferences.

The results showed that strain M6-3 preferred to utilize NH4 + while strain M7-15 preferred to utilize NO3 -. Although both strains shared similar nitrogen metabolic pathways, the differential expression of the glutamine synthetase-coding gene glnA played a crucial role in regulating their inorganic N preferences. This inconsistency in glnA expression may be attributed to GlnR, a global regulator of nitrogen utilization.

This research strengthens the theoretical basis for exploring the underlying causes of differential preferences for inorganic N forms and provided key clues for screening functional microorganisms to ultimately enhance inorganic nitrogen use efficiency.

Background: Portal vein tumor thrombosis (PVTT) is a frequent and serious complication of advanced hepatocellular carcinoma (HCC) that often results in poor prognosis. Although PVTT holds significant clinical relevance, the molecular mechanisms driving its formation are not well understood. Long non-coding RNAs (lncRNAs) have emerged as potential contributors to PVTT progression, prompting this study to explore lncRNAs as potential biomarkers for PVTT. Methods: We analyzed publicly available datasets from the Gene Expression Omnibus to identify differentially expressed lncRNAs and mRNAs across three comparisons: normal vs. HCC, normal vs. PVTT, and HCC vs. PVTT. Transcriptional profiles were characterized, and proteins interacting with HCC- and PVTT-specific lncRNAs were screened using online databases, revealing that all interacting proteins were transcription factors (TFs). We constructed lncRNA-TF-target gene regulatory networks by intersecting TF target genes with differentially expressed genes (DEGs) from each comparison. Protein-protein interaction (PPI) network analysis was performed to identify key clusters and hub genes, with TFs such as AR and ESR1 being highlighted. Gene Ontology analyses were conducted to understand the biological functions of the regulatory networks. Results: The study identified distinct transcriptional profiles for normal, HCC, and PVTT samples. Key regulatory networks, involving lncRNAs, TFs, and target genes, were constructed, and significant hub genes, including AR and ESR1, were identified as potential therapeutic targets. PPI network analysis revealed important clusters associated with PVTT progression, while Gene Ontology analyses provided insights into relevant biological functions. Conclusions: This study presents a novel framework for understanding lncRNA-TF-mediated gene regulation in PVTT. It identifies potential therapeutic targets and prognostic biomarkers that could facilitate the development of targeted therapies for PVTT, offering new opportunities to improve clinical outcomes.

The farnesoid X receptor (FXR) is a nuclear receptor (NR) known to obligately heterodimerize with the retinoid X receptor (RXR). FXR is expressed as four isoforms (α1-α4) that drive transcription from IR-1 (inverted repeat-1) response elements (REs). Recently, we found that FXR isoforms α2/α4 also activate transcription from non-canonical ER-2 (everted repeat-2) REs, mediating most metabolic effects of general FXR activation. Here, we explored molecular determinants of regulation by FXRα2 from ER-2 REs through quantitative interaction proteomics, site-directed mutagenesis and transcriptomics. We discovered FXRα2 binds to and activates ER-2 elements in vitro and in reporter assays independently of RXR. Genome-wide binding analysis in mouse liver revealed higher ER-2 motif enrichment in FXR sites lacking RXR. Abrogation of FXRα2:RXR heterodimerization abolished IR-1, but preserved ER-2 transactivation. Transcriptome-wide, RXR overexpression inhibited 25% of FXRα2 targets in HepG2. These genes were specifically activated by the heterodimerization-deficient mutant FXRα2L434R, enriched for ER-2 motifs at their promoters, and involved in lipid metabolism and ammonia detoxification. In conclusion, RXR acts as a molecular switch, inhibiting FXRα2 activation from ER-2 while enhancing it from canonical IR-1 REs. Our results showcase FXR as the first NR with isoform-specific RXR-independent REs, highlighting a new layer of regulation and complexity for RXR-heterodimerizing NRs.

This study aimed to investigate the role of the autophagy-related long noncoding RNA (lncRNA) MIR210HG in hepatocellular carcinoma and its potential as a therapeutic target.

LncRNA MIR210HG expression and its correlation with survival outcomes in hepatocellular carcinoma patients were analyzed using data from The Cancer Genome Atlas (TCGA). Kaplan-Meier and Cox regression analyses were conducted to assess survival correlations. Quantitative reverse transcription PCR was used to measure lncRNA MIR210HG expression in liver cancer cells and normal liver cells. Functional assays, including CCK-8, Transwell, flow cytometry, and western blot, were performed to evaluate the effects of lncRNA MIR210HG on cell proliferation, invasion, apoptosis, and autophagy in hepatocellular carcinoma.

Elevated lncRNA MIR210HG expression correlated with poor overall survival in hepatocellular carcinoma patients. LncRNA MIR210HG expression was significantly up-regulated in hepatocellular carcinoma cells compared to normal liver cells. Knockdown of lncRNA MIR210HG inhibited cell proliferation and autophagy, while promoting apoptosis in hepatocellular carcinoma cells, findings that were confirmed through both in vitro and in vivo studies.

The findings suggest that lncRNA MIR210HG contributes to hepatocellular carcinoma progression by regulating autophagy and could serve as a promising therapeutic target in hepatocellular carcinoma treatment strategies.

Fibrolamellar Hepatocellular Carcinoma (FLC) is a rare liver cancer characterized by a fusion oncokinase of the genes DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). A few FLC-like tumors have been reported showing other alterations involving PKA. To better understand FLC pathogenesis and the relationships among FLC, FLC-like, and other liver tumors, we performed a massive multi-omics analysis. RNA-seq data of 1412 liver tumors from FLC, hepatocellular carcinoma, hepatoblastoma and intrahepatic cholangiocarcinoma are analyzed, obtaining transcriptomic signatures unrestricted by experimental processing methods. These signatures reveal which dysregulations are unique to specific tumors and which are common to all liver cancers. Moreover, the transcriptomic FLC signature identifies a unifying phenotype for all FLC tumors regardless of how PKA was activated. We study this signature at multi-omics and single-cell levels in the first spatial transcriptomic characterization of FLC, identifying the contribution of tumor, normal, stromal, and infiltrating immune cells. Additionally, we study FLC metastases, finding small differences from the primary tumors.

Hepatocellular carcinoma (HCC) represents a significant health burden in Egypt, largely attributable to the endemic prevalence of hepatitis B and C viruses. Early identification of HCC remains a challenge due to the lack of widespread screening among at-risk populations. The objective of this study was to assess the utility of machine learning in predicting HCC by analyzing the combined expression of lncRNAs and conventional laboratory biomarkers. Plasma levels of four lncRNAs (LINC00152, LINC00853, UCA1, and GAS5) were quantified in a cohort of 52 HCC patients and 30 age-matched controls. The individual diagnostic performance of each lncRNA was assessed using ROC curve analysis. Subsequently, a machine learning model was constructed using Python's Scikit-learn platform to integrate these lncRNAs with additional clinical laboratory parameters for HCC diagnosis. Individual lncRNAs exhibited moderate diagnostic accuracy, with sensitivity and specificity ranging from 60 to 83% and 53-67%, respectively. In contrast, the machine learning model demonstrated superior performance, achieving 100% sensitivity and 97% specificity. Notably, a higher LINC00152 to GAS5 expression ratio significantly correlated with increased mortality risk. The integration of lncRNA biomarkers with conventional laboratory data within a machine learning framework demonstrates significant potential for developing a precise and cost-effective diagnostic tool for HCC. To enhance the model's robustness and prognostic capabilities, future studies should incorporate larger cohorts and explore a wider array of lncRNAs.

Disulfidptosis is a novel programmed cell death mode that has been reported to play a role in oncogenesis. Increasing evidences suggest that the long non-coding RNAs (lncRNAs) play crucial roles in the initiation and progression of bladder cancer (BLCA). However, the role and prognostic value of disulfidptosis-related lncRNAs in BLCA remain unknown.The aim of this study was to construct and validate a disulfidptosis-related lncRNA risk model for predicting the prognosis of BLCA patients. A risk model consisting of 5 disulfidptosis-related lncRNAs was developed to predict the prognosis of BLCA patients. The overall survival (OS) of BLCA patients in the high-risk group was significantly shorter than that in the low-risk group (P < 0.05). The effectiveness of this model was validated using the receiver operating characteristic (ROC) curve analysis, and this model proved superior in prognostic accuracy compared with other clinical features. Furthermore, the tumor immune dysfunction and exclusion (TIDE) score in the high-risk group was significantly higher than that in the low-risk group, suggesting that the high-risk group had a less favorable response to immunotherapy. Simultaneously, patients in the low-risk group exhibited significantly higher sensitivity to CTLA-4 monoclonal antibody therapy compared to those in the high-risk group, suggesting potential benefits of immunotherapy for patients in the low-risk group. The combination of high risk and low tumor mutational burden (TMB) could further shortened the OS of BLCA patients. Lastly, the drug sensitivity analysis revealed that the BLCA cells in the high-risk group showed an increased sensitivity to cisplatin, sunitinib, cetuximab, axitinib, docetaxel, saracatinib, vinblastine and pazopanib compared with those in the low-risk group. According to the Quantitative real time PCR results, we found that five lncRNAs of the risk model were more highly expressed in BCa cell lines than human immortalized uroepithelial cell line. The disulfidptosis-related lncRNA risk model has a valuable effect in assessing the prognosis of BLCA patients.

Triptolide (TP) is involved in the progression of liver cancer. However, the detailed molecular network regulated through TP is still unclear. Long non-coding RNA (LncRNA) SLC9A3 exerts roles in various pathological progresses. Nevertheless, whether SLC9A3 affects the sensitivity of liver cancer cells to TP have not been uncovered. The content of SLC9A3-AS1 and miR-449b-5p was estimated by utilizing quantitative real-time polymerase-chain reaction (qRT-PCR). Cell counting kit 8 (CCK-8) assay was introduced to assess cell viability. Additionally, cell viability as well as invasion was tested via transwell assay. The direct binding between miR-449b-5p and SLC9A3-AS1 or LDHA was confirmed through luciferase reporter gene assay. Moreover, glycolysis rate was tested by calculating the uptake of glucose in addition to the production of lactate in Huh7 cells. LncRNA SLC9A3-AS1 was up-regulated in liver cancer tissue samples and cells. Knockdown of SLC9A3-AS1 notably further inhibited viability, migration as well as invasion in Huh7 cells. MiR-449b-5p was the direct downstream miRNA of SLC9A3-AS1 and was down-regulated by SLC9A3-AS1 in Huh7 cells. In addition, miR-449b-5p was reduced in liver cancer tissues and cells. Overexpressed miR-449b-5p increased the sensitivity of Huh7 cells to TP remarkably. Moreover, miR-449b-5p negatively regulated LDHA expression in Huh7 cells. This work proved that SLC9A3-AS1 increased the sensitivity of liver cancer cells to TP by regulating glycolysis rate mediated via miR-449b-5p/LDHA axis. These findings implied that TP is likely to be a potent agent for treating patients diagnosed with liver cancer.

Portal vein thrombosis, a relatively frequent complication associated with hepatocellular carcinoma (HCC) and liver cirrhosis, is recognized as a significant global health concern. This is mainly due to these conditions' high prevalence and potentially severe outcomes. The aim of our study was to conduct a comprehensive literature review to evaluate the efficacy, accuracy, and clinical implications of 18F-FDG PET-CT in diagnosing and managing portal vein tumor thrombosis (PVTT) in patients with HCC. HCC, which accounts for 80% of liver malignancies, ranks as the fourth most prevalent cancer globally and is a significant contributor to cancer-related mortality. The majority of HCC patients are diagnosed at an advanced stage, leading to a deterioration in patient outcomes. Involvement of the portal vein is also a significant negative factor. This review analyzes the application of 18F-FDG PET-CT in the detection and management of PVTT in patients with HCC, with an emphasis on the importance of the maximum standardized uptake value as an essential diagnostic and prognostic marker. 18F-FDG PET-CT is invaluable for detecting recurrence and guiding management strategies, particularly in patients with high-grade HCC, and plays a pivotal role in differentiating malignant portal vein thrombi from their benign counterparts.

Microorganisms in eutrophic water play a vital role in nitrogen (N) removal, which contributes significantly to the nutrient cycling and sustainability of eutrophic ecosystems. However, the mechanisms underlying the interactions and adaptation strategies of the N removal microorganisms in eutrophic ecosystems remain unclear. We thus analyzed field sediments collected from a eutrophic freshwater ecosystem, enriched the N removal microorganisms, examined their function and adaptability through amplicon, metagenome and metatranscriptome sequencing. We found that the N removal activities could be affected through potential competition and inhibition among microbial metabolic pathways. High-diversity microbial communities generally increased the abundance and expression of N removal functional genes. Further enrichment experiments showed that the enrichment of N removal microorganisms led to a development of simplified but more stable microbial communities, characterized by similar evolutionary patterns among N removal microorganisms, tighter interactions, and increased adaptability. Notably, the sustained provision of NH4+ and NO2- during the enrichment could potentially strengthen the interconnections among denitrification, anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA) processes. Moreover, the identification of shared metabolic traits among denitrification, anammox and DNRA implies important cooperative associations and adaptability of N removal microorganisms. Our findings highlight the microbial interactions affect the adaptive strategies of key microbial taxa involved in N removal.

Patients with primary multifocal hepatocellular carcinoma (HCC) have a poor prognosis and often experience a high rate of treatment failure. Multifocal HCC is mainly caused by intrahepatic metastasis (IM), and though portal vein tumor thrombosis (PVTT) is considered a hallmark of IM, the molecular mechanism by which primary HCC cells invade the portal veins remains unclear. Therefore, it is necessary to recognize the early signs of metastasis of HCC to arrange better treatment for patients.

To determine the differential molecular features between primary HCC with and without phenotype of metastasis, we used the CIBERSORTx software to deconvolute cell types from bulk RNA-Seq based on a single-cell transcriptomic dataset. According to the relative abundance of tumorigenic and metastatic hepatoma cells, VEGFA+ macrophages, effector memory T cells, and natural killer cells, HCC samples were divided into five groups: Pro-T, Mix, Pro-Meta, NKC, and MemT, and the transcriptomic and genomic features of the first three groups were analyzed. We found that the Pro-T group appeared to retain native hepatic metabolic activity, whereas the Pro-Meta group underwent dedifferentiation. Genes highly expressed in the group Pro-Meta often signify a worse outcome.

The HCC cohort can be well-typed and prognosis predicted according to tumor microenvironment components. Primary hepatocellular carcinoma may have obtained corresponding molecular features before metastasis occurred.

Hepatocellular carcinoma (HCC), the most common form of primary liver cancers, accounts for a significant portion of cancer-related death globally. However, the molecular mechanisms driving the onset and progression of HCC are still not fully understood. Emerging evidence has indicated that non-protein-coding regions of genomes could give rise to transcripts, termed non-coding RNA (ncRNA), forming novel functional driving force for aberrant cellular activity. Over the past decades, overwhelming evidence has denoted involvement of a complex array of molecular function of ncRNAs at different stages of HCC tumorigenesis and progression. In this context, several pre-clinical studies have highlighted the potentials of ncRNAs as novel therapeutic modalities in the management of human HCC. Moreover, N6-methyladenosine (m6A) modification, the most prevalent form of internal mRNA modifications in mammalian cells, is essential for the governance of biological processes within cells. Dysregulation of m6A in ncRNAs has been implicated in human carcinogenesis, including HCC. In this review, we will discuss dysregulation of several hallmark ncRNAs (miRNAs, lncRNAs, and circRNAs) in HCC and address the latest advances for their involvement in the onset and progression of HCC. We also focus on dysregulation of m6A modification and various m6A regulators in the etiology of HCC. In the end, we discussed the contemporary preclinical and clinical application of ncRNA-based and m6A-targeted therapies in HCC.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. It is a major public health problem worldwide, and it is often diagnosed at advanced stages, when no effective treatment options are available. Extracellular vesicles (EVs) are nanosized double-layer lipid vesicles containing various biomolecule cargoes, such as lipids, proteins, and nucleic acids. EVs are released from nearly all types of cells and have been shown to play an important role in cell-to-cell communication. In recent years, many studies have investigated the role of EVs in cancer, including HCC. Emerging studies have shown that EVs play primary roles in the development and progression of cancer, modulating tumor growth and metastasis formation. Moreover, it has been observed that non-coding RNAs (ncRNAs) carried by tumor cell-derived EVs promote tumorigenesis, regulating the tumor microenvironment (TME) and playing critical roles in the progression, angiogenesis, metastasis, immune escape, and drug resistance of HCC. EV-related ncRNAs can provide information regarding disease status, thus encompassing a role as biomarkers. In this review, we discuss the main roles of ncRNAs present in HCC-derived EVs, including micro(mi) RNAs, long non-coding (lnc) RNAs, and circular (circ) RNAs, and their potential clinical value as biomarkers and therapeutic targets.

Circular RNAs (circRNAs) play critical roles in the initiation and progression of various cancers. However, the potential functional roles of circSNX14 in hepatocellular carcinoma (HCC) remain largely unknown. CircSNX14 expression pattern was analyzed in HCC tissues and cell lines via qRT-PCR. The effects of circSNX14 on cell proliferation, invasion, angiogenesis, and Epithelial-mesenchymal transition (EMT) were investigated by overexpression experiments. The role of circSNX14 in the tumorigenesis of HCC cells was examined using in vivo xenograft mouse model. The interaction between circSNX14, miR-562, and Large Tumor Suppressor Kinase 2 (LATS2) mRNA was confirmed by Luciferase reporter assay and RNA immunoprecipitation (RIP) analysis. CircSNX14 was significantly down-regulated in HCC tissues and cell lines, and its down-regulation was correlated with a poor prognosis in HCC patients. In the following functional experiments, circSNX14 overexpression remarkably suppressed the proliferation and invasion of HCC cells, and attenuated the mesenchymall status. circSNX14 overexpression also suppressed the tumorigenesis of HCC cells in the mouse model. We further revealed the interaction of circSNX14 and miR-562, and miR-562 could suppress the expression of LATS2 by interacting with its mRNA. The negative correlation of circSNX14 and miR-562, negative correlation of miR-562 and LATS2, and positive correlation of circSNX14 and LATS2 have been confirmed by Pearson correlation in the HCC samples. Collectively, these results reveal a novel role of circSNX14/miR-562/LATS2 axis in regulating the malignant progression of HCC cancer progression, indicating the tumor suppressor role of circSNX14 and its potential as a prognostic biomarker.

Colorectal cancer is one of the most common malignant tumors. At the advanced stage of colorectal cancer, cancer cells migrate with the blood to the liver from the hepatic portal vein, eventually resulting in a portal vein tumor thrombus (PVTT). To date, the progression of the early onset of PVTT [portal vein microthrombus (PVmT) induced by tumors] is unclear. Herein, we developed an on-chip PVmT model by loading the spheroid of colorectal cancer cells into the portal vein of a hepatic lobule chip (HLC). On the HLC, the progression of PVmT was presented, and early changes in metabolites of hepatic cells and in structures of hepatic plates and sinusoids induced by PVmT were analyzed. We replicated intrahepatic angiogenesis, thickened blood vessels, an increased number of hepatocytes, disordered hepatic plates, and decreased concentrations of biomarkers of hepatic cell functions in PVmT progression on a microfluidic chip for the first time. In addition, the combined therapy of thermo-ablation and chemo-drug for PVmT was preliminarily demonstrated. This study provides a promising method for understanding PVTT evolution and offers a valuable reference for PVTT therapy.

Vascular invasion is a major route for intrahepatic and distant metastasis in hepatocellular carcinoma (HCC) and is a strong negative prognostic factor. Circular RNAs (circRNAs) play important roles in tumorigenesis and metastasis. However, the regulatory functions and underlying mechanisms of circRNAs in the development of vascular invasion in HCC are largely unknown.

High throughput sequencing was used to screen dysregulated circRNAs in portal vein tumor thrombosis (PVTT) tissues. The biological functions of candidate circRNAs in the migration, vascular invasion, and metastasis of HCC cells were examined in vitro and in vivo. To explore the underlying mechanisms, RNA sequencing, MS2-tagged RNA affinity purification, mass spectrometry, and RNA immunoprecipitation assays were performed.

circRNA sequencing followed by quantitative real-time PCR (qRT-PCR) revealed that circRNA pleckstrin and Sect. 7 domain containing 3 (circPSD3) was significantly downregulated in PVTT tissues. Decreased circPSD3 expression in HCC tissues was associated with unfavourable characteristics and predicted poor prognosis in HCC. TAR DNA-binding protein 43 (TDP43) inhibited the biogenesis of circPSD3 by interacting with the downstream intron of pre-PSD3. circPSD3 inhibited the intrahepatic vascular invasion and metastasis of HCC cells in vitro and in vivo. Serpin family B member 2 (SERPINB2), an endogenous bona fide inhibitor of the urokinase-type plasminogen activator (uPA) system, is the downstream target of circPSD3. Mechanistically, circPSD3 interacts with histone deacetylase 1 (HDAC1) to sequester it in the cytoplasm, attenuating the inhibitory effect of HDAC1 on the transcription of SERPINB2. In vitro and in vivo studies demonstrated that circPSD3 is a promising inhibitor of the uPA system.

circPSD3 is an essential regulator of vascular invasion and metastasis in HCC and may serve as a prognostic biomarker and therapeutic target.

Long noncoding RNAs (lncRNAs) contribute to hepatocellular carcinoma (HCC) progression and development. However, the function and molecular mechanisms of action of LINC01343 in HCC remain unclear. qRT-PCR and western blotting were performed to assess miR-526b-5p, LINC01343, and ROBO1 levels in HCC cell lines and tissue samples. Flow cytometry, transwell, and cell counting kit-8 assays were conducted in vitro to assess how LINC01343 influences the apoptosis, migration, and proliferation of HCC cells. In addition, the role of LINC01343 in the growth of tumors was verified using an in vivo xenograft tumor assay. Specific binding of miR-526b-5p to LINC01343/ROBO1 was validated using RNA immunoprecipitation and dual-luciferase reporter experiments. LINC01343 was upregulated in HCC cells and tissues. In vitro, LINC01343-knockdown Hep3B and Huh-7 cells exhibited enhanced apoptosis and suppressed proliferation and migration. An in vivo study further validated that LINC01343-knockdown repressed tumor growth. In terms of mechanisms, LINC01343 directly sponged miR-526b-5p, negatively modulating its expression. Moreover, further experiments revealed that inhibiting miR-526b-5p could counteract the tumor-suppressive effects of LINC01343-knockdown in Hep3B and Huh-7 cells. ROBO1 was identified as a direct target of miR-526b-5p. ROBO1 knockdown weakens the migratory and proliferative abilities of Hep3B and Huh-7 cells. Nonetheless, the inhibition of miR-526b-5p mitigated this effect. These findings revealed that LINC01343 serves as a vital oncogene in HCC. Moreover, the LINC01343/miR-526b-5p/ROBO1 axis may be a prospective target for HCC treatment.

The correlations between cuproptosis and long noncoding RNAs (lncRNAs) with the tumor microenvironment (TME), immunotherapy, and some other characteristics of hepatocellular carcinoma (HCC) remain unclear.

Sixteen cuproptosis regulators and 356 cuproptosis-related lncRNAs (CRLnc) were identified from 374 HCC profiles in The Cancer Genome Atlas (TCGA) database. Six differentially expressed CRLnc were selected, and a prognostic risk model based on the CRLnc signature (CRLncSig) was constructed. The prognostic power of the model was verified. Moreover, a cuproptosis-related gene cluster (CRGC) was generated based on six lncRNAs and differentially expressed genes. The relationship between immune cell infiltration in the TME, immunotherapy, CRLncSig, and CRGC was demonstrated through various algorithms, Tumor Immune Dysfunction and Exclusion (TIDE), tumor mutational burden (TMB), etc. Potential drugs and sensitivity to those agents were evaluated for the risk model. LncRNA AL158166.1 was selected and verified in HCC tissues and cell lines, the impact of its knockdown and overexpression in HCC cells was examined, and the copper (Cu) concentration and the cuproptosis-related gene expression were detected.

A CRLncSig prognostic risk model with good predictive ability was constructed. The low-risk group had a longer overall survival (OS), lower tumor purity, more extensive immune cell infiltration, higher immune score, enrichment in immune-activated pathways, and more positive response to immunotherapy versus the high-risk group. CRGC-B exhibited the best OS and the lowest tumor stage; the immune cell infiltration analysis was similar to the low-risk group in CRLncSig. CRGC-B belonged to the "immune-high" group of the TME. The low-risk group had a higher TIDE score and susceptibility to antitumor drugs. The lncRNA AL158166.1 had the highest hazard ratio. The levels of AL158166.1 were higher in HCC tissues versus healthy tissues. Knockdown of AL158166.1 could lead to an increase in intracellular Cu concentration, induce DLAT low expression, and inhibit the proliferation and migration of HCC cells, whereas overexpression of AL158166.1 exerted the reverse effect.

Overall, a new CRLncSig prognostic risk model and a cuproptosis-related molecular signature were constructed and evaluated. The model and signature were associated with the prognosis, immune infiltration, and immunotherapy of HCC. Inhibiting the lncRNA AL158166.1 may induce cuproptosis and showed potential for the inhibition of tumors. Evaluation of the CRLnc, CRLncSig, and CRGC may enhance our understanding of the TME, determine the effectiveness of immunotherapy, and act as a marker for the prognosis of HCC.

Gastrointestinal (GI) cancer includes many cancer types, such as esophageal, liver, gastric, pancreatic, and colorectal cancer. As the cornerstone of personalized medicine for GI cancer, liquid biopsy based on noninvasive biomarkers provides promising opportunities for early diagnosis and dynamic treatment management. Recently, a growing number of studies have demonstrated the potential of cell-free RNA (cfRNA) as a new type of noninvasive biomarker in body fluids, such as blood, saliva, and urine. Meanwhile, transcriptomes based on high-throughput RNA detection technologies keep discovering new cfRNA biomarkers. In this review, we introduce the origins and applications of cfRNA, describe its detection and qualification methods in liquid biopsy, and summarize a comprehensive list of cfRNA biomarkers in different GI cancer types. Moreover, we also discuss perspective studies of cfRNA to overcome its current limitations in clinical applications. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Sepsis is a high mortality disease which seriously threatens human life and health, for which the pathogenetic mechanism still unclear. There is increasing evidence showed that immune and inflammation responses are key players in the development of sepsis pathology. LncRNAs, which act as ceRNAs, have critical roles in various diseases. However, the regulatory roles of ceRNA in the immunopathogenesis of sepsis have not yet been elucidated.

In this study, we aimed to identify immune biomarkers associated with sepsis. We first generated a global immune-associated ceRNA (IMCE) network based on data describing interactions pairs of gene-miRNA and miRNA-lncRNA. Afterward, we excavated a dysregulated sepsis immune-associated ceRNA (SPIMC) network from the global IMCE network by means of a multi-step computational approach. Functional enrichment indicated that lncRNAs in SPIMC network have pivotal roles in the immune mechanism underlying sepsis. Subsequently, we identified module and hub genes (CD4 and STAT4) via construction of a sepsis immune-related PPI network. Then, we identified hub genes based on the modular structure of PPI network and generated a ceRNA subnetwork to analyze key lncRNAs associated with sepsis. Finally, 6 lncRNAs (LINC00265, LINC00893, NDUFA6-AS1, NOP14-AS1, PRKCQ-AS1 and ZNF674-AS1) that identified as immune biomarkers of sepsis. Moreover, the CIBERSORT algorithm and the infiltration of circulating immune cells types were performed to identify the inflammatory state of sepsis. Correlation analyses between immune cells and sepsis immune biomarkers showed that the LINC00265 was strongly positive correlated with the macrophages M2 (r = 0.77).

Collectively, these results may suggest that these lncRNAs (LINC00265, LINC00893, NDUFA6-AS1, NOP14-AS1, PRKCQ-AS1 and ZNF674-AS1) played important roles in the immune pathogenesis of sepsis and provide potential therapeutic targets for further researches on immune therapy treatment in patients with sepsis.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. Portal vein tumour thrombus (PVTT) is considered one of most fearful complications of HCC and is strongly associated with a poor prognosis. Clarification of the mechanisms underlying the formation and development of PVTT is crucial for developing novel therapeutic strategies for HCC patients. Several studies have been made to uncover that tumour microenvironment, stem cells, abnormal gene expression and non-coding RNAs deregulation are associated with PVTT in patients with HCC in the last decade. However, the exact molecular mechanisms of PVTT in patients with HCC are still largely unknown. In the present review, we briefly summarized the molecular mechanisms underlying the formation and development of PVTT in HCC.

Gene expression variance has been linked to organismal function and fitness but remains a commonly neglected aspect of molecular research. As a result, we lack a comprehensive understanding of the patterns of transcriptional variance across genes, and how this variance is linked to context-specific gene regulation and gene function. Here, we use 57 large publicly available RNA-seq data sets to investigate the landscape of gene expression variance. These studies cover a wide range of tissues and allowed us to assess if there are consistently more or less variable genes across tissues and data sets and what mechanisms drive these patterns. We show that gene expression variance is broadly similar across tissues and studies, indicating that the pattern of transcriptional variance is consistent. We use this similarity to create both global and within-tissue rankings of variation, which we use to show that function, sequence variation, and gene regulatory signatures contribute to gene expression variance. Low-variance genes are associated with fundamental cell processes and have lower levels of genetic polymorphisms, have higher gene-gene connectivity, and tend to be associated with chromatin states associated with transcription. In contrast, high-variance genes are enriched for genes involved in immune response, environmentally responsive genes, immediate early genes, and are associated with higher levels of polymorphisms. These results show that the pattern of transcriptional variance is not noise. Instead, it is a consistent gene trait that seems to be functionally constrained in human populations. Furthermore, this commonly neglected aspect of molecular phenotypic variation harbors important information to understand complex traits and disease.

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.

Circular RNAs (circRNAs), which are involved in various human malignancies, have emerged as promising biomarkers. The present study aimed to investigate unique expression profiles of circRNAs in hepatocellular carcinoma (HCC) and identify novel biomarkers associated with HCC development and progression.

CircRNA expression profiles of HCC tissues were jointly analyzed to identify differentially expressed circRNAs. Overexpression plasmid and siRNA targeting candidate circRNAs were used in functional assays in vitro. CircRNA-miRNA interactions were predicted using miRNAs expressed in the miRNA-seq dataset GSE76903. To further screen downstream genes targeted by the miRNAs, survival analysis and qRT-PCR were conducted to evaluate their prognostic role in HCC and construct a ceRNA regulatory network.

Three significantly upregulated circRNAs, hsa_circ_0002003, hsa_circ_0002454, and hsa_circ_0001394, and one significantly downregulated circRNA, hsa_circ_0003239, were identified and validated by qRT-PCR. Our in vitro data indicated that upregulation of hsa_circ_0002003 accelerated cell growth and metastasis. Mechanistically, DTYMK, DAP3, and STMN1, which were targeted by hsa-miR-1343-3p, were significantly downregulated in HCC cells when hsa_circ_0002003 was silenced and were significantly correlated with poor prognosis in patients with HCC.

Hsa_circ_0002003 may play critical roles in HCC pathogenesis and serve as a potential prognostic biomarker for HCC. Targeting the hsa_circ_0002003/hsa-miR-1343-3p/STMN1 regulatory axis could be an effective therapeutic strategy in patients with HCC.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the main cause of cancer-related death globally. Immune dysregulation of CD4⁺ T cells has been identified to play a role in the development of HCC. Nevertheless, the underlying molecular pathways of CD4⁺ T cells in HCC are not completely known. Thus, a better understanding of the dysregulation of the lncRNA-miRNA/mRNA network may yield novel insights into the etiology or progression of HCC. In this study, circulating CD4⁺ T cells were isolated from the whole blood of 10 healthy controls and 10 HCC patients for the next-generation sequencing of the expression of lncRNAs, miRNAs, and mRNAs. Our data showed that there were different expressions of 34 transcripts (2 lncRNAs, XISTs, and MIR222HGs; 29 mRNAs; and 3 other types of RNA) and 13 miRNAs in the circulating CD4⁺ T cells of HCC patients. The expression of lncRNA-XIST-related miRNAs and their target mRNAs was confirmed using real-time quantitative polymerase chain reaction (qPCR) on samples from 100 healthy controls and 60 HCC patients. The lncRNA-miRNA/mRNA regulation network was created using interaction data generated from ENCORI and revealed there are positive correlations in the infiltration of total CD4⁺ T cells, particularly resting memory CD4⁺ T cells, and negative correlations in the infiltration of Th1 CD4⁺ T cells.

Chronic liver diseases usually developed through stepwise pathological transitions under the persistent risk factors. The molecular changes during liver transitions are pivotal to improve liver diagnostics and therapeutics yet still remain elusive. Cumulative large-scale liver transcriptomic studies have been revealing molecular landscape of various liver conditions at bulk and single-cell resolution, however, neither single experiment nor databases enabled thorough investigations of transcriptomic dynamics along the progression of liver diseases. Here we establish GepLiver, a longitudinal and multidimensional liver expression atlas integrating expression profiles of 2469 human bulk tissues, 492 mouse samples, 409,775 single cells from 347 human samples and 27 liver cell lines spanning 16 liver phenotypes with uniformed processing and annotating methods. Using GepLiver, we have demonstrated dynamic changes of gene expression, cell abundance and crosstalk harboring meaningful biological associations. GepLiver can be applied to explore the evolving expression patterns and transcriptomic features for genes and cell types respectively among liver phenotypes, assisting the investigation of liver transcriptomic dynamics and informing biomarkers and targets for liver diseases.

Cuproptosis is an unusual form of cell death caused by copper accumulation in mitochondria. Cuproptosis is associated with hepatocellular carcinoma (HCC). Long noncoding RNAs (LncRNAs) have been shown to be effective prognostic biomarkers, yet the link between lncRNAs and cuproptosis remains unclear. We aimed to build a prognostic model of lncRNA risk and explore potential biomarkers of cuproptosis in HCC. Pearson correlations were used to derive lncRNAs co-expressed in cuproptosis. The model was constructed using Cox, Lasso, and multivariate Cox regressions. Kaplan-Meier survival analysis, principal components analysis, receiver operating characteristic curve, and nomogram analyses were carried out for validation. Seven lncRNAs were identified as prognostic factors. A risk model was an independent prognostic predictor. Among these seven lncRNAs, prostate cancer associated transcript 6 (PCAT6) is highly expressed in different types of cancer, activating Wnt, PI3K/Akt/mTOR, and other pathways; therefore, we performed further functional validation of PCAT6 in HCC. Reverse transcription-polymerase chain reaction results showed that PCAT6 was aberrantly highly expressed in HCC cell lines (HepG2 and Hep3B) compared to LO2 (normal hepatocytes). When its expression was knocked down, cells proliferated and migrated less. PCAT6 might be a potential biomarker for predicting prognosis in HCC.

Long noncoding RNAs (lncRNAs) are prominent as crucial regulators of tumor establishment and are repeatedly dysregulated in multiple cancers. Therefore, lncRNAs have been identified to play an essential function in carcinogenesis and progression of cancer at genetic and epigenetic levels. FENDRR (fetal-lethal noncoding developmental regulatory RNA) as a LncRNA is a hallmark of various malignancies. FENDRR is crucial for multiple organs' development, such as the lung and heart. The effects of FENDRR under signaling pathways in different cancers have been identified. In addition, it has been verified that FENDRR can affect the development and progression of various cancers. In addition, FENDRR expression has been associated with epigenetic regulation of target genes participating in tumor immunity. Furthermore, FENDRR downregulation was observed in various types of cancers, including colorectal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, gallbladder cancer, lung cancer, breast cancer, endometrial cancer, prostate cancer, chronic myeloid leukemia, osteosarcoma, and cutaneous malignant melanoma cells. Here, we review the biological functions and molecular mechanisms of FENDRR in several cancers, and we will discuss its potential as a cancer biomarker and as a probable option for cancer treatment.

Tumor thrombus of bone sarcomas represents a unique reservoir for various types of cancer and immune cells, however, the investigation of tumor thrombus at a single-cell level is very limited. And it is still an open question to identify the thrombus-specific tumor microenvironment that is associated with the tumor-adaptive immune response. Here, by analyzing bulk tissue and single-cell level transcriptome from the paired thrombus and primary tumor samples of osteosarcoma (OS) patients, we define the immunostimulatory microenvironment in tumor thrombus of OS with a higher proportion of tumor-associated macrophages with M1-like states (TAM-M1) and TAM-M1 with high expression of CCL4. OS tumor thrombus is found to have upregulated IFN-γ and TGF-β signalings that are related to immune surveillance of circulating tumor cells in blood circulation. Further multiplexed immunofluorescence staining of the CD3/CD4/CD8A/CD68/CCL4 markers validates the immune-activated state in the tumor thrombus samples. Our study first reports the transcriptome differences at a single-cell level between tumor thrombus and primary tumor in sarcoma.

Transcriptome complexity is emerging as an unprecedented and fascinating domain, especially by high-throughput sequencing technologies that have unveiled a plethora of new non-coding RNA biotypes. This review covers antisense long non-coding RNAs, i.e., lncRNAs transcribed from the opposite strand of other known genes, and their role in hepatocellular carcinoma (HCC). Several sense-antisense transcript pairs have been recently annotated, especially from mammalian genomes, and an understanding of their evolutionary sense and functional role for human health and diseases is only beginning. Antisense lncRNAs dysregulation is significantly involved in hepatocarcinogenesis, where they can act as oncogenes or oncosuppressors, thus playing a key role in tumor onset, progression, and chemoradiotherapy response, as deduced from many studies discussed here. Mechanistically, antisense lncRNAs regulate gene expression by exploiting various molecular mechanisms shared with other ncRNA molecules, and exploit special mechanisms on their corresponding sense gene due to sequence complementarity, thus exerting epigenetic, transcriptional, post-transcriptional, and translational controls. The next challenges will be piecing together the complex RNA regulatory networks driven by antisense lncRNAs and, ultimately, assigning them a function in physiological and pathological contexts, in addition to defining prospective novel therapeutic targets and innovative diagnostic tools.

KIN17 DNA and RNA binding protein (Kin17) is involved in the regulation of tumorigenesis of diverse human cancers. However, its role in the cancer progression and metastasis in hepatocellular carcinoma (HCC) remains largely unknown. Bioinformatics and immunohistochemistry staining were used to investigate the expression pattern of KIN17 and its prognostic value in HCC patients. The transwell, wound-healing assay was employed to determine the effects of KIN17 on migration and invasion of HCC cells in vitro. The tail veins model was employed to determine the effects of KIN17 on lung metastasis in vivo. The biological mechanisms involved in cell migration and invasion regulated by KIN17 were determined with Western blot analysis method. KIN17 expression was significantly increased in HCC tissues compared with adjacent normal tissues, with particularly higher in portal vein tumor thrombus and intrahepatic metastasis tissues. Patients with higher KIN17 expression experienced poor overall and disease free survival. KIN17 knockdown in HuH7 and HepG2 cells significantly reduced cell migration and invasion abilities, whereas its overexpression promoted migration and invasion in MHCC-97L and HepG2 cells in vitro and in vivo. In HuH7 and HepG2 cells, KIN17 knockdown inhibited the TGF-β/Smad2 pathway. In contrast, KIN17 overexpression stimulated TGF-β/Smad2 pathway in MHCC-97L and HepG2 cells, along with the genes involved in the epithelial-mesenchymal transition. These findings suggest that KIN17 promotes migration and invasion in HCC cells by stimulating the TGF-β/Smad2 pathway. KIN17 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.

Hepatocellular carcinoma (HCC) progresses sequentially in a stepwise pattern. Long noncoding RNA (lncRNA) can regulate the complex cascade of hepatocarcinogenesis. Our study aimed to elucidate the expression profile of H19 and MALAT1 during the different stages of hepatocarcinogenesis and the correlation between H19 and MALAT1 with the genes implicated in the carcinogenesis cascade. We employed a chemically induced hepatocarcinogenesis murine model to mimic the successive stages of human HCC development. Using real-time PCR, we analyzed the expression patterns of H19 and MALAT1, as well as the expression of biomarkers implicated in the Epithelial-Mesenchymal transition (EMT). The protein expression of the mesenchymal marker vimentin was also evaluated using immunohistochemistry in the stepwise induced stages. The histopathological evaluation of the liver tissue sections revealed significant changes during the experiment, with HCC developing at the final stage. Throughout the stages, there was a dynamic significant increase in the expression of H19 and MALAT1 compared to the normal control. Nevertheless, there was no significant difference between each stage and the preceding one. The tumor progression biomarkers (Matrix Metalloproteinases, vimentin, and β-catenin) exhibited the same trend of steadily increasing levels. However, in the case of Zinc finger E-box-binding homeobox 1 and 2 (ZEB1 and ZEB2), the significant elevation was only detected at the last stage of induction. The correlation between lncRNAs and the tumor progression biomarkers revealed a strong positive correlation between the expression pattern of H19 and MALAT1 with Matrix Metalloproteinases 2 and 9 and vimentin. Our findings imply that genetic and epigenetic alterations influence HCC development in a stepwise progressive pattern.

During the independent process of evolution in plants, photosynthesis appears to have been under convergent evolution to adapt to specific selection pressure in their geographical regions. However, it is unclear how lncRNA regulation and DNA methylation are involved in the phenotypic convergence in distinct lineages. Here, we present a large-scale comparative study of lncRNA transcription profile and whole-genome bisulfite sequencing (WGBS) data in two unrelated Populus species, selected from three relatively overlapping geographical regions. The results indicated that 39.75% lncRNAs of Populus tomentosa were shown to have homologous sequences in the 46.99% lncRNA of Populus simonii. Evolutionary analysis revealed that lncRNAs showed a rapid gain rate in the Populus lineage. Furthermore, co-expression networks in two Populus species identified eight lncRNAs that have the potential to simultaneously cis- or trans-regulate eight photosynthetic-related genes. These photosynthetic lncRNAs and genes were predominantly expressed in accessions from the southern region, indicating a conserved spatial expression in photosynthetic pathways in Populus. We also detected that most lncRNA targeted photosynthetic genes hypomethylated in promoter regions of Southern accessions compared with Northern accessions. Geographical DMRs correlated with genetic SNP variations in photosynthetic genes among Populus from the three geographic regions, indicating that DNA methylation coordinated with lncRNAs in convergent evolution of photosynthesis in Populus. Our results shed light on the evolutionary forces acting on patterns of lncRNA and DNA methylation, and provide a better understanding of the genetic and epigenetic mechanism in photosynthetic convergence evolution.

Portal vein tumor thrombosis (PVTT), a common complication of advanced hepatocellular carcinoma (HCC), remains the bottleneck of the treatments. Liver cancer cells potentially experienced multi-steps during PVTT process, including cancer cells leave from cancer nest, migrate in extracellular matrix, invade the vascular barrier, and colonize in the portal vein. Accumulated evidences have revealed numerous of molecular mechanisms including genetic and epigenetic regulation, cancer stem cells, immunosuppressive microenvironment, hypoxia, et al. contributed to the PVTT formation. In this review, we discuss state-of-the-art PVTT research on the potential molecular mechanisms and experimental models. In addition, we summarize PVTT-associated clinical trials and current treatments for PVTT and suppose perspectives exploring the molecular mechanisms and improving PVTT-related treatment for the future.

Long non-coding RNAs (LncRNAs) are single-stranded RNAs over 200 nucleotides in length that have no protein-coding function and have long been considered non-functional by-products of transcription. Recent studies have shown that dysregulation of lncRNAs may be involved in the malignant biological behavior of tumors. Targeted regulation of lncRNAs has become a research focus of anti-tumor treatment. LncRNAs heart and neural crest derivatives expressed 2 antisense RNA 1 (HAND2-AS1) was down-regulated in various tumors and can be used as a critical tumor regulator to modulate tumor cells proliferation, apoptosis, metastasis, invasion, metabolism and drug resistance. Additionally, aberrantly expressed HAND2-AS1 was closely related to the clinical pathological characteristics of cancer patients and serve as a promising tumor diagnostic and prognostic biomarker. This article aims to review the roles of HAND2-AS1 in tumorigenesis and development, as well as the underlying molecular mechanisms and clinical significance.

Hepatocellular carcinoma (HCC) remains a global health challenge, causing 600,000 deaths each year. Infectious factors, including hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV), have long been considered the major risk factors for the development and progression of HCC. These pathogens induce hepatocyte transformation through a variety of mechanisms, including insertional mutations caused by viral gene integration, epigenetic changes, and the induction of long-term immune dysfunction. The discovery of these mechanisms, while advancing our understanding of the disease, also provides targets for new diagnostic and therapeutic approaches. In addition, the discovery and research of chronic HEV infection over the past decade indicate that this common hepatitis virus also seems to have the potential to induce HCC. In this review, we provide an overview of recent studies on the link between hepatitis virus and HCC, as well as new diagnostic and therapeutic approaches to HCC based on these findings. Finally, we also discuss the potential relationship between HEV and HCC. In conclusion, these associations will further optimize the diagnosis and treatment of infection-associated HCC and call for better management policies.

Hepatocellular carcinoma (HCC) ranks the most common types of cancer worldwide. As the fourth leading cause of cancer-related deaths, its prognosis remains poor. Most patients developed HCC on the basis of chronic liver disease. Cirrhosis is an important precancerous lesion for HCC. However, the molecular mechanisms in HCC development are still unclear. To explore the changes at the level of transcriptome in this process, we performed RNA-sequencing on cirrhosis, HCC and paracancerous tissues. Continuously changing mRNA was identified using Mfuzz cluster analysis, then their functions were explored by enrichment analyses. Data of cirrhotic HCC patients were obtained from TCGA, and a fatty acid metabolism (FAM)-related prognostic signature was then established. The performance and immunity relevance of the signature were verified in internal and external datasets. Finally, we validated the expression and function of ADH1C by experiments. As a result, 2012 differently expressed mRNA were identified by RNA-sequencing and bioinformatics analyses. Fatty acid metabolism was identified as a critical pathway by enrichment analyses of the DEGs. A FAM-related prognostic model and nomogram based on it were efficient in predicting the prognosis of cirrhotic HCC patients, as patients with higher risk scores had shorter survival time. Risk scores calculated by the signature were then proved to be associated with a tumor immune environment. ADH1C were downregulated in HCC, while silence of ADH1C could significantly promote proliferation and motility of the HCC cell line.

Hepatocellular carcinoma (HCC) has a high degree of malignancy and poor prognosis. N6-methyladenosine (m6A) modifications and microRNAs (miRNAs) play pivotal roles in tumorigenesis and development. However, the role of m6A-related miRNAs in HCC has not been clarified yet. This study aimed to identify the role of m6A-miRNAs in HCC prognosis through bioinformatics analysis.

The clinicopathological information and RNA sequencing data of 369 HCC tumor tissues and 49 tumor-adjacent tissues were downloaded from the TCGA database. A total of 23 m6A regulators were extracted to evaluated the m6A-related miRNAs using Pearson's correlation analysis. Then, we selected prognosis-related m6A-miRNAs using a univariate Cox regression model and used the consensus cluster analysis to explore the characteristics of the m6A-miRNAs. The coefficient of the least absolute shrinkage and selection operator (LASSO) Cox regression was applied to construct a prognostic risk score model. The receiver operated characteristic (ROC) analysis was applied to evaluate the prognostic value of the signature. The biological functions of targeted genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Then, to validate the potential predictive value for prognosis, the miRNA expression profiles from the GSE76903 and GSE6857 were used. Single sample Gene Set Enrichment Analysis (ssGSEA) and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) were applied to assess the immune microenvironment of HCC. Additionally, a meta-analysis was used to verify the prognostic value of the m6A-microRNAs. RT-PCR was applied to validated the expression of miRNAs in HCC tissues. Cell viability, transwell assay and RNA m6A dot blot assays of HCC cells was applied to access the function of miR-17-5p.

The expression of 48 m6A-related miRNAs was identified and 17 prognostic m6A-miRNAs was discovered. The expression profile of those 17 miRNAs was divided into three clusters, and these clusters were associated with the tumor microenvironment (TME) and prognosis. The nine m6A-related miRNA signature was associated with the prognosis of HCC, the AUC of the ROC was 0.771(TCGA dataset), 0.788(GSE76903) and 0.646(GSE6857). The TME and the expression of immune checkpoint molecules were associated with the risk score. The meta-analysis also validated the prognostic value of the m6A-related miRNAs (miR182-5p (HR:1.58, 95%CI:1.04-2.40) and miR-17-5p (HR:1.58, 95%CI: 1.04-2.40)). The expression of miR-17-5p was upregulated in HCC tissues and miR-17-5p showed an oncogenic role in HCC cells.

The clinical innovation is the use of m6A-miRNAs as biomarkers for predicting prognosis regarding immunotherapy response in HCC patients.