RPF2 regulates the protein kinase B/mammalian target of rapamycin pathway in the pathogenesis of Helicobacter pylori

Abstract

BACKGROUND

RPF2 is a crucial factor in ribosome synthesis, which has been linked to the development of several cancers. However, the mechanism of RPF2 in gastric carcinogenesis is unclear.

AIM

To explore the role and mechanism of RPF2 in the pathogenesis of Helicobacter pylori (H. pylori) infection.

METHODS

GES-1 was co-cultured with H. pylori in vitro to detect changes in the expression of RPF2. Overexpression and silencing of RPF2 were performed. Quantitative real-time polymerase chain reaction (q-PCR) and Western blot (WB) were used to determine mRNA and protein expression of RPF2, protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and epithelial-mesenchymal transition-related factors MMP2 and MMP9; cell counting kit 8 and wound healing assays were utilized to evaluate cell viability and migratory capacity; q-PCR, WB, and immunohistochemistry were employed to establish RPF2 expression in cancer tissues.

RESULTS

H. pylori facilitated RPF2 expression and triggered AKT/mTOR signaling pathway. Functional experiments showed that RPF2 overexpression could promote a series of malignant transformations such as cell proliferation, cell migration and invasion, and further enhance AKT/mTOR signaling pathway activation. RPF2 knockdown had the opposite effect. In addition, RPF2 expression was higher in gastric cancer tissues than in adjacent tissues.

CONCLUSION

RPF2 plays a significant role in the pathogenic mechanism of H. pylori infection and may be useful in the detection and management of gastric cancer caused by H. pylori infection.

Key Words: RPF2; Helicobacter pylori; Epithelial-mesenchymal transition; Protein kinase B/mammalian target of rapamycin pathway; Gastric cancer

Core Tip: Helicobacter pylori (H. pylori) has a profound impact on gastric cancer development. RPF2 is overexpressed in cancer and can enhance cancer cell proliferation. However, the mechanism of RPF2 in the pathogenesis of H. pylori infection remains largely unknown. We found that H. pylori infection increased RPF2 expression, affecting the malignant behavior of gastric epithelial cells by regulating the protein kinase B/mammalian target of rapamycin pathway. This study is the first to explore the mechanism of RPF2 in the pathogenesis of H. pylori infection.

INTRODUCTION

Gastric cancer is one of the most common tumors in human beings with the fifth highest incidence and mortality rate among the causes of death worldwide and the highest prevalence in eight countries[1,2]. Among them, Helicobacter pylori (H. pylori) infection can be effectively prevented and treated as one of the main causes of gastric cancer development[3,4].

H. pylori is a microaerobic bacterium that stimulates leukocytes to secrete large amounts of cytokines to participate in immune and inflammatory responses. When an immune or inflammatory response occurs, the expression and function of the relevant inhibitory factors may become dysregulated and cause the persistence of H. pylori-induced gastric inflammation, which can lead to the development of gastric cancer[5]. The colonization of H. pylori in human stomach tissue is affected by many factors. Interactions between virulence factors, phase, variable genes, and host genetics collectively determine the outcome of infection, with specific bacterial virulence factors more likely to cause severe gastric disease when co-occurring with the genetic background of susceptible hosts, and this complex interplay makes the outcome of H. pylori infection significantly different among individuals[6].

Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells acquire mesenchymal characteristics[7,8]. The occurrence of EMT contributes to embryogenesis and is essential for physiological processes such as wound healing; its pathologic reactivation plays a vital role in diseases such as organ fibrosis and cancer progression to metastasis[9]. In addition, H. pylori often leads to pathogenesis through progressive EMT[4,10]. H. pylori infection can up-regulate transforming growth factor-β 1 to activate EMT[11]. CagA, a cytotoxin released during H. pylori infection, triggers phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathway activation and enhances fat mass and obesity-associated protein (FTO) transcription through proto-oncogenes; elevated FTO promotes EMT in gastric cancer cells[12,13].

The AKT/mammalian target of rapamycin (mTOR) pathway is intricately involved in EMT progression[14,15]. In the pathogenic process of H. pylori infection, it can induce Dickkopf-related protein (DKK) 1 activation, promote the interaction of DKK1 and cytoskeleton-related protein 4, and promote gastric cancer through the PI3K/AKT/mTOR pathway[16]. During gastric cancer development, aberrant AKT/mTOR pathway activation can induce increased cell viability and enhanced migration, and can cause cancer cells to evade apoptosis through the dysregulation of anti-apoptotic and pro-apoptotic genes, thereby promoting carcinogenesis[17].

RPF2, a protein-coding gene belonging to the BRIX family, can participate in the assembly of ribosomal large subunits and is required for the maturation of the 60S ribosomal subunit[18-20]. Ribosome biosynthesis is closely related to the development of many cancers, including gastric cancer[21-23]. Studies have shown that increased ribosome content in epithelial cells can promote cell migration after cancerous transformation[24]. RRS1, which forms a ribosomal subunit together with RPF2, is positively correlated with the development of various cancers[25-31]. In addition, studies have shown that RPF2 is highly expressed in patients with hepatocellular carcinoma, and its expression level correlates with hepatocellular carcinoma tumor node metastasis stage, tumor size, tumor differentiation, and vascular invasion[32]. In colorectal cancer, RPF2 triggers EMT by activating the AKT/GSK-3β signaling pathway in colon cancer cells, thereby enhancing the migratory and invasive capabilities of these cells[33]. However, the role and mechanism of RPF2 in H. pylori pathogenesis are still unclear.

Therefore, we investigated the role and mechanism of H. pylori infection on RPF2, the AKT/mTOR pathway, and EMT in gastric epithelial cells. This paper establishes the foundation for further defining the pathogenic mechanism of H. pylori infection and the identification of biological targets for early diagnosis and treatment.

MATERIALS AND METHODS

Strain culture and cell culture

H. pylori strain (MEL-HP27, Hp27) was isolated and cultured from the gastric tissue specimens of patients with chronic atrophic gastritis in Zhengzhou, identified for preservation, and cultured with Columbia blood agar medium in a constant temperature incubator at 37 °C, 50 mL/L carbon dioxide. The gastric mucosal epithelial cell line GES-1 was cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin in a constant temperature incubator at 37 °C, 50 mL/L carbon dioxide.

Cell infection with H. pylori

H. pylori Hp27 was co-cultured with GES-1 for 24 hours at a multiplicity of infection of 100:1.

RNA extraction and quantitative real-time polymerase chain reaction

Total RNA was extracted from cells using Trizol reagent according to the manufacturer’s instructions. SYBR quantitative real-time polymerase chain reaction (qPCR) master mix was used to conduct q-PCR following reverse transcription per the manufacturer’s guidelines.

Western blot

Proteins were isolated from cell lines using radio immunoprecipitation assay buffer with protease and phosphatase inhibitors. Protein samples were characterized using a bicinchoninic acid assay kit and then heated at 98 °C for 15 minutes. Gel electrophoresis was performed using 30 μg protein and then transferred to a polyvinylidene difluoride membrane. The membranes were labeled with primary antibody and incubated in a blocking buffer containing secondary antibody for 1 hour.

Transfection of the GES-1 cell line

The RPF2 overexpression plasmid was constructed (by Shenzhen BGI Gene Technology Co., LTD, China) and transfected into GES-1 cells. The overexpression cell model was divided into four groups: PcDNA3.1 (+), pcDNA3.1 (+) + Hp27, pcDNA-RPF2 and pcDNA-RPF2 + Hp27. RPF2 small interfering (si) RNA was designed (by Heyuan Biotechnology Co., LTD, China) and transfected. The silencing cell model was divided into four groups: Si-negative control (NC), si-NC + Hp27, si-RPF2 and si-RPF2 + Hp27. Plasmids and siRNAs were transfected into GES-1 cells using Lipofectamine 3000 Reagent (Invitrogen) and opti-MEM (Gibco).

Cell counting kit 8

A 96-well plate was inoculated with 1500 cells/well. Cell viability was assayed by mixing cell counting kit 8 (CCK8) reagent with 100 μL of 10% DMEM.

Wound healing assay

According to the experimental grouping, a 200 μL sterile lance tip was used in the middle of each well to make a hard scratch along the vertical direction of the straightedge, and 2 mL of 2% antiserum-free cell culture medium was added to each well and incubated.

Immunohistochemistry

Gastric cancer tissues and the paracancerous tissues were sent to Aifang Biological Company for detection.

Statistical analyses

ImageJ was used to process imaging data. Differences between treatments were analyzed by t-test or analysis of variance. All statistical analyses were performed using GraphPad Prism 9.0, and the data were expressed as the mean ± SE. Differences were considered statistically significant when P < 0.05.

RESULTS

H. pylori infection elevates RPF2 expression in GES-1 cells

To verify the difference in RPF2 expression after H. pylori infection, we co-cultured H. pylori with GES-1 cells and performed q-PCR and immunoblotting. H. pylori infection resulted in increased RPF2 mRNA and protein expression compared to the control group (Figure 1A-C).

Figure 1 RPF2 and protein kinase B/mammalian target of rapamycin expression in Helicobacter pylori-infected cells. A: Western blots of RPF2 and protein kinase B (AKT)/mammalian target of rapamycin (mTOR); B: RPF2 mRNA level (1.00 ± 0.02 vs 5.10 ± 0.23); C: RPF2 protein levels (1.00 ± 0.05 vs 1.70 ± 0.12); D: Phospho-AKT protein levels; E: Phospho-mTOR protein levels. Mean ± SE. ^aP < 0.05. ^bP < 0.01. ^dP < 0.0001. AKT: Protein kinase B; p-AKT: Phospho-protein kinase B; mTOR: Mammalian target of rapamycin; p-mTOR: Phospho-mammalian target of rapamycin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H. pylori: Helicobacter pylori.

H. pylori infection activates the AKT/mTOR pathway in GES-1 cells

To test the effect of H. pylori infection on the AKT/mTOR pathway, we performed immunoblotting. H. pylori infection resulted in increased phospho-AKT (p-AKT)/p-mTOR expression compared to the uninfected group (Figure 1A, D and E).

Down-regulation of RPF2 inhibits H. pylori-mediated AKT/mTOR activation and regulates EMT and c-Myc expression

To verify the role of RPF2 in the AKT/mTOR pathway, we generated a cell model for silencing RPF2. We confirmed knock down at the protein level by Western blotting. We divided the cells into four groups: si-NC, si-NC + Hp27, si-RPF2, and si-RPF2 + Hp27. We performed Western blotting to detect protein expression of E-cadherin, N-cadherin, and vimentin and q-PCR to measure ZEB1, and c-Myc mRNA expression. H. pylori infection activated the AKT/mTOR pathway, promoted EMT, down-regulated E-cadherin, and up-regulated N-cadherin, vimentin, ZEB1, and c-Myc compared to the control group (Figure 2 and Figure 3A-F); si-RPF2 inhibited the AKT/mTOR expression, which resulted in the upregulation of E-cadherin expression and the down-regulation of the EMT components N-cadherin, vimentin, ZEB1 and c-Myc expression. To test whether EMT was indeed inhibited, we performed scratch assays and found that si-RPF2 inhibited cell migration into the scratch area (Figure 2 and Figure 3A-F). Infection of the si-RPF2 group with H. pylori inhibited H. pylori-induced AKT/mTOR expression and prevented the decrease of EMT (Figure 2 and Figure 3A-F).

Figure 2 Protein kinase B/mammalian target of rapamycin and epithelial-mesenchymal transition protein expression in RPF2 gene silencing cells. A-C: RPF2 and protein kinase B (AKT)/mammalian target of rapamycin (mTOR) protein levels; D: Western blots of RPF2 and AKT/mTOR; E: Western blots of epithelial-mesenchymal (EMT) transition and c-Myc; F-J: EMT and c-Myc protein levels. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. AKT: Protein kinase B; p-AKT: Phospho-protein kinase B; mTOR: Mammalian target of rapamycin; p-mTOR: Phospho-mammalian target of rapamycin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H. pylori: Helicobacter pylori; si-NC: Small interfering-negative control; si-RPF2: Small interfering-RPF2.

Figure 3 Changes in mRNA expression of epithelial-mesenchymal transition related factors, MMP2 and MMP9 in silencing/overexpressing RPF2 cells. A-H: RPF2, epithelial-mesenchymal transition (EMT), c-Myc, MMP2, and MMP9 mRNA levels in silencing RPF2 cells; I-P: RPF2, EMT, c-Myc, MMP2, and MMP9 mRNA levels in overexpressing RPF2 cells. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. si-NC: Small interfering-negative control; si-RPF2: Small interfering-RPF2.

Silencing of RPF2 inhibits proliferation and migration of GES-1 cells

Using the same treatment groupings as above, we detected differences in the expression of RPF2, MMP2, and MMP9 at the mRNA and protein levels. Relative to the control group, the H. pylori-infected group exhibited upregulated mRNA and protein expressions of RPF2, MMP2, and MMP9, whereas knockdown of RPF2 resulted in decreased expression of RPF2, MMP2, and MMP9 (Figure 3G and H and Figure 4A-C); treatment of si-RPF2 cells with H. pylori resulted in increased RPF2, MMP2, and MMP9 expression compared to the si-RPF2 group alone (Figure 3G and H and Figure 4A-C).

Figure 4 Effect of RPF2 knockdown on MMP2 and MMP9 expression, cell viability and migration. A: Western blots for MMP2 and MMP9; B and C: MMP2 and MMP9 protein levels; D: Cell counting kit 8 in silencing cells; E: Pictures of wound healing assay in silencing cells; F: Gap closure rate after wound healing assay in knockdown cells. Purple represents small interfering-negative control + Hp27 vs small interfering-negative control. Blue represents small interfering-RPF2 vs small interfering-negative control. Gray represents small interfering-RPF2 + Hp27 vs small interfering-RPF2. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. si-NC: Small interfering-negative control; si-RPF2: Small interfering-RPF2; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H. pylori: Helicobacter pylori.

CCK8 assays demonstrated that cell viability was elevated in the H. pylori group compared to the control group and decreased in the si-RPF2 group (Figure 4D); cell viability was significantly elevated in the si-RPF2 + Hp27 group compared with the si-RPF2 group (Figure 4D).

The results of scratch experiments showed that the migration ability of cells after H. pylori infection was significantly elevated compared to the control group, and the migration ability of cells in the si-RPF2 group was reduced (Figure 4E and F); the migration ability of the si-RPF2 + H. pylori group showed an upward trend compared to the si-RPF2 group (Figure 4E and F).

Overexpression of RPF2 promotes H. pylori-mediated activation of the AKT/mTOR pathway and EMT and c-Myc expression

We generated cell models overexpressing RPF2 and divided cells into four groups: pcDNA3.1 (+), pcDNA3.1 (+) + Hp27, pcDNA-RPF2, and pcDNA-RPF2 + Hp27. We detected the expression of E-cadherin, N-cadherin, and vimentin by Western blotting and ZEB1 and c-Myc by q-PCR. RPF2 overexpression promoted the expression of p-AKT/p-mTOR proteins and EMT, and resulted in decreased E-cadherin expression and increased N-cadherin, vimentin, ZEB1, and c-Myc expression compared to the control group (Figure 5 and Figure 3I-N).

Figure 5 Changes in the expression of protein kinase B/mammalian target of rapamycin and epithelial-mesenchymal transition proteins in RPF2-overexpressing cells. A-C: RPF2 and protein kinase B (AKT)/mammalian target of rapamycin (mTOR) protein levels; D: Western blots for RPF2 and AKT/mTOR; E: Western blot pictures of epithelial-mesenchymal transition (EMT) and c-Myc; F-J: EMT and c-Myc protein levels. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. AKT: Protein kinase B; p-AKT: Phospho-protein kinase B; mTOR: Mammalian target of rapamycin; p-mTOR: Phospho-mammalian target of rapamycin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H. pylori: Helicobacter pylori.

Overexpression of RPF2 promotes proliferation and migration of GES-1 cells

We examined the differences in MMP2 and MMP9 the mRNA and protein expression levels between each group. MMP2 and MMP9 mRNA and protein levels increased in the RPF2 group relative to the control group (Figure 3O and P and Figure 6A-C). The results of CCK8 and wound healing assays showed that cell viability was increased and migration was significantly increased after RPF2 overexpression compared to the control group (Figure 6D-F).

Figure 6 Effect of RPF2-overexpression on MMP2 and MMP9 expression, cell viability and migration. A: Western blots of MMP2 and MMP9; B and C: MMP2 and MMP9 protein levels; D: Cell counting kit 8 in overexpressing cells; E: Pictures of wound healing assay in overexpressing cells; F: Gap closure rate after wound healing assay in overexpressing cells. Purple represents small interfering-negative control + Hp27 vs small interfering-negative control. Blue represents small interfering-RPF2 vs small interfering-negative control. Gray represents small interfering-RPF2 + Hp27 vs small interfering-RPF2. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H. pylori: Helicobacter pylori.

Higher expression of RPF2 in gastric cancer tissues

To verify the higher expression of RPF2 in the pathogenic process of H. pylori, we isolated mRNA and protein from cancer tissues and paracancerous tissues and found that RPF2 expression was significantly elevated in cancer tissues (Figure 7A-C). Immunohistochemistry results indicated that RPF2 was overexpressed in gastric cancer tissues, and RPF2 protein was primarily located in the nucleus, with a small cytoplasmic pool (Figure 7D and E).

Figure 7 RPF2 expression in clinical gastric cancer tissues. A: Western blots of RPF2 in clinical gastric cancer tissues; B: mRNA levels of RPF2 in clinical gastric cancer tissues (1.01 ± 0.08 vs 2.19 ± 0.18); C: Protein levels of RPF2 in clinical gastric cancer tissues (0.11 ± 0.03 vs 1.02 ± 0.03); D and E: Localization and expression of RPF2 protein in gastric cancer tissues (0.03 ± 0.01 vs 0.51 ± 0.07). Mean ± SE. N: Normal tissues; T: Tumor tissues.

DISCUSSION

H. pylori is one of the major causes of gastric carcinogenesis, and the increase of its drug resistance urges the need for more sensitive assays and markers to H. pylori infectious gastric cancer. This research represents the initial exploration into how H. pylori infection affects RPF2 expression and influences the proliferative and metastatic behaviors of gastric epithelial cells. Our data suggest that RPF2 may activate the AKT/mTOR pathway in the context of H. pylori infection, thereby facilitating EMT progression and increased tumor marker expression. These data might offer valuable insights for investigations into the early diagnosis and intervention of H. pylori-related gastric cancer.

Co-culturing H. pylori with gastric epithelial cells revealed that the RPF2 mRNA expression level significantly increased after H. pylori intervention. Immunoblotting results demonstrated that RPF2 protein levels increased after H. pylori infection and activated the AKT/mTOR pathway.

Numerous studies have shown that the AKT/mTOR pathway is a common oncogenic pathway indispensable for many biological processes such as cell growth, metastasis, survival, and metabolism[34-36]. mTORC2 can regulate cellular actin skeleton remodeling and EMT by regulating the phosphorylation status of protein kinase C and activating AKT. In addition, AKT activation leads to the accumulation of Snail in the nucleus, which triggers Twist expression, decreasing E-cadherin expression, which further contributes to actin cytoskeleton remodeling[14]. Studies have shown that CCNB2 is highly expressed in gastric cancer patients, and RNA sequencing results show that CCNB2 is related to PI3K/AKT signaling pathway activation[37]. The polyamidoamine (PAMAM)-wrapped miR-144 (PAMAM/miR-144) nanoparticle system significantly reduced EMT by targeting the mTOR signaling pathway in HGC-27 cells and significantly inhibited the growth of subcutaneous gastric cancer xenografts in nude mice[38]. These findings align with the results of our study on the AKT/mTOR pathway. This paper, for the first time, establishes the role of RPF2 in the AKT/mTOR pathway activation by H. pylori. Constructing a silencing/overexpression model of RPF2 found that RPF2 activated the AKT/mTOR pathway to induce gastric mucosal lesions in H. pylori; caused differential expression of EMT-related proteins, including E-cadherin, N-cadherin, vimentin, ZEB1, and c-Myc; and facilitated the development of EMT. The development of EMT is widely regarded as a fundamental mechanism for the development of malignant tumors including gastric, lung, and breast cancers. In this context, E-cadherin, a critical intercellular adhesion molecule, helps preserve the epithelial cell phenotype and promotes cell-cell adhesion. Additionally, N-cadherin, vimentin, and ZEB1, which are key regulators of the EMT process in tumor cells, can facilitate EMT by downregulating E-cadherin expression[7].

Ribosomes function as translators, and their biogenesis is a multistep process that begins in the nucleolus and ends in the cytoplasm. Any alteration in this process may lead to overactivation of ribosome biogenesis[32,39,40]. RPF2 promotes ribosome synthesis and enhances glycolysis through activation of the AKT/GSK-3β signaling pathway in colorectal cancer tissues and cell lines, promotes EMT, and enhances cell migration and invasion in vitro and in vivo, which leads to colon carcinogenesis[33,41]. In addition, RPF2 may promote drug resistance in colorectal cancer through the CARM1-MYCN pathway[42]. For intracellular RPF2 silencing/overexpression, q-PCR and immunoblotting showed that overexpression of RPF2 increased MMP2 and MMP9 expression, and CCK8 and wound healing assay showed that RPF2 overexpression \ significantly enhanced the proliferation ability and migration ability of gastric epithelial cells, which was enhanced after H. pylori infection, whereas RPF2 downregulation had the opposite effect. In clinical gastric cancer tissues, RPF2 expression was increased, and this finding is consistent with our in vitro studies suggesting that RPF2 may be a pro-cancer factor.

In summary, the present study demonstrated that RPF2 was highly expressed after H. pylori infection and promoted EMT through activation of the AKT/mTOR pathway, which enhanced cell proliferation and migration. RPF2 could serve as a potential biomarker for evaluating gastric cancer cell viability and migration capacity after H. pylori infection. Additionally, it might represent a novel therapeutic target for curbing the local invasion and distant metastasis of H. pylori-infected gastric cancer via small molecule inhibitors or siRNA.

CONCLUSION

RPF2 exhibited elevated expression in gastric cancer tissues, which activated the AKT/mTOR pathway in gastric epithelial cells, facilitated EMT in H. pylori-infected cells, and improved cell viability and migration. This study suggests for the first time that RPF2 has a vital role in the pathogenic mechanism of H. pylori infection, which may be of reference value for early diagnosis and therapeutic research of H. pylori-infected gastric cancer.

ACKNOWLEDGEMENTS

We would like to thank Fan XS, Liang TY and Cheng C (All from the Department of Epidemiology, School of Public Health, Zhengzhou University) for their assistance in experimental manipulation and valuable comments in this study.