Integrated diagnostic model based on serum exosomal microRNAs and functional mechanism of miR-200b-3p in hepatocellular carcinoma

Figure 1 Study flowchart. miRNA: MicroRNA; HCC: Hepatocellular carcinoma; qRT-PCR: Quantitative real-time polymerase chain reaction; RFE: Recursive feature elimination; NGS: Next-generation sequencing.

Figure 2 Expression levels of six key differentially expressed microRNAs in serum exosomes between hepatocellular carcinoma and non-hepatocellular carcinoma groups. A: MiR-200b-3p; B: MiR-215-5p; C: MiR-335-5p; D: MiR-378a-3p; E: MiR-452-5p; F: MiR-501-3p. ^dP < 0.0001. HCC: Hepatocellular carcinoma.

Figure 3 Area under the receiver operating characteristic curve values of six candidate microRNAs in diagnostic models. A: MiR-200b-3p; B: MiR-215-5p; C: MiR-335-5p; D: MiR-378a-3p; E: MiR-452-5p; F: MiR-501-3p. ^cP < 0.001, ^dP < 0.0001. AUC: Area under the curve.

Figure 4 Construction and variable assessment of the hepatocellular carcinoma-3miRNA diagnostic model. A: Graphical abstract illustrating the model construction workflow; B: Forest plot displaying the odds ratios and confidence intervals of each variable included in the final multivariate logistic regression model. RT-qPCR: Real-time quantitative polymerase chain reaction; HCC: Hepatocellular carcinoma; CI: Confidence interval.

Figure 5 Diagnostic performance of the 3-miRNA model. A: The receiver operating characteristic curve and area under the curve of the training set; B: The validation set; C: The total development set. AUC: Area under the curve.

Figure 6 MiR-200b-3p directly targets and suppresses phosphoserine aminotransferase 1 by binding to its 3’ untranslated region. A: Venn diagram showing the four microRNAs that were significantly differentially expressed in both serum exosomes and liver tissues of hepatocellular carcinoma (HCC) and non-HCC cases; B-F: Expression levels of the five predicted target mRNAs that are differentially expressed in both serum exosomes and liver tissues of HCC and non-HCC cases; G: Analysis of phosphoserine aminotransferase 1 expression levels by immunohistochemical staining; H and I: Quantitative real-time polymerase chain reaction validation of miR-200b-3p overexpression efficiency in HepG2 and MHCC97H cells; J and K: Western blot analysis validating the efficiency of miR-200b-3p knockdown and overexpression in HepG2 and MHCC97H cells; L: Dual-luciferase reporter assay validates phosphoserine aminotransferase 1 as a direct target of miR-200b-3p. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. PSAT1: Phosphoserine aminotransferase 1; NC: Normal control; HCC: Hepatocellular carcinoma; OE: Overexpression; SP: Sponge; NS: Not significant.

Figure 7 MiR-200b-3p suppresses the malignant phenotypes of hepatocellular carcinoma cells. A-D: CCK-8 assays and cell colony formation assay (n = 3) indicated that overexpression of miR-200b-3p suppressed hepatocellular carcinoma cell proliferation, whereas its knockdown enhanced proliferative capacity; E and F: Wound healing assays (n = 3) demonstrated that miR-200b-3p knockdown accelerated wound closure, while its overexpression impeded cell migration; G: In Matrigel invasion assays (n = 3), silencing of miR-200b-3p promoted cellular invasion, in contrast to the suppressive effect observed upon miR-200b-3p overexpression. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. NC: Normal control; OE: Overexpression; SP: Sponge.