miR-9-5p expression is associated with vascular invasion and prognosis in hepatocellular carcinoma, and in vitro verification

Abstract

Purpose: Hepatocellular carcinoma (HCC) is a common liver malignancy. Early vascular invasion (VI) has been associated with poor prognosis in HCC patients. MicroRNAs (miRNAs) play a significant role in the emergence and development of many tumor types.

Methods: Differential expression analysis of miRNAs related to VI was performed based on data from the TCGA database, and survival-associated miRNAs identified. We identified miR-9-5p as a survival-related miRNA and verified its expression in 61 clinical samples using quantitative real-time PCR. We further performed functional enrichment analysis, protein-protein interaction analysis, univariate and multivariate analysis of the survival-related miRNAs, and cell function assays.

Results: In this study, we identified miR-9-5p that could predict VI and prognosis in HCC patients. Cellular experiments demonstrated that downregulation of miR‑9‑5p inhibits migration, invasion, and angiogenesis of HCC cells. Further, we explored and verified the possible mechanism through which miR-9-5p is involved in HCC progression. Univariate and multivariate analysis revealed that miR-9-5p was an independent risk factor for HCC. Finally, the nomogram based on miR-9-5p showed a good predictive value of HCC survival.

Conclusions: MiR-9-5p is associated with VI in HCC, and higher expression of miR-9-5p indicates poor prognosis in HCC.

Keywords: Hepatocellular carcinoma; Prognosis; TCGA; Vascular invasion; miRNAs.

Fig. 1

Identification of differentially expressed miRNAs. a Kaplan–Meier survival analysis of VI (+) group and VI (−) group. b Volcano plot showing miRNAs expression. Blue, low expression; red, high expression

Fig. 2

Kaplan–Meier survival analysis of the identified high expression miRNAs (miR-9-5p, miR-9-3p, miR-196a-5p, miR-1270, and miR-2144-5p) and low expression miRNAs (miR-375-3p and miR-483-3p). a miR-9-5p, p = 0.0164. b miR-9-3p, p = 0.0242. c miR-196a-5p, p = 0.1845. d miR-375-3p, p = 0.793. e miR-483-3p, p = 0.9513. f miR-1270, p = 0.8331. g miR-2144-5p, p = 0.1119

Fig. 3

The expression of the selected miRNAs. a Expression of miR-9-5p in the VI (+) group (n = 81), VI (−) group (n = 160), and normal group (n = 50). b Expression of miR-9-3p in the VI (+) group (n = 81), VI (−) group (n = 160), and normal group (n = 50). c The expression level of miR-9-5p in 61 HCC tissues by RT-qPCR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Downregulation of miR‑9‑5p inhibits migration, invasion, and angiogenesis of HCC cells. a Expression of miR-9-5p in five different HCC cells. b Tube formation assay was performed after HUVEC cells were co-cultured with HCC-LM3 cells transfected with different plasmids. c Representative images of HCC-LM3 migration using wound healing assay. d Cell migration ability in HCC-LM3 was assessed by transwell migration assay. e Cell invasion ability in HCC-LM3 was assessed by transwell invasion assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Risk score analysis of miR-9-5p in the TCGA cohort. a The expression heatmap of miR-9-5p in HCC. b The miR-9-5p distribution in the TCGA-LIHC cohort. c The survival time and survival status with increasing miR-9-5p expression

Fig. 6

Clinical value of miR-9-5p. a Univariate Cox analysis of miR-9-5p in HCC. b Multivariate Cox analysis of miR-9-5p in HCC. c ROC curves for mir-9-5p expression and clinical variables (gender, age, grade, stage, T, and AFP)

Fig. 7

Functional analysis of the target genes of miR-9-5p. a Identification of target genes in three databases. b Circle graph of KEGG pathway analysis. c Bar plot for GO analysis (BP biological process, CC cell component, MF molecular function)

Fig. 8

Protein–protein interaction analysis. a PPI network of the target genes. b Identified hub target genes of the PPI network. c The expression of hub genes in TCGA Database

Fig. 9

Nomogram for predicting the survival rate of patients with HCC. a A nomogram containing age, grade, stage, T, and miR-9-5p expression. b–d Calibration curves of the nomogram for predicting the 1-, 2-, and 3-year survival. e ROC curve analyses for the nomogram in predicting the 1-, 2-, and 3-year survival