miR-106b-5p promotes aggressive progression of hepatocellular carcinoma via targeting RUNX3

Abstract

Background and objectives: The roles of microRNA(miR)-106b-5p in hepatocellular carcinoma (HCC) remain unclear. We aimed here to investigate the clinical significance of miR-106b-5p expression in HCC and its underlying mechanisms.

Methods: Expression levels of miR-106b-5p in 108 HCC clinical samples by quantitative real-time reverse transcription PCR. Associations of miR-106b-5p expression with various clinicopathological features and patients' prognosis were evaluated by Chi-square test, Kaplan-Meier, and Cox proportional regression analyses, respectively. The target gene of miR-106b-5p and their functions in HCC cells were investigated by luciferase reporter, CCK-8, and Transwell Matrigel invasion assays.

Results: miR-106b-5p expression was markedly higher in HCC tissues than in noncancerous adjacent liver tissues (P < .001). miR-106b-5p upregulation was significantly associated with advanced TNM stage (P = .02), short recurrence-free (P = .005), and overall (P = .001) survivals. Importantly, miR-106b-5p expression was an independent predictor of poor prognosis (P < .05). RUNX3 was identified as a direct target gene of miR-106b-5p in HCC cells. Functionally, miR-106b-5p upregulation promoted the viability and invasion of HCC cells, while enforced RUNX3 expression reversed the oncogenic effects of miR-106b-5p overexpression.

Conclusions: miR-106b-5p may serve as a potent prognostic marker for tumor recurrence and survival of HCC patients. miR-106b-5p may exert an oncogenic role in HCC via regulating its target gene RUNX3.

Keywords: cell viability; hepatocellular carcinoma; invasion; microRNA-106b-5p; overall survival; recurrence-free survival.

Figure 1

Expression patterns and prognostic value of miR‐106b‐5p in HCC. A, The relative expression levels of miR‐106b‐5p were significantly higher in HCC tissues compared with paired noncancerous adjacent liver tissues measured by quantitative real‐time reverse transcription PCR analysis (P < .001). B, Kaplan‐Meier survival curves for recurrence‐free survival are plotted according to miR‐106b‐5p expression (compared with log‐rank test). C, Kaplan‐Meier survival curves for overall survival are plotted according to miR‐106b‐5p expression (compared with log‐rank test)

Figure 2

MiR‐106b‐5p promotes cell viability and invasion of SMMC7721 cell line. A, Expression levels of miR‐106b‐5p in SMMC7721 cells transfected with mimic‐miR‐106b‐5p or mimic‐NC was detected by quantitative real‐time reverse transcription PCR. The results are the mean ± SD of triplicate experiments. B, miR‐106b‐5p overexpression significantly enhanced the viability of SMMC7721 cells (P < .05). C, miR‐106b‐5p overexpression significantly promoted the invasion ability of SMMC7721 cells (P < .001). The results are the mean ± SD of triplicate experiments

Figure 3

Increased expression of miR‐106b‐5p promotes cell viability and invasion of HepG2 cell line. A, Expression levels of miR‐106b‐5p in HepG2 cells transfected with mimic‐miR‐106b‐5p or mimic‐NC was detected by quantitative real‐time reverse transcription PCR. The results are the mean ± SD of triplicate experiments. B, miR‐106b‐5p overexpression significantly enhanced the viability of HepG2 cells (P < .05). C, miR‐106b‐5p overexpression significantly promoted the invasion ability of HepG2 cells (P < .001). The results are the mean ± SD of triplicate experiments

Figure 4

Reduced expression of miR‐106b‐5p suppresses cell viability and invasion of HepG2 cell line. A, Expression levels of miR‐106b‐5p in HepG2 cells transfected with anti‐miR‐106b‐5p or anti‐NC was detected by quantitative real‐time reverse transcription PCR. The results are the mean ± SD of triplicate experiments. B, Reduced expression of miR‐106b‐5p significantly suppressed the viability of HepG2 cells (P < .05). C, Reduced expression of miR‐106b‐5p significantly suppressed the invasion ability of HepG2 cells (P < .05). The results are the mean ± SD of triplicate experiments

Figure 5

RUNX3 was a direct target gene of miR‐106b‐5p in SMMC7721 cell line. A, A putative binding site for miR‐106b‐5p identified in the 3'‐UTR of RUNX3. B, The enforced expression of miR‐106b‐5p decreased the luciferase activity associated with the RUNX3‐WT‐3'‐UTR (**P < .01) but did not affect that with the RUNX3‐MUT‐3'‐UTR. C, Western blot analysis demonstrated that the expression level of RUNX3 protein was significantly suppressed in the SMMC7721 cells overexpressed miR‐106b‐5p relative to those transfected with miR‐NC (**P < .01). D, Quantitative real‐time reverse transcription PCR analysis demonstrated that the expression level of RUNX3 mRNA was significantly suppressed in the SMMC7721 cells overexpressed miR‐106b‐5p relative to those transfected with miR‐NC (**P < .01)

Figure 6

RUNX3 was a direct target gene of miR‐106b‐5p in HepG2 cell line. A, The enforced expression of miR‐106b‐5p decreased the luciferase activity associated with the RUNX3‐WT‐3'‐UTR (**P < .01) but did not affect that with the RUNX3‐MUT‐3'‐UTR. B, Western blot analysis demonstrated that the expression level of RUNX3 protein was significantly suppressed in the HepG2 cells overexpressed miR‐106b‐5p relative to those transfected with miR‐NC (**P < .01). C, Quantitative real‐time reverse transcription PCR analysis demonstrated that the expression level of RUNX3 mRNA was significantly suppressed in the HepG2 cells overexpressed miR‐106b‐5p relative to those transfected with miR‐NC (**P < .01)

Figure 7

RUNX3 over‐expression reverses the oncogenic effects of miR‐106b‐5p in SMMC7721 cell line. A, Western blot analysis confirmed that the downregulation of RUNX3 protein expression caused by the upregulation of miR‐106b‐5p was recovered by the co‐transfection with RUNX3‐vector in HSMMC7721 cells (** P < .01). B and C, The restored RUNX3 expression significantly rescued the effects of exogenous miR‐106b‐5p on the viability (* P < .05) and invasion (** P < .01) of SMMC7721 cells

Figure 8

RUNX3 over‐expression reverses the oncogenic effects of miR‐106b‐5p in HepG2 cell line. A, Western blot analysis confirmed that the downregulation of RUNX3 protein expression caused by the upregulation of miR‐106b‐5p was recovered by the co‐transfection with RUNX3‐vector in HepG2 cells (** P < .01). B and C, The restored RUNX3 expression significantly rescued the effects of exogenous miR‐106b‐5p on the viability (* P < .05) and invasion (** P < .01) of HepG2 cells