MicroRNA-18a promotes hepatocellular carcinoma proliferation, migration, and invasion by targeting Bcl2L10 - PubMed (original) (raw)
MicroRNA-18a promotes hepatocellular carcinoma proliferation, migration, and invasion by targeting Bcl2L10
Xiaodong Wang et al. Onco Targets Ther. 2018.
Abstract
Background: Hepatocellular carcinoma (HCC) is known to feature several microRNA dysregulations. This study aimed to determine and investigate the prognostic value of microRNA (miRNA/miR)-18a and its role in regulating the progression of HCC.
Methods: miR-18a expressions in human HCC tissues, pair-matched adjacent normal liver tissues as well as in HCC cell lines were determined by quantitative real-time PCR. The prognostic value of miR-18a was determined using Kaplan-Meier survival analysis and multivariable Cox regression assay. The ability of miR-18a in promoting HCC progression was verified in vitro.
Results: miR-18a expressions in HCC tissues and cells were more than twice those of the normal control group (P<0.05). miR-18a expression was associated with the alpha-fetoprotein (AFP) level, TNM stage, tumor size, and intrahepatic vascular invasion (P<0.05). Kaplan- Meier survival analysis revealed that HCC patients with high expression of miR-18a possessed a more unfavorable prognosis (log-rank P<0.001). Overexpression of miR-18a promoted cell apoptosis and proliferation, induced S phase transition, as well as enhanced the migration and invasion ability of HCC cells. miR-18a was found to directly target the downstream molecule Bcl2L10. Furthermore, overexpressing Bcl2L10 was able to partly reverse the promoting effects of miR-18a on HCC cell progression.
Conclusion: miR-18a may serve as a prognostic biomarker of HCC as it is demonstrated to carry out a decisive role in HCC progression by promoting HCC cell invasion, migration, and proliferation through targeting Bcl2L10.
Keywords: Bcl2L10; apoptosis; cell cycle; hepatocellular carcinoma; miR-18a.
Conflict of interest statement
Disclosure The authors report no conflicts of interest in this work.
Figures
Figure 1
Differential expression of miR-18a in HCC tissues and HCC cell lines. Notes: (A) miR-18a expression was much higher in HCC tissues compared with adjacent non-tumor tissues (P<0.001). (B) miR-18a expression was much higher in HepG2 and SMMC7721 cell lines compared with normal liver cell LO2 (P<0.001). (C) Kaplan–Meier analyses of the correlations between miR-18a expression and overall survival of 123 patients with HCC (log-rank P<0.001). Data were represented as mean ± SD. ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a.
Figure 2
Effect of miR-18a on the proliferation of HCC cells. Notes: (A) miR-18a mimic increased miR-18a level (P<0.01 in the HepG2 cell and P<0.001 in the SMMC7721 cell) and miR-18a inhibitor decreased miR-18a level (P<0.05) in HCC cell lines. (B) Upregulation of miR-18a in HCC cells facilitated cell proliferation while downregulation of miR-18a inhibited cell proliferation. (C) Cell apoptosis rate was detected by flow cytometry after transfection with miR-18a inhibitor or miR-18a mimic. (D) The proportion of cells in each cell cycle phase was detected using flow cytometry after the cells were transfected with miR-18a inhibitor or miR-18a mimic. Data were reported as mean ± SD. *P<0.05, **P<0.01, and ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; NC, negative control; PI, propidium iodide.
Figure 3
Effect of miR-18a on migration and invasion of HCC cells in vitro. Notes: (A) The effect of miR-18a on cell migration was determined by a wound scratching assay. Cell migration capacity of miR-18a mimic transfected HCC cells was promoted (P<0.05), whereas cell migration capacity of miR-18a inhibitor transfected HCC cells was inhibited (P<0.05). Scale bars =200 µm; magnification 40×. (B) After transfection of cells with miR-18a, the ability of cells to pass through the basement membrane was enhanced (P<0.001). In contrast, after transfecting cells with miR-18a inhibitor, the ability was inhibited (P<0.001). Scale bars =20 µm; magnification 100×. Data were reported as mean ± SD. ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; NC, negative control.
Figure 4
Bcl2L10 was a direct target of miR-18a. Notes: (A) The predicted binding site of miR-18a in the 3′-UTR of Bcl2L10. (B) miR-18a mimic significantly inhibited the luciferase activities of Bcl2L10-3′-UTR-Wt reporter in both cell lines (P<0.001), while it exhibited no inhibitory effects on the luciferase activities of Bcl2L10-3′-UTR-Mut reporter in HCC cells (_P_>0.05). (C) The mRNA expression of Bcl2L10 was much lower in HCC tissues compared with adjacent non-tumor tissues (P<0.001). (D) Immunochemistry showed that the protein expression of Bcl2L10 was much lower in HCC tissues than that in adjacent non-tumor tissue (P<0.01). Scale bars =20 µm. (E) Western blot analysis revealed that the protein expression of Bcl2L10 was higher in adjacent non-tumor tissues than in HCC tissues (P<0.01). (F) miR-18a expression negatively correlated with Bcl2L10 mRNA expression in HCC tissues (R=0.405, P<0.01, correlation coefficient =−0.636). Data were reported as mean ± SD. **P<0.01 and ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10; Wt, wild-type; Mut, mutant.
Figure 5
Effect of miR-18a on the expression of Bcl2L10 in vitro. Notes: (A) Upregulation of miR-18a decreased Bcl2L10 mRNA level, while downregulation of miR-18a increased Bcl2L10 mRNA level in HCC cells (P<0.001). (B) Western blot analysis revealed that the protein expression of Bcl2L10 was decreased in the miR-18a mimic transfected HCC cells (P<0.05) and was increased in the miR-18a inhibitor transfected cells (P<0.05). Data were reported as mean ± SD. *P<0.05 and ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10; NC, negative control.
Figure 6
Overexpression of Bcl2L10 partially reversed the function of miR-18a on HCC cell proliferation in vitro. Notes: (A) The Bcl2L10 plasmids promoted the mRNA and protein expression of Bcl2L10 in HCC cells (P<0.001). (B) Overexpression of Bcl2L10 partially reversed the effect of miR-18a on HCC cell proliferation (P<0.05). (C) Cell apoptosis rate was detected by flow cytometry after transfection with miR-18a mimic or miR-18a mimic + Bcl2L10 plasmids. (D) The proportion of cells in each cell cycle phase was detected using flow cytometry after cells were transfected with miR-18a mimic or miR-18a mimic + Bcl2L10 plasmids. Data were reported as mean ± SD. *P<0.05 and ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10; PI, propidium iodide.
Figure 6
Overexpression of Bcl2L10 partially reversed the function of miR-18a on HCC cell proliferation in vitro. Notes: (A) The Bcl2L10 plasmids promoted the mRNA and protein expression of Bcl2L10 in HCC cells (P<0.001). (B) Overexpression of Bcl2L10 partially reversed the effect of miR-18a on HCC cell proliferation (P<0.05). (C) Cell apoptosis rate was detected by flow cytometry after transfection with miR-18a mimic or miR-18a mimic + Bcl2L10 plasmids. (D) The proportion of cells in each cell cycle phase was detected using flow cytometry after cells were transfected with miR-18a mimic or miR-18a mimic + Bcl2L10 plasmids. Data were reported as mean ± SD. *P<0.05 and ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10; PI, propidium iodide.
Figure 7
Overexpression of Bcl2L10 partially reversed the function of miR-18a on HCC cell migration and invasion in vitro. Notes: (A) Bcl2L10 decreased the migration ability of miR-18a transfected HCC cells in vitro. Scale bars =200 µm; magnification 40×. (B) Bcl2L10 inhibited the invasion capacity of miR-18a transfected HCC cell in vitro. Scale bars =20 µm; magnification 100×. Data were reported as mean ± SD. **P<0.01 and ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10.
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