MiR-491 Blocks Activation Of STAT3 Via EGFR And Inhibits Cancer Stem Cell-like Properties In Hepatocellular Carcinoma Cells

ObjectiveTo expound the molecular regulation mechanism of mi R-491 on characterizing of cancer stem cells(CSCs) in hepatocellular carcinoma(HCC), as well as its effects on HCC development, metastasis and recurrence, which have provided new approaches and research strategies for diagnosis of HCC development, metastasis and prognosis recurrence, targeted therapy and patient prognosis prediction.Methods1. Expression of mi R-491 on tumor tissue and normal tissue, as well as the correlativity with metastatic potentiality of hepatocellular carcinoma cells.(1). Detection of miRNA-491 expressive discrepancy between liver carcinoma tissues and normal tissues: Detect expression of mi RNA-491 on tumor tissues and normal tissues by adopting real-time quantitative transcription PCR(q RT-PCR);(2). Detection of miR-491 expressive in hepatocellular carcinoma cells: Detect expression level of mi R-491 at different metastatic potentiality hepatocellular carcinoma cells lines by adopting real-time quantitative transcription PCR(q RT-PCR).Detection on effects of mi R-491 expression level variation on hepatocellular carcinoma cells colony-forming and CSCs sphere formation ability: separate transfection of mi R-491-mimic/inhibitor in hepatocellular carcinoma cellsMHCC97H and HepG2, hepatocellular carcinoma cells colony forming experiment and suspension cell cloning ball forming experiments to detect CSCs variation.3. STAT3 regulation of mi R-491 in hepatocellular carcinoma cells lines through EGFR(1). Detection on mi R-491 expression level variation on hepatocellular carcinoma cells EGFR and STAT3 expression: separate transfection of mi R-491-mimic/inhibitor in hepatocellular carcinoma cells MHCC97 H and Hep G2; detection on EGFR and STAT3 protein phosphorylation level variation by adopting Western blot;(2). Detection on the effects of mi R-491 expression level variation on hepatocellular carcinoma cells EGFR expression: cotransfection of p EGFP-EGFR 3’UTR-Luc and mi R-491-mimic in hepatocellular carcinoma cell MHCC97 H, cotransfection of p EGFP-EGFR 3’UTR-Luc and mi R-491-inhibitor in hepatocellular carcinoma cell Hep G2, detection on EGFR luciferase activity variations by adopting luciferase assay;(3). Detection on the effects of EGFR expression level variation on hepatocellular carcinoma cell STAT3 level: 24 h after transfection of mi R-491-inhibitor in hepatocellular carcinoma cell Hep G2, continuously use EGFR si RNA to treat cells for 12 h and detect EGFR and STAT3 protein level variation on by adopting Western blot; 24 h after transfection of mi R-491-mimic in hepatocellular carcinoma cell MHCC97 H, continuously use EGFR high expression plasmid to treat cells for 12 h and detect EGFR and STAT3 protein level variation on by adopting Western blot.4. Effects of EGFR/STAT3 on cancer stem cell-like properties, invasion and metastasis in HCC cells(1). Detection on effects of EGFR/STAT3 expression level variation on sphere formation and tumor cell colony-forming ability in HCC: cotransfection of mi R-491-mimic and STAT3 high expression plasmid in hepatocellular carcinoma cell MHCC97H; cotransfection of mi R-491-inhibitor and STAT3 si RNA in hepatocellularcarcinoma cell Hep G2; detection of CSCs sphere formation of suspension cell cloning sphere formation experience and colony-forming ability.(2). Detection on effects of EGFR/STAT3 expression level variation on hepatocellular carcinoma cells metastasis potential: cotransfection of mi R-491-mimic and STAT3 high expression plasmid in hepatocellular carcinoma cell MHCC97 H and cotransfection of mi R-491-inhibitor and STAT3 si RNA in hepatocellular carcinoma cell Hep G2. As well as Detection on tumor cell invasion and metastasis ability variation by adopting transwell experiment.Results1. mi R-491 is related to hepatocellular carcinoma cells metastatic potentiality and tumor tissuesThe results of detection on mi R-491 expression level on hepatocellular carcinoma cells line and liver carcinoma tissues, normal tissues by adopting of q RT-PCR revealed that: mi R-491 expression and metastatic potentiality present negative correlation. It is far lower than normal tissues in terms of liver carcinoma tissues expression.2. mi R-491 affects characterizing of CSCs in HCCThe results revealed that sphere formation of MHCC97 H transfect with mi R-491-mimic is significantly lower than that of control group; however, the sphere formation of Hep G2 cell transfect with mi R-491-inhibitor is significantly high than that of control group.3. STAT3 regulation of mi R-491 in hepatocellular carcinoma cells lines through EGFR(1). After up-regulation of mi R-491 in hepatocellular carcinoma cell MHCC97 H, the results revealed that the EGFR protein level and STAT3 protein phosphorylation level have obviously decreased; while after down-regulation of hepatocellular carcinoma cell Hep G2, EGFR protein level and STAT3 protein phosphorylation levelhave obviously increased;(2). Cotransfection of pEGFP-EGFR 3’UTR-Luc and mi R-491-mimic in hepatocellular carcinoma cell MHCC97 H, the results revealed that EGFR and mi R-491 promoter region can combine. Up-regulation of mi R-491 level and decreasing of EGFR luciferase activity. Cotransfection of p EGFP-EGFR-3’UTR-Luc and mi R-491-inhibitor in hepatocellular carcinoma cell Hep G2, the results revealed that EGFR and mi R-491 promoter can combine. Down-regulation of mi R-491 level, EGFR luciferase activity increasing, which indicate that mi R-491 can directly target EGFR in hepatoma carcinoma cells;(3). After treatment of HepG2 by mi R-491-inhibitor and treatment of HepG2 by EGFR si RNA, the results revealed that inhibition of EGFR can make rising STAT3 protein phosphorylation level of Hep G2 resulted from mi R-491 dropping. After treatment of MHCC97 H by mi R-491-mimic and treatment of EGFR high expression plasmid, the results revealed that high expression of EGFR can make dropping STAT3 protein phosphorylation level of MHCC97 H resulted from mi R-491 rising, which indicate that hepatocellular carcinoma cells STAT3 expression level variation resulted from mi R-491 is completed by EGFR.4. Effects of mi R-491/STAT3 on cancer stem cell-like properties and metastasis potential in Hepatocellular Carcinoma cells(1). After transfection of mi R-491-mimic in hepatocellular carcinoma cell MHCC97 H, the results revealed that CSCs sphere formation and colony-forming ability have weakened as well. Cotransfection of mi R-491-mimic and STAT3 high expression plasmid. the results revealed that high expression of STAT3 can make CSCs sphere formation and colony formation resulted from mi R-491-mimic weaken rising. After transfection of mi R-491-inhibitor in hepatocellular carcinoma cell Hep G2, the results revealed that CSCs sphere formation and colony-forming ability has obviously increased. Cotransfection of mi R-491-inhibitor and STAT3 si RNA, the results revealed that low expression of STAT3 can make CSCs sphere formation and colony-forming ability resulted from mi R-491-inhibitor weaken dropping.(2). After transfection of mi R-491-mimic in hepatocellular carcinoma cell MHCC97 H, the results revealed that tumor cell metastasis potential has obviously weakened. Cotransfection of mi R-491-mimic and STAT3 high expression plasmid, the results revealed that high expression of STAT3 can make tumor cell metastasis potential resulted from mi R-491-mimic weaken rising. After transfection of mi R-491-inhibitor in hepatocellular carcinoma cell Hep G2, the results revealed that tumor cell metastasis potential has obviously increased. Cotransfection of mi R-491-inhibitor and STAT3 si RNA, the results revealed that low expression of STAT3 can make tumor cell metastasis potential resulted from mi R-491-inhibitor weaken dropping.Conclusion1. mi R-491 is related to tumor cell malignant degree and hepatocellular carcinoma pathological stage;2. mi R-491 inhibiting CSCs characterizing in hepatocellular carcinoma cells;3.STAT3 regulation of miR-491 through target EGFR;4. EGFR/STAT3 participates in characterizing of CSCs in HCC as well as metastasis potential.In conclusion, we indicate that mi R-491 can reduce STAT3 phosphorylation level by target EGFR, and inhibit characterizing of CSCs in HCC thus to affect metastasis potential of tumor cell. Research results indicate that pathway control of mi R-491 and characterizing of CSCs in HCC plays a vital role in hepatocellular carcinoma occurrence, development and prognosis relapse.