Hsa-miR-4282 Inhibits The Proliferation, Invasion And Migration Of Liver Cancer Cells By Regulating GRB2

CHAPTER1:THE EXPRESSION OF HSA-MIR-4282 IN HEPATOCELLULAR CARCINOMAOBJECTIVE: To investigate whether there is differences in the expression level of mi R-4282 among hepatocellular carcinoma tissues,adjacent tissues,hepatocellular carcinoma cell lines and normal liver cell lines.METHODS: The surgical specimens of patients with liver cancer were collected to extracte total RNA by Trizol.The q RT-PCR assay was used to quantitate the expression level of mi R-4282 in hepatocellular carcinoma tissues,adjacent tissues,hepatocellular carcinoma cell line MHCC97-H,SMMC-7721 and normal liver cell line HL-7702.RESULTS: The expression level of Mi R-4282 was significantly lower expressed in hepatocellular carcinoma tissues than adjacent tissues(0.680 ±0.350 vs 1.006 ± 0.108,P=0.001).Similarly,the expression level in liver cancer cell lines MHCC97-H and SMMC-7721 were lower than that in normal liver cell line HL-7702(0.520 ± 0.050 vs 0.410 ± 0.070 vs 1.003 ±0.079,P=0.005,P=0.001).CONCLUSION: The expression of mi R-4282 in liver cancer cells and lhepatocellular carcinoma tissues was lower than that in normal liver cells and adjacent tissues.CHAPTER 2 : THE EFFECT OF DIFFERENT EXPRESSION LEVELS OF HSA-MIR-4282 ON BIOLOGICAL BEHAVIOR OF HEPATOCELLULAR CARCINOMA CELLSOBJECTIVE: The low expression and high expression of mi R-4282 were transfected,and the cell function of the liver cancer cell line SMMC-7721 was confirmed by in vitro cell function experiments after increase or decrease of mi R-4282.METHODS: mi R-4282 was up-regulated and down-regulated in hepatocellular carcinoma cell line SMMC-7721 by transient transfection of mi R-4282 mimics and inhibitor.Cell total RNA was extracted after transfection48 hours,and the transfection expression was verified by q RT-PCR after reverse transcription.Then the effect of high expression and low expression of mi R-4282 on the proliferation of SMMC-7721 cells was verified by MTT assay.The effect of mi R-4282 expression on cell clone formation ability was verified by plate cloning assay.The effect of mi R-4282 transfection on apoptosis rate was verified by flow cytometry apoptosis assay;Transwell migration assay and cell scratch assay confirmed the effect of mi R-4282 on lateral and longitudinal migration of cells;Transwell invasion assay verified invasive ability after mi R-4282 was transfected in cell line.Finally,statistical analysis showed that if the high and low expression of mi R-4282 had an effect on the invasion,proliferation,cloning and apoptosis of SMMC-7721 cells.RESULTS: After transfection of mi R-4282 mimics/inhibitor,the expression of the mimics group was higher than that of the control group(28.326±2.301 vs1.052±0.0854,P=0.004),and the expression of the inhibitor group was lower than that of the control group(0.287±0.037 vs 1.075±0.193,P = 0.001).The MTT assay showed that the OD value of the mi R-4282 mimics group was lower than that of the negative control group(P<0.01),and the OD value of the inhibitor group was higher than that of the negative control group(P<0.01).In the plate colony formation experiment,the number of clones in mi R-4282 mimics group was lower than that in the control group(146±10 vs 193±12,P=0.013),and the number of clones in the inhibitor group was higher than that in the control group(240±7 vs 191±10,P=0.005).In the apoptosis experiment,the apoptosis rate of mi R-4282 mimics group was increased compared with the control group(23.887%±1.885% vs 16.600%±1.141%,P=0.009),while the apoptosis rate of the inhibitor group decreased(14.977%±0.460% vs 17.790%± 0.725%,P=0.010).In the cell scratch test,the healing rate of the mi R-4282 mimics group was slowed down and the healing rate of the inhibitor group was significantly accelerated(P<0.05).In the Transwell migration experiment,the number of mi R-4282 mimics group cell migration was decreased compared with the control group(8 ± 3 vs 30 ± 4,P<0.001),and the number of inhibitor migration was higher than that of the control group(45±5 vs 31±9,P<0.001).Transwell invasion assay the number of cells passing through the ventricular membrane in mi R-4282 mimics group was reduced compared with the control group(12±7 vs 24±2,P<0.001),and the number of cells passing through the chamber after mi R-4282 down-regulation was increased compared with the control group(34±8 vs 20±3,P<0.001).CONCLUSION: Low expression of mi R-4282 in hepatocellular carcinoma enhanced the proliferation,invasion and migration of SMMC-7721 cells by mi R-4282-GRB2-Ras acting on ERBB signal transduction pathway and mi R-4282-GRB2-EGFR pathway.CHAPTER 3:HSA-MIR-4282 INTERACTION GENE PREDICTION AND IDENTIFICATION AND ITS MECHANISM OF ACTIONOBJECTIVE: To study the mechanism of mi R-4282-induced proliferation,invasion and metastasis of Hepatocellular carcinoma cells.METHODS: Bioinformatics analysis was used to predict the genes involved in the interaction with mi R-4282 using Targetscan et al.,and to select appropriate related genes and pathways according to the functions of the above two parts in KEGG PATHWAY,and to pass the dual luciferase reporter system.Verify interactions.q RT-PCR was used to verify the up-regulation and down-regulation of the expression of mi R-4282 on the expression of this gene,and then the protein interacting with the target gene was searched by STRING and previous bioinformatics analysis,and verified by Western blot and q RT-PCR.The expression of this gene and the same name protein was up-regulated and down-regulated while up-regulated and down-regulated mi R-4282.RESULTS: Through bioinformatics analysis,we found the gene GRB2 as a research object.The results of dual luciferase assay showed that the GRB2 gene interacted with mi R-4282,and the expression of GRB2 was verified by q RT-PCR.The results showed that when mi R-4282 was up-regulated,the expression of GRB2 decreased(0.499 ± 0.150 vs 1.397 ± 0.632,P=0.009),whereas the expression of GRB2 increased(1.726 ± 0.198 vs 0.965 ± 0.129,P=0.035).After analyzing the STRING website and KEGG PATHWAY,we found that EGFR interacts with GRB2 and can affect cell proliferation.The results show that the expression of EGFR is down-regulated when mi R-4282 is up-regulated(0.288 ± 0.115 vs 1.097 ± 0.120,P=0.013),on the contrary,the expression of EGFR increased(1.726±0.198 vs 0.965±0.129,P=0.035),the expression of the same name protein was consistent with the gene,and the up-regulated group(0.500±0.121 vs 1.160±0.246,P=0.027);down-regulated group(1.605±0.256 vs 0.958±0.110,P=0.030).GRB2 can affect the invasion and migration ability of cells through ERBB pathway.We selected Ras protein as a downstream protein validation target in the pathway.The results showed that Ras protein was highly expressed when mi R-4282 was lowly expressed(1.503 ± 0.187 vs 0.994 ± 0.918,P=0.026),the expression level decreased significantly when mi R-4282 was highly expressed(0.639±0.075 vs 1.164±0.193,P=0.023).CONCLUSION: Mi R-4282,which has low expression level in Hepatocellular carcinoma cells,promotes cell proliferation,invasion and migration.mi R-4282 regulates the expression of Ras/EGFR through GRB2 and enhances the proliferation,invasion and migration of SMMC-7721 cells.