The Effect Of MicroRNA-7 Regulating Epidermal Growth Fator Receptor On The Proliferation And Migration Of Hepatocellular Carcinoma Cells

Major: Imaging and Nuclear Medicine Student ID:1210660180496 Postgraduate:Yang Peng Supervisor:Xu Guo Hui Objective:In vitro study of micro RNA-7(miR-7)to understand whether it can regulatet the epidermal growth factor receptor(EGFR),and observe that the EGFR gene is in miR-7 Corresponding changes under regulation,to explore the effect of overexpression or silent expression of miR-7 on the migration and proliferation of human liver cancer cells,and seeking potential new targets for the treatment of liver cancer.Methods:(1)Human HepG2 hepatocellular carcinoma cells were routinely cultured anddivided into 2 subgroups of mir-7 up-regulation(mir-7 mimic group)and mir-7down-regulation(mir-7 inhibitor group).Each subgroup contained its own negativecontrol group(mimic negative group,inhibitor negative group)and blank controlgroup(mock group),a total of 6 groups.The corresponding overexpressed andsilenced plasmids were transfected by liposome transfection method,and 6 groups ofcells were collected by centrifugation 48 h after transfection.After RNA extraction,real-time PCR was used to detect the expression level of EGFR mRNA in eachgroup to determine the transfection efficiency.At the same time,the total protein ofeach group was extracted.After protein quantification by BCA method,the EGFRprotein expression level of each group was detected by Western Blot afteroverexpression or silencing of mir-7,confirming the regulatory relationship ofmirna-7 to EGFR gene targeting.(2)The six groups of transfected cells in the logarithmic growth period were selected,and the proliferation ability of cells in each group was detected by MTT method at24h,48 h,72h and 96 h,and the migration ability of cells in each group was detectedby cell scratch experiment at 24 h,48h and 72 h.To assess changes in cellproliferation and migration after mir-7 silencing or overexpression.All adoptsSPSS19.0 statistical software package for statistical analysis on the experimentaldata,accord with normal distribution of measurement data to mean ± standarddeviation,said more than mean comparison between groups using single factoranalysis of variance,said components compared by chi-square test,with P < 0.05 forthe difference is statistically significant.Results:(1)After cell culture,cell RNA was extracted and showed by fluorescence quantitative PCR.In the mir-7 subgroup,the relative expression level of EGFR mRNA in the mir-7 mimic group(0.09±0.89)was significantly lower than that in the mimic negative group(0.40±0.09)and the mock group(0.61±0.29),and the difference was statistically significant(P<0.05).However,in the mir-7 expression silencing subgroup,the relative expression level of EGFR mRNA in the mir-7inhibitor group(4.0±0.49)was significantly higher than that in the inhibitor negative group(0.39±0.40)and the mock group(0.40±0.61),with a statistically significant difference(P<0.05).This suggests that mirna-7 can regulate the expression of EGFR,and the two are negatively correlated.(2)The results of western blot showed that the relative expression of EGFR protein in the mimic group(0.12±0.33)was significantly lower than that in the mimic negative group(0.25±0.19)and mock group(0.25±0.12)in the over-expressed mir-7subgroup(P<0.05).However,in the mir-7 silencing subgroup,EGFR protein expression in the inhibitor group(0.51±0.03)was significantly higher than that in the inhibitor negative group(0.23±0.13)and the mock group(0.18±0.10),with a statistically significant difference(P<0.05).(3)MTT experiment results showed that in the mir-7 overexpressed subgroup,the mimic group significantly slowed down the cell proliferation rate at 24,48,72 and 96h after transfection compared with the mimic negative group and mock group.On the contrary,in the mir-7 over-expression subgroup,the proliferation rate of cells in the Inhibitor group at the above four time points was significantly faster than that in the Inhibitor negative group and mock group,with statistically significant differences(P<0.05).There was no significant difference between the mock group and the transfected control group(P>0.05).(4)The scratch experiment results showed that in the mir-7 overexpressed subgroup,the mimic group significantly slowed down the cell migration rate at 24,48 and 72h after transfection compared with the mimic negative group and mock group,with statistically significant differences(P<0.05).On the contrary,in the mir-7overexpression subgroup,the cell migration rate in the Inhibitor group at the above three time points was significantly faster than that in the Inhibitor negative group and mock group,with statistically significant differences(P<0.05).There was no significant difference between the mock group and the transfected control group(P>0.05).Conclusions:By up-regulating the expression of miRNA-7,the proliferation and migration of liver cancer cells can be slowed down,and the expression of EGFR was down-regulated;By down-regulating the expression of miRNA-7,the proliferation and migration of liver cancer cells can be accelerated,and the expression of EGFR was up-regulated,suggesting that miRNA-7 could regulate the proliferation and migration of HCC cells by targeting EGFR gene—miRNA-7 may become a potential target for HCC in the future.