Study On The Correlation Between The Expression Of COTE1 And Autophagy And Its Effects On Chemotherapy Sensitivity In Liver Cancer

Objective:To study the correlation between the expression of COTE1 in liver cancer tissues and autophagy,and the effect of interfering with the expression of COTE1 in liver cancer cells PLC/PRF/5 on the sensitivity of chemotherapy drugs adriamycin and 5-fluorouracil.Methods:(1)We selected 25 liver cancer tissues to detect the expression of COTE1 protein and autophagy related protein LC3 by immunohistochemistry,and analyzed their correlation.(2)We used RNAi to interferenced the expression of COTE1 in liver cancer cells PLC/PRF/5 and selected a better siRNA sequences.Then the sequence of COTE1-shRNA was packaged into the lentivirus vector.We got the COTE1 silent stable expression cell lines by lentivirus transfection into PLC/PRF/5 liver cancer cells and puro screening.Lentivirus interference effect was proved by QPCR and Western Blot.Meanwhile,western blot was used to detect the expression of autophagy related protein LC3 after down-regulating the expression of COTE1 protein in liver cancer cells,so as to further analyze their correlation.(3)COTE1-shRNA group and its comparison group were treated with different concentrations of chemotherapeutic drugs doxorubicin and 5-fluorouracil.CCK-8 assay was used to detected the change of the sensitivity to chemotherapy drugs after the down-regulation of COTE1.Western Blot and flow cytometry were used to detected cell autophagy and apoptosis level,respectively.Results:(1)Immunohistochemical results showed that among the 25 liver cancer tissues,20 cases had positive COTE1 expression and 8 cases had positive LC3 expression.Kappa analysis showed that the expression of COTE1 in liver cancer was significantly correlated with the expression of autophagy related protein LC3(P < 0.05).(2)By siRNA transfection liver cancer cells,Western Blot results show two siRNA sequences all can interfered the expression of COTE1 effectively.We selected one packaging into lentivirus.After transfection two days later,we can see a large number of green fluorescence under the fluorescence microscope,and no fluorescence was observed in transfection virus blankgroup,which prompting the successful transfection.The QPCR and Western blot results showed that the COTE1 was significantly down-regulated in mRNA level and protein level after screening in 0.1?g/ml puro for a week(P < 0.05),which indicated the stable down-regulation of COTE1 expression was successfully established.In addition,Western Blot showed that the expression of LC3-II/LC3-I decreased after interfering of the expression of COTE1(P < 0.05),which further proving the correlation between the expression of COTE1 and autophagy in liver cancer cells.(3)CCK-8 assay showed that the inhibitory rate of chemotherapy drugs on the proliferation of HCC cells increased as the concentration of chemotherapy drugs increased,and the inhibitory rate on the proliferation of HCC cells transfected with COTE1-shRNA was significantly higher than that of the control group(P < 0.05).Western Blot and flow cytometry results showed that the autophagy level of liver cancer cells was inhibited and the apoptosis level was increased after down-regulating the expression of COTE1(P < 0.05).Conclusion:(1)The expression of COTE1 in liver cancer is correlated with autophagy,and down-regulating the expression of COTE1 in liver cancer cells can inhibit the autophagy level of liver cancer cells.(2)Interference with the expression of COTE1 in liver cancer cells can enhance the sensitivity of tumor cells to the chemotherapy drug adriamycin or 5-fluorouracil by inhibiting autophagy or enhancing apoptosis.