Down-regulation of Notch1 gene promotes autophagy of human liver cancer cell line HepG2

Abstract

Objective To investigate the effect of Notch1 gene on the proliferation, apoptosis, autophagy and Akt/mTOR signaling pathway of HepG2 liver cancer cells. Methods Thirty-two cases of liver cancer tissues from patients undergoing liver cancer surgery in the Third Affiliated Hospital of Xinxiang Medical College were selected, and the expression of Notch1 mRNA was detected by fluorescence quantitative PCR. Notch1 siRNA eukaryotic expression vector was constructed, transfected into HepG2 cells by liposome, RT-qPCR and Western blot were applied to check the interference effect of Notch1, MTT method was used to detect cell proliferation and flow cyto- metry was used to detect cell apoptosis, Western blot was also applied in the detection of apoptosis-related proteins BCL-2, BAX, cleared-caspase-3, autophagy-related proteins LC3B, Beelinl, ATG7, ATG5 and Akt/mTOR signaling pathway related proteins Akt, p-Akt, p-mTOR, p-P70S6K. Results The relative expression of Notch1 gene in primary hepatocellular carcinoma tissue was significantly higher than that in adjacent tissues (1.865±0.791 vs 0.709±0.414) (P＜0.001). After Notch1 siRNA was transfected into HepG2 cells, the expression of Notch1 mRNA and protein in Notch1 siRNA group was significantly lower than that in NC siRNA group and in control group (P＜0.001). The cell proliferation rate of Notch1 siRNA group at 24, 48, 72 and 96 h was significantly lower than that of NC siRNA group and control group (P＜0.001). Forty-eight hours after transfection, the apoptosis rate and Bax level of Notch1 shRNA group were significantly higher than that of NC siRNA and control group, and the expression of Bcl-2 and cleaved caspase-3 were significantly lower than that of NC siRNA group and control group (P＜0.001). The expression of autophagy-related proteins LC3B, Beelinl, ATG7 and ATG5 in Notch1 siRNA group was significantly higher than that of NC siRNA group and control group (P＜0.001). The expression level of p-Akt, p-mTOR, and p-P70S6K in Notch1 siRNA group was significantly lower than those in NC siRNA group and control group (P＜0.001). Conclusions Down-regulation of expression of Notch1 gene can significantly inhibit the proliferation of liver cancer HepG2 cells, induce cell apoptosis, and increase cell autophagy. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway.

Keywords

• liver cancer|notch1 gene|akt/mtor signal pathway|autophagy