Abstract:

Objective To discuss the effect of thioredoxin domain-containing protein 9 （TXNDC9） gene knockdown on the apoptosis and autophagy of colorectal cancer （CRC） cells， and to clarify its potential molecular mechanism based on the p53-dependent pathway. Methods The human normal colonic epithelial cell line NCM460 and colon cancer cell lines HCT116， SW620， SW480， and LoVo were selected. The HCT116 cells were divided into NC group （transfected with negative control si-NC）， si-TXNDC9 group （transfected with si-TXNDC9）， and p53 pathway inhibitor PTF-α group （PTF-α group， transfected with si-TXNDC9 and treated with 5 μmol·L^-1 PTF-α for 24 h）. Plate colony formation assay and Transwell chamber assay were used to detect the numbers of clone formation and invasion cells in various groups. Western blotting method was used to detect the expression levels of autophagy， apoptosis， and p53 pathway-related proteins in the cells in various groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was used to detect the apoptotic rates of the cells in various groups.Transmission electron microscope （TEM） was used to observe the numbers of autophagosomes in the cells in various groups. The xenograft tumor models in nude mice were established， and the mice were randomly divided into control group （inoculated with si-NC-transfected HCT116 cells）， knockdown group （inoculated with si-TXNDC9-transfected HCT116 cells）， and inhibitor group （inoculated with si-TXNDC9-transfected HCT116 cells and intraperitoneally injected with PTF-α） （n=5）. The tumor volumes of nude mice in various groups were measured and recorded every 7 days， and the mice were sacrificed on the 28th day， and the tumors were stripped and weighed. Results In cell experiments， compared with the NCM460 cells， the expression levels of TXNDC9 protein in the HCT116， SW620， SW480， and LoVo cells were significantly increased （P<0.05）. Compared with NC group， the numbers of clone formation and invasion cells of HCT116 cells in si-TXNDC9 group were significantly decreased （P<0.05）， the apoptotic rate of the cells was significantly increased （P<0.05）， the expression levels of B-cell lymphoma 2（Bcl-2） and B-cell lymphoma-extra large （Bcl-XL） proteins in the cells were significantly decreased （P<0.05）， the expression level of Bcl-2-associated X protein （BAX） was significantly increased （P<0.05）， the number of autophagosomes was significantly increased （P<0.05）， the expression levels of autophagy-related protein 7 （ATG7） and Beclin-1 proteins and the ratio of microtubule-associated protein 1 light chain 3 （LC3）-Ⅱ/LC3-Ⅰ were significantly increased （P<0.05）， the expression level of P62 protein was significantly decreased （P<0.05）， and the expression levels of P53 and P21 proteins were significantly increased （P<0.05）. Compared with si-TXNDC9 group， the numbers of clone formation and invasion cells of HCT116 cells in PTF-α group were significantly increased （P<0.05）， the apoptotic rate of the cells was significantly decreased （P<0.05）， the expression levels of Bcl-2 and Bcl-XL proteins in the cells were significantly increased （P<0.05）， the expression level of BAX protein was significantly decreased （P<0.05）， the number of autophagosomes was significantly decreased （P<0.05）， the expression levels of ATG7 and Beclin-1 proteins and the LC3-Ⅱ/LC3-Ⅰ ratio in the cells were significantly decreased （P<0.05）， the expression level of P62 protein was significantly increased （P<0.05）， and the expression levels of P53 and P21 proteins were significantly decreased （P<0.05）. In animal experiments， compared with control group， the tumor weights and tumor volumes at each time point in knockdown group were significantly decreased （P<0.05）； compared with knockdown group， the tumor weights and tumor volumes at each time point in inhibitor group were significantly increased （P<0.05）. Conclusion TXNDC9 knockdown can promote apoptosis and autophagy in the CRC cells by regulating the p53-dependent pathway， thereby inhibiting their proliferation and invasion abilities.

Key words: Colorectal cancer, Thioredoxin domain-containing protein 9, P53 protein, Apoptosis, Autophagy