Journal of Jilin University(Medicine Edition) ›› 2022, Vol. 48 ›› Issue (3): 657-667.doi: 10.13481/j.1671-587X.20220314

• Research in basic medicine • Previous Articles    

Effect of silencing helicase BLM gene on chemotherapy sensitivity of irinotecan in colorectal cancer cells and its mechanism

Qiuting CAO,Jingchun HAN,Xiaofei ZHANG()   

  1. Department of Oncology,Affiliated Xinhua Hospital,Dalian University,Dalian 116021,China
  • Received:2021-09-03 Online:2022-05-28 Published:2022-06-21
  • Contact: Xiaofei ZHANG E-mail:274365378@qq.com

Abstract: Objective

To investigate the effect of silencing the Bloom’s syndrome helicase(BLM) expression on the chemotherapy sensitivity of irinotecan (CPT-11) in the colorectal cancer (CRC) cells, and to elucidate its related mechanism.

Methods

The HT-29, Lovo, HCT-116, RKO, and DLD1 cells were cultured in vitro. The RKO and DLD1 cells with high expressions of BLM mRNA and protein were screened by real-time fluorescence quantitative PCR(RT-qPCR) and Western blotting methods. The cells were infected with lentivirus vector carrying shRNA to silence the expression of BLM in the RKO and DLD1 cells. The survival rates of the CRC cells and the half inhibitory concentration (IC50) values of CPT-11 in various groups after transfection were detected by CCK-8 assay. The RKO or DLD1 cells were divided into LV-shNC group (infected with LV-shNC), CPT-11+LV-shNC group ( infected with LV-shNC and treated with CPT-11) and CPT-11+LV-shBLM group (infected with LV-shBLM and treated with CPT-11). The percentages of the cells in various groups at different cell cycles were detected by flow cytometry. The expression levels of BCL2 associated death promoter(Bad),cleaved of cysteinyl aspartate specific proteinase(cleaved caspase-3), cyclin-dependent protein kinase 4(CDK4),cyclin-dependent protein kinase 6 (CDK6),and cyclin-dependent kinase inhibitor 1A(p21) proteins in the CRC cells in various groups were detected by Western blotting method; the apoptotic rates of the cells in various groups were detected by flow cytometry.The tumor volume and positive expression rates of BLM, Ki-67,and TUNEL in tumor tissue of the CRC-bearing mice were detected by constructing the allograft tumor model.

Results

Compared with the HT-29, Lovo or HCT-116 cells, the expression levels of BLM mRNA and protein in the RKO and DLD1 cells were increased (P<0.05);compared with RKO or DLD1 cells infected with LV-shNC, the expression levels of BLM mRNA in the RKO or DLD1 cells infected with LV-shBLM lentivirus were decreased(P<0.05). Compared with LV-shNC group, the survival rates and IC50 values of the cells in CPT-11+LV-shNC group and CPT-11+LV-shBLM group were decreased(P<0.05).Compared with LV-shNC group, the percentages of the cells at G0/ G1 phase and S phase and the apoptotic rates of the cells in CPT-11+LV-shNC group and CPT-11+LV-shBLM group were increased (P<0.05), while the percentages of the cells at G2 / M phase were decreased (P<0.05),and the expression levels of Cyclin D1, CDK4, CDK6 and Bcl-2 proteins were decreased(P<0.05), while the expression levels of p21, Bax, Bad,and cleaved caspase-3 proteins were increased (P<0.05);compared with CPT-11+LV-shNC group, the percentages of the cells at G0/G1 phase and S phase and the apoptotic rate of the cells in CPT-11+LV-shBLM group were increased (P<0.05), while the percentage of the cells at G2/M phase was decreased (P<0.05),and the expression levels of Cyclin D1,CDK4, CDK6,and Bcl-2 proteins were decreased(P<0.05), while the expression levels of p21, Bax, Bad,and cleaved caspase-3 proteins were increased (P<0.05). The nude mice transplanted tumor experiment results showed that compared with LV- shNC group, the tumor volumes of the nude mice and the positive expression rates of BLM and Ki-67 in the cells in CPT-11+LV-shNC group and CPT-11+LV-shBLM group were decreased (P<0.05), and the positive expression rates of TUNEL were increased(P<0.05); compared with CPT-11+LV-shNC group, the tumor volume of the nude mice in CPT-11+LV -shBLM group was decreased (P<0.05), and the positive expression rates of BLM and Ki-67 in tumor tissue were decreased (P<0.05), and the positive expression rate of TUNEL was increased(P<0.05).

Conclusion

Silencing BLM expression in the CRC cells may promote CPT-11-induced apoptosis by inhibiting the cycle progression of tumor cells, and ultimately improve chemotherapy resistance of CRC cells in vivo and in vitro.

Key words: Colorectal neoplasms, Bloom’s syndrome helicase, Cell cycle, Chemosensitivity, Apoptosis

CLC Number: 

  • R735.3