The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance.

Changping Deng,Xingyuan Ma,Fabiao Hu,Zhangting Zhao,Yuping Liu,Wenliang Zhang,Tianwen Wang,Tong Zhang,Shihui Li,Yiwen Zhou,Wenyun Zheng

doi:10.3389/fcell.2021.797005

Abstract

Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.

Highlights

Quantitative analysis and controllable expression of genes is a novel method for studying key genes in tumor
Several studies had shown that the expression of Survivin in drug-resistant tumor cells was significantly higher than that of normal cells (Lamers et al, 2012), and inhibition of Survivin could increase the sensitivity of resistant cells to chemotherapy drugs (Lamers et al, 2012)
These findings proved that Survivin was involved in the multi-drug resistance (MDR) of tumors

Summary

Introduction

Quantitative analysis and controllable expression of genes is a novel method for studying key genes in tumor. This method helps to analyze the biological functions of specific genes themselves, and better explains the biological phenomena related to them (Ede et al, 2016). The dominant-negative mutant of Survivin could increase the sensitivity of breast cancer cells to DOX, which suggested that Survivin was associated with tumor multi-drug resistance (MDR) (Xu et al, 2012). In order to deeply explore the role of Survivin in MDR, it would be very helpful to develop a system that regulates the expression of BIRC5 in real time. The link between Survivin and chemotherapy resistance can be further explored

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Conclusion