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

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.

Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells

“…That is, amino acids 2−713 of Cas9 are known as N‐Cas9 and 714–1368 as C‐Cas9. Also, sgRNAs were selected according to previous reports 32,43 . This method was named paCas9‐GOI.…”

“…The membranes were later incubated with horseradish peroxidase‐conjugated secondary antibody (Goat Anti‐Rabbit IgG (H+L) or Goat Anti‐Mouse IgG (H+L); 1:6000; Proteintech) for 2 h on a horizontal oscillator at room temperature. Products were generated on film by the chemiluminescence kit purchased from Sangon Biotech and quantitated by ImageJ software 32,33,39 …”

“…Hu et al 33 established a degradation platform that could constrain Survivin at both transcriptional and protein levels based on ferritin nanocage. Meanwhile, Deng et al 32 used the Tet-off system to regulate Survivin expression precisely and found that Survivin might mediate the development of chemotherapy resistance. All the above studies showed that inhibiting the expression of Survivin could be used to treat cancers and reduce the generation of chemoresistance.…”

“…established a degradation platform that could constrain Survivin at both transcriptional and protein levels based on ferritin nanocage. Meanwhile, Deng et al 32 . used the Tet‐off system to regulate Survivin expression precisely and found that Survivin might mediate the development of chemotherapy resistance.…”

“…Survivin (also called BIRC5), a strategic and fundamental protein molecule in cancer, mainly plays the role of anti-apoptosis and is exceedingly apparent in its expression in all tumour cells including bladder cancer. [31][32][33] Therefore, Survivin has been projected as a striking target for innovative anti-cancer interventions. Lactose-derived branched cationic biopolymer delivery system emerged from the work of Qi et al 34 in transmitting Cas9 for editing the Survivin gene to cure hepatocellular carcinoma.…”