saKLK1-374 is More Difficult to Induce KLK1 Expression in Normal Cell Lines Than in Tumor Cell Lines And Inhibits the Growth of Prostate Cancer Cells Not via Induction of KLK1 Expression

Abstract

\n Background: RNA activation, as a method of regulating gene expression at the transcriptional level, is far less widely used than RNA interference because of the insufficient understanding of the mechanism and the unstable success rate. It is necessary to analyze the failure cases of RNA activation to promote the application of RNA activation. When we validated the saRNAs designed to induce KLK1 expression, we found that saKLK1-374 can up-regulate KLK1 expression in prostate tumor cell lines, but failed in normal prostate cell lines. In addition, we also found that saKLK1-374 inhibited the growth of prostate cancer cells, which seems to be the opposite of the function of KLK1. This article is about experimental research and analysis of these two issues.Methods: To determine whether the phenomenon that the RNA activation of normal cells is difficult to succeed is only valid when the target gene is KLK1, we used p21WAF1/CIP1 as the target gene to perform RNA activation experiments in normal prostate cells and prostate cancer cells. Next, to determine whether the above phenomenon exists in other tissues, we also performed RNA activation experiments with KLK1 and p21WAF1/CIP1 as target genes in normal cell lines and tumor cell lines derived from the bladder. We have also extended the time from transfection to the detection of target gene expression to evaluate whether a longer saRNA action time can change the phenomenon that saRNA fails to up-regulate target gene expression in normal cells. In terms of mechanism research, we used fluorescently labeled dsRNA to evaluate the transfection efficiency, and also detected the expression of Ago2 and IPO8 proteins. In another issue of saKLK1-374 inhibiting prostate cancer cells, we tested the ROS content and apoptosis levels of prostate cancer cells after saKLK1-374 transfection. We used recombinant KLK1 protein to directly interfere with prostate cancer cells as a positive control for KLK1 function research. In turn, we also used siRNA to inhibit the expression of KLK1 in prostate cancer cells to compare the growth of prostate cancer cells when KLK1 mRNA was up-regulated and reduced.Results: The p21WAF1/CIP1 gene could be significantly upregulated by saRNA in prostate cancer cell lines, but not in normal prostate cell lines. The expression of KLK1 in bladder-derived cell lines was extremely low and could not be induced by saRNA. The p21WAF1/CIP1 gene could be up-regulated by saRNA to a higher extent in bladder cancer cell lines, while it was up-regulated by saRNA in normal urothelial cell line to a lower extent. Prolonging the action time of saRNA could not change that saRNA failed to induce the expression of target genes in normal cell lines. Compared with tumor cell lines, normal cell lines had lower transfection efficiency or lower expression of Ago2 and IPO8. After being transfected with saKLK1-374, prostate cancer cells had increased ROS and increased levels of apoptosis. The recombinant KLK1 protein did not increase ROS in prostate cancer cells, nor did it inhibit their growth. Even though saKLK1-374 up-regulated the expression of KLK1 in prostate cancer cells, siRNA still suppressed the expression of KLK1 below the baseline level, and in this case, the growth of prostate cancer cells was still at a suppressed level.Conclusion: Normal cell lines may be more difficult to be successfully induced target gene expression than tumor cells due to low transfection efficiency or low Ago2 and IPO8 expression. In addition, although saKLK1-374 is designed to up-regulate the expression of KLK1, the reason that it inhibits the proliferation of prostate cancer cells is irrelevant to the up-regulated expression of KLK1.