The Expression And Target Microrna Of KLK5in Breast Cancer

Objective：Kallikrein-related peptidases (KLKs) are secreted trypsin-like protease, encoded bythe Kallikrein gene family (KLKs), and shown to be dysregulated in several malignanciesincluding breast cancer. KLK5is a member of KLKs, but the expression and the control ofKLK5in breast cancer are still not well understood. The objectives of the study are asfollows: detecting the expression of KLK5in breast cancer cell lines and tissues; exploringthe microRNA that targets on the KLK5; revealing the function of the microRNA on thecytobiological characteristics of breast cancer cell.Mathods：1. Realtime-PCR, Western Blot, and ELISA were employed to detect the expressionof KLK5in breast cancer tissue and cell lines.2. Screening of microRNAs that targeting on KLK5mRNA3’-UTR based on theresults of bioinformatics softwares, relative references and realtime-PCR. And then, weexperimentally verified the predicted miR–KLK5interaction using two independenttechniques, measuring KLK5level after transfection with microRNA, and a luciferaseassay with a construct containing the KLK53’-untranslated region (UTR), pmir–KLK5.3. Flow cytometry was used to detect the change of cell cycle, scratch assays toexamine cell migration.Results：1. KLK5expression level was significantly lower in breast cancer tissue, comparedwith that from tumor-surrounding tissue, and the expression of KLK5in early invasivecarcinoma was significantly higher than in local-advanced breast cancer. Furthermore,expression of KLK5was shown to be significantly decreased in high-invasive breast cancercell line MDA-MB-231compared with low-invasive MCF-7cells.2.10microRNAs were predicted by bioinformatics softwares, and miR-125b was finally selected to be the potential microRNA that can target on KLK5mRNA3’-UTR.After transfection with miR-125b, there was a significant decrease in KLK5expression.Cotransfection of miR-125b and the pmir-KLK5led to a significant suppression of theluciferase signal. These data together show that miR-125b can specifically target the KLK5mRNA3’-UTR.3. Treatment of MCF-7cells with miR-125b could significantly increase G0/G1ratio,and decrease S stage and G2/M stage ratio, and much more cells were arrested cell cycle inG0/G1phase. Moreover, breast cancer MCF-7cells with miR-125b over-expression moreslowly closed the scratch wounds compared with the controls.Conclusion：1. Our results, showing differential expression of KLK5in breast cancer, suggest thatKLK5could be a new potential diagnostic marker for breast cancer subtypes and indicate adistinct pathogenesis in different stages.2. miR-125b can target KLK5mRNA3’-UTR, suggesting a novel post-transcriptionalregulatory mechanism for KLKs, and could have potential therapeutic applications.3. Over-expression of miR-125b in MCF-7resulted in suppression cell proliferationand invasion, shows that miR-125b maybe a new target of gene therapy for breast cancer.