Knockdown of GRP۷۸ gene expression by over expression of miR-۱۸۱a in human bladder cancer cell line; ۵۶۳۷

Background and Aim: Invasion and metastasis of the bladder tumor to other organs significantly reduce the survival of patients. Investigating the molecular and cellular mechanisms of cancer that are responsible for cellular invasion is one of the most promising ways to invent new therapies. Some intracellular molecules, such as GRP۷۸, MMPs, microRNA, which play key roles in promoting tumor growth, metastasis, invasion and tissue degradation have been identified as molecular targets. Previous researches have indicated that miR-۱۸۱a down regulates GRP۷۸ mRNA by targeting its ۳′UTR. The aim of this study was to investigate the effect of miR-۱۸۱a construct on targeting GRP۷۸ and consequently studying its effects on growth, migration and metastasis in ۵۶۳۷ cells line.Methods: The sequence of microRNA was designed and then synthesized and cloned into the AAV vector. Then, successful clones were transferred to the ۵۶۳۷ cell line by lipofectamin ۲۰۰۰, expression of GRP۷۸, MMP-۲, MMP-۹, TIMP-۱, TIMP-۲ genes and ۵۶۳۷ cell’s proliferation and migration rate were measured by RT-qPCR, MTT and scratch assay methods respectively. Moreover gelatin zymography was performed to investigate MMP-۲ and MMP-۹ gelatinolytic activities.Results: Data showed that miR-۱۸۱a, GRP۷۸ and MMP-۲ transcripts increased and expression of MMP-۹, TIMP-۱ and TIMP-۲ genes decreased, when compared to controls. The results of wound scratch assay and its analysis showed that miR-۱۸۱a significantly reduced migration of ۵۶۳۶ bladder cancer cells. There was no toxicity associated with miR-۱۸۱a in cultures. Gelatin zymography analysis revealed that transfection of miR-۱۸۱a caused meaningful increase in the activities of MMP-۲ and MMP-۹.Conclusion: Our data clearly demonstrated that the miR-۱۸۱a reduced invasiveness and migration of ۵۶۳۷ bladder cancer cells regardless of increased activities of MMP-۹ and MMP-۲.

کلیدواژه ها:

۵۶۳۷ cancer cell ، miRNA-۱۸۱a ، GRP۷۸ ، MMP-۲ ، ۹