Impact And Mechanism Of MiR-143 On Biological Behavior And Chemosensitivity Of Bladder Cancer Cells

BackgroundAmong microRNAs,miR-143,which plays a tumor suppressive role,is constantly down-regulated in many kinds of malignant tumors.In vitro experiments confirmed that there is a potential targeting site of miR-143 in IGF-IR-3'UTR region.Overexpression of miR-143 was sufficient to inhibit tumor cell proliferation,invasion and metastasis through suppression of IGF-IR.MicroRNA in the regulation of gene expression plays an increasingly important role.However,miR-143-mediated IGF-IR regualtion influence chemosensitivity to gemcitabine has yet to be elucidated in bladder cancer cells.We found that miR-143 may play an important role in the regulation of IGF-IR in bladder cancer cells.So to explore the mechanism of miR-143-mediated IGF-IR regualtion involved in the biological behavior and chemosensitivity of bladder cancer cells is very important.ObjectiveIn this study,we aims to investigate the expression of miR-143 in bladder cancer tissues and cells,as well as its relationship with the growth of bladder cancer cells,and also explores the molecular mechanism of miR-143 regulating malignant behavior and chemosensitivity of bladder cancer.Methods1.Real time Polymerase Chain Reaction assay were performed to detect the expression of miR-143 in bladder cancer tissue specimens and matched adjacent normal tissues.The expression level of miR-143 were detected in bladder cancer cell line T24 and 5637 cells.The relevance of miR-143 involved in the stage of bladder cancer was also analyzed.2.Real time Polymerase Chain Reaction assay were performed to detect the expression of IGF-IR in bladder cancer tissue specimens and matched adjacent normal tissues.The relevance of miR-143 and IGF-IR was also analyzed.Lentiviral plasmids were made to upregulate the expression of miR-143 in bladder cancer cells.Real time Polymerase Chain Reaction assay were performed to assess the expression levels of IGF-IR in T24 and 5637 cells with overexpression of miR-143.3.CCK-8 assay were performed to detect cell proliferation after the transfection of lentivirus and knocking down IGF-IR in 5637 cells.The effects of overexpressed miR-143 and si-IGF-IR on cell proliferation in 5637 cells were examined by using CCK-8 assay.4.Western blotting assay were performed to analyzed the protein expression of p-AKT,AKT,p-ERK and ERK in 5637 cells with overexpressed miR-143 and si-IGF-IR.Results1.The expression levels of miR-143 were significantly lower in bladder cancer tissues than the corresponding adjacent tissues in 23 bladder cancer patients,and its expression was significantly correlated to tumor grade.The expression levels of miR-143 were markedly lower in bladder cancer line T24 and 5637 cells.2.The expression levels of miR-143 and IGF-IR were reversely correlated in bladder cancer tissue specimens.IGF-IR protein expression was markedly inhibited by overexpression of miR-143 in T24 and 5637 cells.3.In CCK-8 assay,the increased expression of miR-143 and small interfering of IGF-IR significantly inhibited the growth of 5637 cells.The chemosensitivity to gemcitabine was improved by up-regulating miR-143 and knocking down IGF-IR in 5637 cells.4.Overexpression of miR-143 was able to suppress P-AKT and P-ERK expression levels.Knockdown of IGF-IR greatly decreased AKT and ERK phosphorylation levels in 5637 cells.Overexpression of miR-143 and si-IGF-IR was all able to regulate AKT and ERK pathway activation in 5637 cells.Conclusion1.The expression of miR-143 is significantly lower in bladder cancer tissues than the corresponding adjacent tissues,and its expression is significantly related with tumor grade.2.MiR-143 can suppress the proliferation ability in bladder cancer cells,and regulate AKT and ERK pathway activation through inhibiting IGF-IR,suggesting a role of tumor suppressor gene.3.MiR-143 and gemcitabine have synergistic inhibition of bladder cancer cell growth.The chemosensitivity to gemcitabine was enhanced by overexpression of miR-143 in 5637 cells.