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The Research of miR-381and mIR-424in Human Renal Cell Cancer

Author: ChenBingHai
Tutor: JiangXianZhen
School: Central South University
Course: Clinical
Keywords: miR-381 miR-424 WEE1 miRNA Cdc2 5-FU renalcell carcinomamiR-381 dual-luciferasereporter assaymiR-381 Cdc2miR-381 renal cell cancer 786-O
CLC: R737.11
Type: PhD thesis
Year: 2013
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Abstract


Renal cell cancer (RCC) is considered as one of the most common neoplasms in the urinary system. Its morbidity and mortality increase year by year. Fewer effective therapies were confirmed in advanced RCC, moreover, only part response of targeted therapy is shown in some patients. Advanced RCC is in most majorities. What’s more, recurrence and metastasis of RCC is very common nowadays. So we believe that there is great significance to pursue effective inhibition of RCC and improvement of chemotherapy resistance.The research of microRNA (miR) attracts increasing attention in recent years. MiR, a kind of single-stranded RNA, has no open reading framework; however, it can regulate expression of target protein by binding the3’UTR of the mRNA. It inhibits either the mRNA expression or protein translation. Increasing evidence showed that many cancers were characterized by abnormal expression of miR, which played important roles in the initiation and progression of carcinoma. MiR-381and miR-424are no exception. Both miR-381and miR-424have roles in carcinomas and researches about the two miRs have been reported. We have made some progress on miR in the RCC. However, the expression, function and regulation of miR in RCC are complicated. So far the role of neither miR-381nor miR-424in RCC has been reported.We believe miR-381and miR-424share the same target of WEE1, which is one of the WEE families. WEE1is highly conserved and it widely exists in eukaryote. WEE1, a serine threonine kinase, is a crucial factor in the regulation of G2/M. In addition, WEE1takes the responsibilities of DNA replication and DNA damage repair before mitotic entry. The deregulation of WEE1was reported recently in various cancers, however, not in RCC.It was reported that WEE1depletion induced apoptosis and mitosis catastrophe by regulating G2/M and leaving the DNA damage (caused by chemotherapy or radiation) not repaired. We speculated that miR-381, an intrinsic WEE1inhibitor, may augment the chemotherapy sensitivity of5-FU.In this research, we firstly investigated the expressions of miR-381, miR-424and their target gene in RCC cell lines. We also explored and validated the direct relationship among miR-381, miR-424and their target gene (WEE1). In addition, we firstly detected the cell proliferation, apoptosis, cell cycle as well as caspase3/7activity after the transfection of miR-381and miR-424. Finally, we investigated the IC50of5-FU when combined with miR-381and studied the mechanism of miR-381and miR-424in RCC.The innovations of this study are as follows:1) The miR-381and miR-424were firstly reported to inhibit proliferation of renal cancer cells and induce apoptosis. We also figure out the exact mechanism of miR-381and miR-424;2) The synergistic effect of miR-381and miR-424was firstly reported in renal cancer cells, moreover, the synergistic effects of all miRs were seldom studied;3) the enhancement of sensitivity of5-FU caused by miR-381was also firstly analyzed in RCC, which may supply a novel therapy method in RCC;4) the direct target relationship among miR-381, miR-424and WEE1was demonstrated at the first time. There are57figures,19tables, and86references. Chapter One:The research on expression characteristics of miR-381, miR-424and their target geneObjective:To investigate and validate the target gene of miR-381and miR-424; To study the expression characteristics of miR-381and miR-424in RCC cell lines and renal tubular epithelial cell line. Methods: We predicted the target gene of miR-381by bioinformatics software and miR-424and validate it by dual-luciferase reporter assay. In addition, we determined the expressions of miR-381, miR-424and their target gene by means of western blot. Result:WEE1is considered as the common target of miR-381and miR-424. The3’ UTR of WEE1is highly conserved, which stably binds to miR-381and miR-424. It is showed in dual-luciferase reporter assay that both miR-381and miR-424directly target WEE1. Overexpressed miR-381and miR-424induced a significant decrease of WEE1. What’s more, miR-381and miR-424are low-expressed in RCC cell lines, while WEE1is overexpressed in RCC cell lines. Conclusion:The abnormal expressions of miR-381, miR-424and their target gene are confirmed in RCC. MiR-381and miR-424share a target gene of WEE1. It may lay a foundation for the further research on the roles of miR-381and miR-424in initiation and progression of RCC. Chapter Two:Research on the role of miR-381and miR-424in RCC and their mechanismObjective:To investigate the roles and mechanism of miR-381and miR-424in RCC cell lines after over-expression of the two miRs. Methods:We overexpressed miR-381and miR-424in786-0, ACHN and HK-2by means of transient transfection. After that we determined cell cycle, apoptosis, cell survival as well as caspase activity. Finally we studied the mechanism by determining the expressions of WEE1, associated cell cycle protein kinase, apoptosis protein and Cdc2with western blot assay. Result:Simultaneous transfection of miR-381and miR-424inhibited cell proliferation and induced apoptosis in786-O. It also increased activity of caspase3/7. In addition, it decreased the cell portion of G2/M. However, no similar effect was found in another RCC cell line ACHN. Monotherapy with either miR-381or miR-424was not as effective as the combination treatment. We believe that miR-381and miR-424work in a synergistic manner. Conclusion:Overexpression of miR-381and miR-424synergistically inhibit RCC cell line786-O. It cannot inhibit ACHN due to the P53activity. It regulates Cdc2as well as induces apoptosis and mitosis catastrophe by regulating Cdc2independent of MYT1and Cdc25. Chapter Three:The research on the enhancement and mechanism of chemotherapy sensitivity of5-FU induced by miR-381Objective:To investigate the role and mechanism of miR-381and exogenous DNA damage caused by5-FU in RCC cell lines. To study the synergistic effects of miR-381and5-FU. Methods:5-FU in different concentrations was added in786-O, ACHN and HK-2before overexpression of miR-381by transient transfection. We determined cell survival in different time point. In addition, we also detected the cell cycle, apoptosis and caspase3/7activity. We used western blot to analyze the expression of WEE1, downstream target gene and associated apoptosis protein and studied the mechanism of miR-381and5-FU. We then rescued WEE1by means of siRNA before MTT determination. Result:786-0cells treated with miR-381were inhibited more effectively than that with control in various concentrations of5-FU. The inhibition efficiency reached maximum in48hours. Overexpression of miR-381significantly decreased the IC50of5-FU. Combination of miR-381and5-FU increased caspase3/7activity, cell apoptosis and regulated cell cycle. Unavailable effect in ACHN may be due to P53activity. Combination of miR-381and5-FU decreased expression of WEE1and inhibited the phosphorylation of Cdc2. After the combination treatment of miR-381and5-FU in786-O, extrinsic siRNA of WEE1can partly rescue the inhibition of786-O cells induced by miR-381and5-FU. Conclusion:In RCC cell line786-0, overexpression of miR-381combined with5-FU effectively inhibit the cell survival and augment chemotherapy sensitivity. IC50of5-FU decreased after transfection of miR-381. It made it possible for5-FU in lower concentration to treat RCC. The mechanism lies as follows. First, G2check point is abrogated in cells with DNA damage caused by5-FU and the DNA damage is not repaired before mitosis catastrophe. Second, it is due to the Bcl-2apoptosis way independent of PARP.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor
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