Histone Deacetylase5Promotes Wilms Tumor Cell Proliferation Through The Upregulation Of C-Met

Objectives:Nephroblastoma also calling Wilms tumor is the commonest primary malignant renal neoplasm in children,accounting for more than 90%of all children’s renal tumors.Hitherto,the molecular mechanism of the occurrence and development of nephroblastoma is unclear,and more than 40 related susceptibility genes have been reported.Epigenetic silencing is a marker of cancer cells.Two important epigenetic changes are DNA methylation and histone modification.The histone deacetylase 5(HDAC5)which belongs to histone deacetylase(HDACs)Ⅱα family is involved in modulating the majority of critical cellular processes,such as transcriptional regulation,apoptosis,proliferation and cell cycle progression.HDAC5 has become a potential therapeutic target due to its epigenetic role in tumorigenesis.Preclinical studies have demonstrated the therapeutic application of HD AC inhibitors as potential anticancer drugs.Therefore,it is of great clinical significance to study the biological function of HDACs in nephroblastoma.However,the biological function of HDAC5 in Wilms tumor remains to be fully elucidated.The present study aimed to investigate the expression and function of HDAC5 in Wilms tumor,how the differential expression of HDAC5 influence on the viability and proliferation of tumor cells in vitro,and to further explore the regulatory effect of HDAC5 on c-Met in nephroblastoma and its molecular mechanism.Methods:The mRNA and protein of HDAC5 were analyzed by RT-qPCR and Western Blot in 23 cases of the primary nephroblastoma and 23 cases of the normal renal adjacent tissues,who had been treated in Children’s hospital of Soochow University.The plasmids encoding HDAC5-flag and HDAC5 specific siRNA were transfected into nephroblastoma cell line G401 cells respectively.The expression of HDAC5 in nephroblastoma cell line G401 cells was determined by RT-qPCR.The proliferation activity of nephroblastoma cell line G401 cells was determined by CCK-8 and BrdU analysis.The protein expression of HDAC5 was analyzed by Western blot.The expression of c-Met was analyzed by RT-qPCR and Western blot in nephroblastoma cell line G401 cells which were overexpressing HDAC5 and silencing HDAC5.The c-Met specific siRNA was transfected into nephroblastoma cell line G401 cells overexpressing HDAC5.The expression of c-Met was analyzed again,the proliferation activity of nephroblastoma cell line G401 cells was determined by CCK-8 and BrdU analysis.Results:RT-qPCR showed that the mRNA of HD AC5 in tumor tissues was significantly higher than that in normal renal adjacent tissues(p<0.05).Western blot analysis showed that the protein of HDAC5 have the same trend.RT-qPCR and Western blot confirmed that overexpression of HDAC5 and targeted silencing of HDAC5 could up-regulated and down regulated the target HDAC5 genes respectively.CCK-8 analysis showed that overexpression of HDAC5 significantly promoted the proliferation of nephroblastoma cell line G401 cells.BrdU method showed that compared with the control group,nephroblastoma cell line G401 cells overexpressing HDAC5 had faster growth rate(p<0.05),and targeting silencing HDAC5 reduced the proliferation rate(p<0.05).Compared with normal nephroblastoma cell line G401 cells,the expression of c-Met mRNA in nephroblastoma cell line G401 cells which overexpressing HDAC5 increased more than 4 times(p<0.05).Western blot analysis also confirmed the upregulation of c-Met in nephroblastoma cell line G401 cells which overexpressing HDAC5.The expression of c-Met also can be down regulated by silencing HDAC5.Targeted silencing of c-Met can lead to decrease expression of c-Met in nephroblastoma cell line G401 cells which overexpressing HDAC5.CCK-8 and BrdU analysised the downregulation of c-Met inhibited the proliferative effects of HDAC5 in nephroblastoma cell line G401 cells.Conclusions:In the present study,the expression of HD AC5 in Wilms tumor has been the first reported,we found HDAC5 was increased in Wilms tumor specimens.And try to explore the mechanism of HDAC5.In vitro investigation demonstrated that overexpression of HDAC5 in nephroblastoma cell line G401 cells significantly increased the proliferation of cell,compared with normal nephroblastoma cell line G401 cells.By contrast,HDAC5 knockdown using siRNA reduced cell growth.The upregulation of HDAC5 was observed to promote the mRNA and protein expression levels of c-Met in nephroblastoma cell line G401 cells.In addition,c-Met depletion significantly reversed the proliferation-promoting function of HDAC5 in human WT cells.