| BackgroundRenal cell carcinoma(RCC)is the third most common tumor in genitourinary system,representing 3%of all cancers worldwide,and the incidence has increased annually by 2%during the past decades,resulting in an estimated 400000 new RCC cases and 175000 cancer-related deaths worldwide in 2018.Approximately 70%of RCC are localized or locally advanced at the time of diagnosis and could be cured by radical or partial nephrectomy.However,30-35%of patients undergoing surgery will develop distant metastatic disease and eventually lead to death.Although remarkable progress has been made in the clinical application of targeted therapy and immunotherapy in recent years,the 5-year overall survival rate of metastatic RCC remains as low as 10-20%.Therefore,identifying new tumor biomarkers and exploring the molecular mechanisms involved in RCC tumorigenesis would be beneficial for developing new diagnostic and therapeutic strategies.CHD5(chromodomain helicase DNA binding protein 5)gene is located at 1p36.31,which was first found due to its frequent deletion in neuroblastoma.The protein encoded by this gene is a member of the ATP-dependent chromatin remodeling protein SNF2 superfamily,which is involved in the regulation of chromatin structure and transcription.According to its dual inhibition of clonality and tumorigenicity in neuroblastoma cells and xenografts,the researchers identified it as a tumor suppressor gene.Subsequent studies have shown that CHD5 also plays a tumor suppressing role in other types of tumors,including glioma,breast cancer,colon cancer and ovarian cancer,and the expression level of CHD5 is closely related to clinical and biological characteristics.Therefore,downregulation of CHD5 might be a key molecular initiating event in tumorigenesis and could serve as a potential therapeutic target.It has been reported that the expression of CHD5 is downregulated due to hypermethylation of the promoter in RCC,but the clinical significance of CHD5 expression in RCC and the underlying molecular mechanism of CHD5 in the tumorigenesis of RCC are still unclear.Therefore,we carried out this study.Objective1.To analyze the expression difference of CHD5 in RCC and adjacent tissues,and explore the correlation between its expression level and clinicopathological characteristics and prognosis.2.To explore the effect of CHD5 overexpression on the biological function of RCC and the molecular mechanism underlying tumorigenesis,so as to provide a new insight for early diagnosis and treatment of RCC.Method1.RT-qPCR was used to detect the expression of CHD5 in 24 RCC and adjacent tissues,and the relationship between CHD5 and clinicopathological characteristics was analyzed.2.Immunohistochemical staining was performed on the tissue microarray containing the complete survival data of 90 RCC patients to analyze the relation between the expression level and the prognosis.3.Overexpression of CHD5 in ACHN and 769-P cells using the CRISPR/Cas9-SAM system.Subsequently,Cell Counting Kit-8(CCK-8)analysis,Plate colony formation assays,and xenograft mouse tumor models were performed to determined the effects of CHD5 on the proliferation of ACHN and 769-P cells in vitro and in vivo;flow cytometry was used to detect changes in cell cycle and apoptosis;wound healing and transwell assay were carried out to assess migration and invasion ability,respectively.4.WB detected the changes of p16INK4a/Rb and p14ARF/p53 pathway-related protein levels after CHD5 overexpression.Result1.Analysis of the expression of CHD5 in 24 cases of RCC and adjacent tissues found that the expression level of CHD5 in tumor tissues was significantly lower than that of adjacent tissues,and the low expression of CHD5 was closely related to advanced TNM stage,high Fuhrman grade and lymph node metastasis.Nevertheless,there was no relationship to patients’gender,age,and tumor size.2.Kaplan-Meier survival analysis of tissue array immunohistochemical staining results showed that patients with low CHD5 expression had a shorter overall survival than patients with high CHD5 expression.3.Overexpression of CHD5 inhibited proliferation,migration,and invasion in vitro,prompted cell cycle G1 phase arrest,induced apoptosis,and suppressed tumor growth in vivo.4.Overexpression of CHD5 in ACHN and 769-P cells increased expression of p14 ARF,MDM2,p53,p21,Bax,Caspase-9,p16 INK4a,and Rb,and decreased expression of P-Rb,CyclinDl,and CDK4.Conclusion1.CHD5 was downregulated in tumor tissues and that low CHD5 expression was correlated with adverse clinicopathological features and poor survival.2.Overexpression of CHD5 inhibited the proliferation,migration and invasion of ACHN and 769-P cells.3.CHD5 positively regulated p14ARF/p53 and p16INK4a/Rb pathways to inhibit tumorigenesis in RCC... |
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