| The roles of histone methyltransferases(HMTases)NSD(nuclear receptor binding SET domain)proteins in malignant tumors have received extensive attention,and have become one of the focus areas of oncology research.The NSD protease family consists of three members: NSD1,NSD2(WHSC1/MMSET)and NSD3(WHSC1L1).NSD2,an vital NSD protease family member,has been found to be significantly elevated in multiple myeloma,prostate cancer,breast cancer,lung cancer,gastric cancer,endometrial cancer,head and neck squamous cell carcinoma,osteosarcoma and other malignant tumor tissues.However,the biological function of NSD2 in cc RCC(clear cell renal cell carcinoma)remains unknown.The topic is divided into three parts based on the background.In the first part,the expression differences of NSD2 between various renal cancer tissues and normal kidney tissues were analyzed through the secondary analysis of bioinformatics.Explore the value of NSD2 expression in clinical diagnosis and prognosis evaluation of cc RCC patients.NSD2 expression level was further verified between cc RCC specimens,cell lines and normal kidney specimens,cell line by advanced techniques.In the second part,the biological functions and possible molecular signaling pathways of NSD2 on cell proliferation and apoptosis were investigated after knocking down or overexpressing NSD2.In the third part,the effects and potential mechanism of NSD2 on cell invasion and migration were explored after down-regulating NSD2 expression.Part Ⅰ: The expression and clinical significance of NSD2 in cc RCCObjective: To investigate the expression differences of NSD2 among cc RCC tissues,cell lines and normal kidney tissues,cell line.To explore the value of NSD2 level inclinical diagnosis and overall survival of cc RCC patients.Methods: The secondary analysis was performed on the gene chip data obtained from the Gene Expression Omnibus(GEO)database in renal cancers.To compare NSD2 m RNA level among various types of renal cancer and normal kidney tissues.To compare NSD2 m RNA level between cc RCC and adjacent tissues,and evaluate NSD2 efficacy in the diagnosis.To compare NSD2 m RNA level between non-metastatic cc RCC and metastatic cc RCC tissues.The secondary analysis of clinical data obtained from the Onco Lnc website was performed to evaluate the effects of NSD2 expression on overall survival of cc RCC patients.To compare NSD2 protein level between cc RCC and normal kidney specimens by immunohistochemistry.Quantitative real-time polymerase chain reaction(q RT-PCR)and western blot assays(WB)were used to verify the expression differences of NSD2 in cc RCC cells and normal renal tubular epithelial cell line.Results: The secondary analysis of gene chip data from GEO database showed that in various types of renal cancer tissues,NSD2 m RNA level was higher than that in normal kidney tissues,and cc RCC was the highest one among that in RCC(renal cell carcinoma).In cc RCC,NSD2 m RNA level was higher than that in adjacent tissues.ROC analysis showed that NSD2 level could be used in the diagnosis and the area under the curve(AUC)was 0.9026.In metastatic cc RCC,NSD2 m RNA level was higher than that in non-metastatic cc RCC tissues.The secondary analysis of clinical data from the Onco Lnc website showed that the overall survival of cc RCC patients with high NSD2 expression was shorter.By immunohistochemistry,NSD2 protein level in cc RCC specimens was higher than that in normal kidney specimens.Quantitative real-time polymerase chain reaction showed that NSD2 m RNA expression was higher in cc RCC cell lines than that in normal renal tubular epithelial cell line.Western blot assays showed that NSD2 protein level in 786-O and ACHN cells was higher than that in normal HK-2 cells.However,there was no obvious change in NSD2 protein expression between Caki-1 cells and normal HK-2 cells.Conclusions: The NSD2 expression was higher in cc RCC and highest in metastatictissues.The overall survival of cc RCC patients with high NSD2 expression was shorter.NSD2 m RNA expression was higher in cc RCC cell lines and protein level was higher in786-O and ACHN cells.Part Ⅱ: The role of NSD2 in cc RCC cell proliferation and apoptosisObjective: To explore the effects of NSD2 on cc RCC cell proliferation,apoptosis,tumor formation and the possible molecular signaling pathway.Methods: To knock down NSD2 expression in cc RCC 786-O and ACHN cells and increase NSD2 level in cc RCC Caki-1 cells.The effects of NSD2 on cc RCC cell proliferation and apoptosis were detected by MTT assay and flow cytometry.The effects of NSD2 on AKT/ERK signaling pathway were determined by western blot assays.To construct the virus vector that can stably knock down NSD2 expression in cc RCC 786-O cells,the effect of NSD2 expression on tumor formation was detected in tumor formation assays.Results: MTT assays showed that after knocking down NSD2 level,the proliferation of 786-O and ACHN cells was reduced compared with NC group(P < 0.05).And after overexpression of NSD2,the proliferation of Caki-1 cells was improved compared with NC group(P < 0.05).Flow cytometry showed that after knocking down NSD2 level,the apoptosis rate of 786-O and ACHN cells was increased compared with NC group(P <0.05).And after overexpression of NSD2,the apoptosis rate of Caki-1 cells was decreased compared with NC group(P < 0.05).Western blot assays showed that after knocking down NSD2 level in 786-O and ACHN cells,the activation of AKT/ERK signaling pathway was inhibited compared with NC group,and bcl-2 level was decreased,while Bax level was increased(P < 0.05).And after overexpression of NSD2 in Caki-1 cells,the activation of AKT/ERK signaling pathway was promoted compared with NC group,and bcl-2 level was increased,while Bax level was decreased(P < 0.05).The tumor formation assays showed that after stably knocking down NSD2 expression in 786-O cells,compared with NC group,the tumor formation ability was significantly suppressed,and the volume and weight of in vivo graft tumors were obviously decreased(P < 0.05).Conclusions: In cc RCC,NSD2 can activate AKT/ERK signaling,which inhibits cell apoptosis and promotes cell proliferation in vivo and in vitro.Part Ⅲ: The role of NSD2 in cc RCC cell invasion and migrationObjective: To explore the effects of NSD2 on cell invasion,migration and the potential mechanism.Methods: After down-regulating NSD2 expression,the effects of NSD2 on cell invasion and migration were detected by transwell invasion and migration assays.The effect of NSD2 on epithelial-mesenchymal transition(EMT)was determined by western blot assays.Results: Transwell invasion assays showed that after down-regulating NSD2 expression,the cell invasion ability was reduced compared with NC group(P < 0.05).Transwell migration assays showed that after down-regulating NSD2 expression,the cell migration ability was reduced compared with NC group(P < 0.05).Western blot assays showed that after down-regulating NSD2 expression,epithelial-mesenchymal transition was inhibited and E-cadherin level was increased,while N-cadherin and Vimentin level was decreased compared with NC group(P < 0.05).Conclusions: Down-regulation of NSD2 expression inhibited epithelial-mesenchymal transition and weakened cell invasion and migration in cc RCC. |
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