The Animal Study Of Effects Of HMGA2 On ACHN Cells

[Objective]Our previous showed that HMGA2 was highly expressed in renal cancer tissues and was closely related to TNM staging and lymph node metastasis,which could be used as a diagnostic indicator of renal cancer and an important indicator of prognosis of patients with renal cancer.In addition,the down-regulation of HMGA2 expression in renal cancer cell lines can significantly inhibit the proliferation and invasion of cancer cells,promote the apoptosis of cancer cells,and inhibit the occurrence and development of renal cancer.However,the effect of HMGA2 on kidney cancer in vivo is still unknown.Therefore,this study investigates the effect of HMGA2 on ACHN cell tumorigenesis of kidney cancer through animal experiments to provide a basis for the study of the pathogenesis of kidney cancer and targeted therapy in vivo.[Methods]1.Culture ACHN cells of renal carcinoma and transfect ACHN cells with lentiviral vector LV-HMGA2-shRNA to establish HMGA2 gene silencing stable cell lines.2.SPF BABLc/nude mice are randomly divided into 3 groups with 4 mice in each group,namely:ACHN group,ACHN-NC group,and ACHN-siRNA group.After one week of adaptive feeding,untreated ACHN cells and ACHN cells transfected with the blank viral vector LV-CN or the lentiviral vector LV-HMGA2-shRNA are subcutaneously injected into the right axilla of nude mice,respectively,and the injection dose was 4×10~6 cells.After injection,the tumor volume is measured once every other day until the end of the experiment.3.Observation and measurement of transplanted tumor:tumor mass is taken and weighed to compare whether there are differences in tumor volume,weight and tumor formation time between each group.4.Western blot and qRT-PCR are used to detect the expression of E-cadherin,N-cadherin and Snail and their mRNA level respectively.[Results]1.Three different HMGA2 gene fragments were selected,HMGA2-siRNA1,HMGA2-siRNA2 and HMGA2-siRNA3,respectively.After interfering with ACHN cells,the relative expression levels of HMGA2 mRNA in each HMGA2-siRNA group were up-regulated compared with those in the LV-NC group and the non-transfected group(P<0.001).In addition,WB results showed that the expression of HMGA2 protein was down-regulated after HMGA2-siRNA interference.It was confirmed that HMGA2-siRNA can interfere with the expression of HMGA2 gene and protein synthesis in ACHN cells.2.Compared with the ACHN group and the ACHN-NC group,the tumor volume and weight of the HMGA2-siRNA group were significantly reduced,and the tumor formation time was significantly prolonged,with statistically significant differences(P<0.001).3.The expression of E-cadherin,an epithelial marker protein,increased after HMGA2expression was down-regulated,while the expression of interstitial marker proteins,N-cadherin and Snail,decreased.4.After the down-regulation of HMGA2 expression,the E-cadherin mRNA level increased while the levels of N-cadherin and Snail mRNA decreased,which was significantly different from the control group(P<0.001).[Conclusion]1.Interference ACHN cells with HMGA2-siRNA can achieve the purpose of silencing HMGA2 gene with good specificity.2.HMGA2 can promote the occurrence of EMT in renal cell carcinoma and participate in the occurrence and development of renal cell carcinoma.3.HMGA2 is an important nuclear transcription factor upstream of Snail protein,and TGF-βsignaling pathway is the main signaling pathway that promotes the generation and maintenance of Snail,suggesting that the mechanism of HMGA2 biological function is related to TGF-βsignaling pathway.