The Effect Of Circ＿0054537 On The Occurrence And Development Of Renal Cell Carcinoma And Its Mechanism

Renal cell carcinoma,referred to as renal cell carcinoma,is a common malignant tumor of the urinary system.renal cell carcinoma is insidious and lacks typical clinical symptoms in the early stage.Therefore,the diagnosis rate is low.Some patients with renal cell carcinoma are already in the middle and advanced stages at the time of diagnosis,and the prognosis is poor.Exploring the pathogenesis of renal cell carcinoma and finding biomarkers and therapeutic targets for early diagnosis are of great significance for improving the prognosis of renal cell carcinoma.Circular RNA（circRNA）and micro（miRNA）are two types of small non-coding RNA,and circRNA can play the role of micro RNA（miRNA）molecular sponge to regulate the expression of miRNA target genes,and then play a biological regulatory function.circ＿0054537 is a member of the circRNA family,but its effect on the occurrence and development of renal cancer and its molecular mechanism are not yet clear.The circular RNA Interactome target gene online software predicts that circ＿0054537 may target miR-640;the micro T CDS target gene online software predicts that neuron pentameric protein 2（NPTX2）may be the target gene of miR-640.This study first detected the expression of circ＿0054537 in renal cell carcinoma tissues,and analyzed the relationship between the expression of circ＿0054537 and the clinical characteristics of renal cancer patients;secondly,explored the effect of circ＿0054537 on the malignant biological behavior of renal cancer cell lines in vitro and tumor growth in vivo;Finally,using the miR-640/NPTX2 axis as the starting point to explore the molecular mechanism of circ＿0054537 affecting the malignant biological behavior of renal cancer cellsPart One The expression and clinical significance of circ＿0054537 in renal cell carcinomaObjective:To explore the expression and clinical significance of circ＿0054537 in renal cell carcinoma tissues.Methods:1.The cancer tissues and adjacent normal tissues of 70 renal cell carcinoma patients who underwent radical resection in the Fourth Hospital of Hebei Medical University from November 2016 to November 2019 were collected,and then the expression of circ＿0054537 in the tissues were detected by q RT-PCR.2.According to the mean value of circ＿0054537 expression in renal cell carcinoma tissues,renal cell carcinoma patients were divided into circ＿0054537 high expression group and circ＿0054537 low expression group.Chi-square test analyzed the relationship between the expression of circ＿0054537 in renal cell carcinoma tissue and the clinical characteristics of patients.Results:The expression of circ＿0054537 in renal cell carcinoma tissue was significantly higher than that in adjacent tissues（P<0.05）,and the expression of circ＿0054537 in renal cell carcinoma tissue was closely related to TNM staging and lymph node metastasis（P<0.05）.The expression of circ＿0054537 in the cancer tissues of renal cell carcinoma patients with high TNM stage and lymph node metastasis was higher than that in patients with low TNM stage and no lymph node metastasis（P<0.05）.Conclusions:circ＿0054537 was highly expressed in renal cell carcinoma tissues,and the expression of circ＿0054537 was closely related to the TNM stage and lymph node metastasis of patients.Part Two The effect of circ＿0054537 on the malignant biological behavior of renal cell carcinoma cells and tumor growth in vivoObjective:To explore the expression of circ＿0054537 in renal cell carcinoma cell lines and its influence on the malignant biological behavior of renal cell carcinoma cell lines.Methods:1.Using human renal tubular epithelial cells HK-2 as a control,qq RT-PCR method was used to detect the expression of circ＿0054537 in renal cell carcinoma cell lines（786-O,A498）.2.Nuclear and cytoplasmic separation test was use to detect the localization of circ＿0054537 in renal cell carcinoma cell lines（786-O,A498）,and RNase R test was use to detect the stability of circ＿0054537.3.si-con,si-circ＿0054537^#1,si-circ＿0054537^#2or si-circ＿0054537^#3was transfected into renal cell carcinoma cell lines（786-O,A498）.In order to verify transfection effect,the expression of circ＿0054537 in the cells was detected by q RT-PCR.4.renal cell carcinoma cell lines（786-O,A498）in si-con group and si-circ＿0054537^#1group were inoculated into cell culture plates,and then CCK-8 method and Ed U test were used to detect cell proliferation;Transwell chamber method was used to detect cell migration and invasion;Flow cytometry was used to detect cell apoptosis;Western blotting method was used to detect the expression of apoptosis-related proteins BAX and BCL2 in cells.5.renal cell carcinoma cells A498 infected with sh-con（sh-con group）and sh-circ＿0054537（sh-circ＿0054537 group）lentivirus were injected subcutaneously into the left hind limb of nude mice.At the same time,a control group（Mock group,injected with the same amount of physiological brine）.The volume of the transplanted tumor was observed,and the transplanted tumor was stripped and weighed 4 weeks later.The expression of circ＿0054537 in the tumor tissue was detected by q RT-PCR.Results:1.Compared with the HK-2 cells,the expression of circ＿0054537 in renal cell carcinoma cell lines（786-O,A498）was significantly increased（P<0.05）.2.circ＿0054537 was mainly located in the cytoplasm of renal cell carcinoma cell lines（786-O,A498）;the expression of circ＿0054537 in renal cell carcinoma cell lines（786-O,A498）was not affected by RNase R（P>0.05）,which proved that circ＿0054537 was a closed-loop structure and not easily degraded by exonuclease.3.Compared with the si-con group,the expression of circ＿0054537 in the renal cell carcinoma cell lines（786-O,A498）in the si-circ＿0054537^#1group,si-circ＿0054537^#2group,and si-circ＿0054537^#3group were significantly reduced（P<0.05）,and the expression of circ＿0054537 in the renal cell carcinoma cell lines（786-O,A498）transfected with si-circ＿0054537^#1was the most significant.Therefore,si-circ＿0054537^#1was selected for follow-up experiments.4.Compared with the si-con group,the OD value,Ed U positive rate,the number of migrated cells,the number of invaded cells,and the expression of BCL2 protein of renal cell carcinoma cell lines（786-O,A498）in si-circ＿0054537^#1group were significantly reduced（P<0.05）,but the rate of apoptosis and the expression of BAX protein in cells increased（P<0.05）.5.Compared with the Mock group or sh-con group,the size of transplanted tumor in sh-circ＿0054537 group was significantly reduced（P<0.05）,tumor weight was reduced（P<0.05）,and the expression of circ＿0054537 in transplanted tumor tissue was reduced（P<0.05）.There was no significant difference in transplanted tumor volume,tumor and the expression of circ＿0054537 in transplanted tumor tissue between the Mock group and the sh-con group（P>0.05）.Conclusions:The expression of circ＿0054537 was increased in renal cell carcinoma cell lines.Knockdown of circ＿0054537 can inhibit the proliferation,migration,and invasion of renal cell carcinoma cell lines in vitro,and induce cell apoptosis.At the same time,knock down circ＿0054537 inhibits tumor growth in vivo.Part Three Study on the mechanism of circ＿0054537 influencing the malignant biological behavior of renal cell carcinoma cells by targeting miR-640/NPTX2 axisObjective:To explore the molecular mechanism of circ＿0054537influencing the malignant biological behavior of renal cell carcinoma cell lines.Methods:1.The GSE61741 data set in the GEO database was used to analyze the expression of miR-640 in renal cell carcinoma tissues.The GEPIA database was used to analyze the expression of NPTX2 in renal cell carcinoma tissues.q RT-PCR method was used to detect the expression of miR-640 and NPTX2m RNA in 70 cases of renal cell carcinoma tissues and normal tissues adjacent to the cancer.Western blotting method was used to detect the expression of NPTX2 protein in the tissues.Pearson correlation analysis was used to analyze the correlation between circ＿0054537 and miR-640 or NPTX2 m RNA,or miR-640 and NPTX2 m RNA in renal cell carcinoma tissues.2.With human renal tubular epithelial cells HK-2 as a control,the expression of miR-640 in renal cell carcinoma cell lines（786-O,A498）was detected by q RT-PCR,and the expression of NPTX2 protein in renal cell carcinoma cell lines（786-O,A498）was detected by Western blotting.3.The circular RNA Interactome target gene online software predicts that circ＿0054537 may have a targeted regulation relationship with miR-640;the micro T CDS target gene online software predicts that NPTX2 may be the target gene of miR-640.Dual luciferase reporter gene experiment and RIP experiment were used to verify the targeting relationship between circ＿0054537 and miR-640,miR-640 and NPTX2.4.renal cell carcinoma cell lines（786-O,A498）were divided into si-con group,si-circ＿0054537^#1group,si-circ＿0054537^#1+in-miR-con group,si-circ＿00545-37^#1+in-miR-640 group,miR-con group,miR-640 group,miR-640+pcDNA group,miR-640+NPTX2 group.q RT-PCR method was used to detect the expression of miR-640 in cells,and Western blotting was used to detect the expression of NPTX2 protein.CCK-8 method and Ed U test were used to detect cell proliferation;Transwell chamber method was used to detect cell migration and invasion;Flow cytometry was used to detect cell apoptosis;Western blotting method was used to detect the expression of apoptosis-related proteins BAX and BCL2 in cells.Results:1.The analysis of the GSE61741 data set in the GEO database shows that the expression of miR-640 in renal cell carcinoma tissues was lower than that of normal tissues.GEPIA database analysis showed that the expression of NPTX2 in renal cell carcinoma tissues was higher than that in normal tissues.q RT-PCR results showed that the expression of miR-640 in 70 cases of renal cell carcinoma tissues was significantly lower than that of normal tissues adjacent to cancer（P<0.05）,and the expression of NPTX2 m RNA and protein were higher than those of tissues adjacent to cancer（P<0.05）.Pearson correlation analysis showed that circ＿0054537 was negatively correlated with the expression of miR-640（r=-0.5853,P<0.05）,and circ＿0054537 was positively correlated with the expression of NPTX2 m RNA（r=0.5166,P<0.05）,miR-640 There was a negative correlation between miR-640 and NPTX2 m RNA（r=-0.6802,P<0.05）.2.Compared with the HK-2 cells,the expression of miR-640 in renal cell carcinoma cell lines（786-O,A498）were significantly reduced（P<0.05）,and the expression of NPTX2 protein were significantly increased（P<0.05）.3.The results of the dual luciferase activity test showed that the luciferase activity of renal cell carcinoma cell lines（786-O,A498）co-transfected with miR-640 mimics and circ＿0054537 or NPTX2 wild-type luciferase reporter carrier were reduced（P<0.05）.The results of the RIP experiment showed that the enrichment of circ＿0054537 and miR-640increased significantly（P<0.05）,and the enrichment of miR-640 and NPTX2m RNA increased significantly（P<0.05）.circ＿0054537 could negatively regulate the expression of miR-640,miR-640 could negatively regulate the expression of NPTX2,and circ＿0054537 could positively regulate the expression of NPTX2 through miR-640.4.Compared with the si-circ＿0054537^#1+in-miR-con group,the expression of miR-640 in the renal cell carcinoma cell lines（786-O,A498）in the si-circ＿0054537^#1+in-miR-640 group were significantly reduced（P<0.05）,but the expression of NPTX2 protein were significantly increased（P<0.05）,the OD value,Ed U positive rate,the number of migrated cells,the number of invaded cells,and the expression of BCL2 protein were significantly increased（P<0.05）,but the rate of apoptosis and the expression of BAX protein in cells reduced（P<0.05）.5.Compared with the miR-con group,the expression of miR-640 in the renal cell carcinoma cell lines（786-O,A498）in the miR-640 group were significantly increased（P<0.05）,but the expression of NPTX2 protein was significantly reduced（P<0.05）,the OD value,Ed U positive rate,the number of migrated cells,the number of invaded cells,and the expression of BCL2protein were significantly reduced（P<0.05）,but the rate of apoptosis and the expression of BAX protein in cells increased（P<0.05）.Compared with the miR-640+pc DNA group,the expression of NPTX2 protein in the renal cell carcinoma cell lines（786-O,A498）in the miR-640+NPTX2 group was significantly increased（P<0.05）,the OD value,Ed U positive rate,the number of migrated cells,the number of invaded cells,and the expression of BCL2protein were significantly increased（P<0.05）,but the rate of apoptosis and the expression of BAX protein in cells reduced（P<0.05）.Conclusions:Knockdown of circ＿0054537 could inhibit the proliferation,migration,and invasion of renal cell carcinoma cells by targeting miR-640 and then inhibit the expression of NPTX2,and promote the apoptosis of renal cell carcinoma cells.