| Objective: In human cancers,renal carcinoma ranks the ninth.Its etiology is extremely complex,both internal genetic factors,and external environmental factors.Long-term smoking,obesity,hypertension and antihypertensive treatment are clear pathogenic factors.The incidence of renal cell carcinoma is increasing year by year,but its mortality is also increasing obviously.The reason is that the mechanism of its occurrence and development is not clear.Renal cell carcinoma(RCC),also known as renal adenocarcinoma or renal cancer for short,accounts for 80% to 90% of all renal malignancies.Clear cell renal cell carcinoma(ccRCC)accounts for 85% of all cases of RCC,and is the most common and aggressive subtype of RCC.Non-coding RNA is a type of RNA that exists widely in human cells and cannot be translated into proteins.Studies have shown that long non-coding RNAs(LncRNAs)and short micro RNAs(miRNAs)are closely related to cancer.In particular,the interaction between LncRNA and miRNA has become a focus of research on the mechanism of tumor genesis and development in recent years.However,the regulatory mechanism of non-coding RNA in renal cell carcinoma remains unclear.Therefore,by exploring the mechanism of LncRNA/miRNA/ target gene signaling pathway on ccRCC,this study is expected to provide a new idea for targeted therapy of renal cancer.Methods: 1.Studying the identification of LncRNA and its expression conditions in tumor and its adjacent tissues in clear cell renal cell carcinomaThe LncRNA SNHG12 to be studied was searched through Enco RI,GEPIA,TCGA and other online databases to check its expression in ccRCC and the survival of patients in the database,and the LncRNA to be studied was determined based on literature.90 pairs of carcinomas and paracancerous tissues that were pathologically diagnosed as ccRCC were selected from the laboratory specimen bank,and tissue RNA was extracted.The differences in the expression levels of SNHG12 in ccRCC and paracancerous tissues were detected by q RT-PCR.According to the expression of SNHG12 in ccRCC,SNHG12 was divided into high expression group and low expression group,and the relationship between the two groups and clinical characteristics was analyzed to further clarify the significance of SNHG12 in ccRCC.2.Functional verification of SNHG12 in clear cell renal cell carcinoma cell lines The ccRCC cells of multiple cell lines were cultured,and the RNA of each cell line was extracted respectively.The expression level of SNHG12 in different cell lines was detected by q RT-PCR technology,and the two cell lines with higher content were selected as the subsequent research objects.SiRNA was transfected into two cell lines to observe the changes of proliferation and migration ability of ccRCC cells after down-regulation of SNHG12.3.Screening and detection of miRNAs interacting with SNHG12 and validation of their interactionsThe miRNAs that interact with LncRNA SNHG12 were found through Lnc ACTdb2.0,Target Scan,Diana and other online databases.Online databases such as ENCORI,GEPIA and TCGA were used to search miRNAs to check their expression in ccRCC and the survival of patients in the database,and the miRNA to be studied was finally determined based on literature review.Tissue RNA was extracted from the paired ccRCC carcinomas and paracancerous tissues selected from the laboratory specimen library before,and the differences in miRNA expression levels in ccRCC and paracancerous tissues were detected by q RT-PCR.The correlation between SNHG12 and miRNA was determined by correlation analysis.The possible binding sites between SNHG12 and miRNA were predicted through online databases such as Target Scan and Lnc ACTdb.The combination of the two was verified by double luciferase reporter gene assay.At the same time,it also revealed that SNHG12,as an endogenous competitive RNA,specifically binds to miRNA through sponge adsorption,inhibits the function of miRNA,indirectly upregulates m RNA,promotes the expression of protein and promotes the development of tumors.4.Screening of downstream target genes and verification of the regulatory mechanism of SNHG12 and miRNA towards target genesThe downstream target genes were searched through Lnc ACTdb2.0,Target Scan,DIANA and other online databases.Online databases such as ENCORI,GEPIA and TCGA were used to search the downstream key genes to check the expression of these proteins in ccRCC and the survival of patients,and the downstream target genes to be studied were finally determined based on literature review.SNHG12 was down-regulated and the protein levels of downstream target genes were detected by Western Blot.The miRNA was up-regulated,and the protein levels of downstream target genes were detected by Western Blot.Online databases such as Target Scan and Lnc ACTdb2.0 were used to predict the possible binding sites between miRNA and the m RNA 3’UTR region of downstream target genes.The combination of the two was verified by double luciferase reporter gene assay.Western Blot recovery experiments were designed to further verify the regulation effect of SNHG12 and miRNA on the expression of downstream target genes.Ed U recovery experiment was designed to further verify the effect of SNHG12 and miRNA on the proliferation ability of ccRCC cells after regulating the expression of downstream target genes.Transwell recovery experiments were designed to further verify the effect of SNHG12 and miRNA on migration ability of ccRCC cells after regulating the expression of downstream target genes.5.Further verification of the interactive regulation among SNHG12,miRNA and target protein in nude mice tumorigenesis experimentThe xenograft experiments in nude mice were conducted to observe the growth of the transplanted tumor with different treatment methods,and further verify the regulatory effects of SNHG12 and miRNA on target proteins.Immunohistochemical tests were performed on the transplanted tumor tissues to further verify the influence of SNHG12 and miRNA on the proliferation ability of ccRCC cells after the regulation of the expression of downstream target proteins.Results:1.Based on the expression level and survival analysis of the target LncRNA in ccRCC and adjacent tissues in the database,and combined with the literature,SNHG12 was finally determined as an LncRNA to be studied.In the database,SNHG12 was highly expressed in ccRCC tissues.Survival analysis showed that the higher the expression of SNHG12,the shorter the overall survival time of patients.RNA from 90 cases of carcinomas and paracancerous tissues that were pathologically diagnosed as ccRCC were extracted from the laboratory specimen library,and the expression level of SNHG12 was detected by q RT-PCR.Compared with paracancerous tissues,the expression level of SNHG12 in ccRCC tissues was significantly increased,which verified the information in the database.Subsequent SNHG12 expression in clear cell renal cell carcinoma quantity of the correlation between clinical features and analysis,found SNHG12 expression of high and low and sex,age,tumor stage(TNM staging)has nothing to do,but related to the pathological nuclear grading,SNHG12 expression ccRCC of the higher,the higher the nuclear grade,the higher malignant degree,the tumor is more likely to occur local invasion and distant metastasis and recurrence.2.Five ccRCC lines,786-O,769-P,OSRC-2,Ka KI-1 and ACHN,were cultured,and the RNA of each cell line was extracted respectively.The expression level of SNHG12 in different cell lines was detected by q RT-PCR,and it was found that the content of SNHG12 in 769-P and OSRC-2 cell lines was relatively high,so these two cell lines were selected as the follow-up research objects.SNHG12 silencer was transfected into two cell lines to observe the changes of cell proliferation and migration ability after down-regulation of SNHG12.Ed U proliferation assay showed that the proliferation ability of SNHG12 cell lines was decreased after down-regulation.Transwell assay showed that down-regulated SNHG12 cell lines had reduced cell migration.3.Through database screening,survival analysis and correlation analysis,and combined with literature,miR-30a-3p was finally identified as the miRNA interacting with SNHG12.In the database,miR-30a-3p was low expressed in ccRCC.Survival analysis showed that the lower the expression level of miR-30a-3p,the shorter the overall survival time.RNA from 72 cases of cancer and paracancerous tissues that were pathologically diagnosed as ccRCC pairs was extracted from the laboratory specimen bank,and the expression level of miR-30a-3p was detected by q RT-PCR.MiR-30a-3p showed low expression in ccRCC tissues,which verified the information in the database.Further analysis of the correlation between the expression levels of SNHG12 and miR-30a-3p in corresponding cancer tissues showed that the two were negatively correlated.The possible binding sites of the two were predicted by the website,plasmids were constructed and transfected into two cell lines,769-P and OSRC-2.By double luciferase reporter gene assay,it was found that the fluorescence intensity of miR-30a-3p and wild-type SNHG12 reporter gene plasmids cotransfected with miR-30a-3p and wild-type SNHG12 reporter gene plasmids was the lowest,confirming the specific binding of SNHG12 and miR-30a-3p.4.RUNX2 was finally identified as the downstream target gene through database screening,survival analysis and expression correlation analysis combined with literature.In the database,RUNX2 was highly expressed in the ccRCC organization.Survival analysis showed that the level of RUNX2 expression was associated with overall survival.The higher the expression of RUNX2,the shorter the overall survival.Studies have clearly indicated that RUNX2 is highly expressed in renal cell carcinoma and promotes the progression of RCC by inducing EMT.The expression level of RUNX2 protein decreased after SNHG12 down-regulation.The possible binding sites of miR-30a-3p and the 3’UTR region of RUNX2 gene were predicted by the website,plasmids were constructed and transfected into two cell lines.The fluorescence intensity of the co-transfected miR-30a-3p and wild-type RUNX2 reporter gene plasmids was found to be the weakest by double luciferase reporter gene assay,which confirmed that RUNX2 could bind to miR-30a-3p.MiR-30a-3p was upregulated,and RUNX2 protein expression was decreased.Western Blot results showed that the expression of RUNX2 protein decreased after the down-regulation of SNHG12,but the simultaneous silencing of miR-30a-3p partially reversed the inhibitory effect of the down-regulation of SNHG12 on the expression of RUNX2 protein.It has been reported that IGF-1R and WNT2 are directly regulated by miR-30a-3p and act as oncogenes in clear cell renal cell carcinoma to promote the progression of renal cancer.Therefore,in the recovery experiment,we also detected the changes of IGF-1R and WNT2 proteins,and the protein change trend of both was the same as that of RUNX2.The results of Ed U recovery experiment showed that downregulation of SNHG12 inhibited the migration ability of clear cell renal cell carcinoma cells,while the silencing of miR-30a-3p partially reversed the inhibitory effect of downregulation of SNHG12 on the proliferation ability of clear cell renal cell carcinoma cells.Transwell recovery assay results showed that downregulation of SNHG12 inhibited the migration ability of ccRCC cells,while silencing miR-30a-3p partially reversed the inhibitory effect of downregulation of SNHG12 on the migration ability of ccRCC cells.The above experimental results suggested that SNHG12 and miR-30a-3p could promote the proliferation and migration of ccRCC cells by regulating the expression of RUNX2 and other onco genes.5.The experimental results of xenograft vertified that,silence of SNHG12 inhibited the growth of xenograft in SNHG12-KD group,while simultaneously silenced miR-30a-3p could partially reverse the inhibitory effect of downregulation of SNHG12 alone on the growth of xenograft.We further stained the transplanted tumor tissues,and the results of immunohistochemical test showed that the down-regulation of SNHG12 effectively inhibited the positive rate of Ki-67 in the transplanted tumor,while the silence of miR-30a-3p partially reversed the down-regulation of SNHG12 alone on the positive rate of Ki-67.The above experimental results proved the regulatory relationship between the three.Conclusions: SNHG12 plays a cancer-promoting role in ccRCC,while miR-30a-3p plays a tumor suppressive role in ccRCC.As an endogenous competitive RNA,SNHG12 plays the role of "sponge adsorption",specifically binding to miR-30a-3p,inhibiting the function of miR-30a-3p,indirectly upregulating the expression of RUNX2 and other onco genes,and promoting the proliferation and migration of ccRCC cells. |
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