| Objective:To detect the expression of Lnc RNA PIK3CD-AS1 in renal cancer cells,to study the effect of PIK3CD-AS1 on the proliferation and apoptosis of renal cancer cells,and then to explore the possible molecular mechanisms.Methods:Real-time fluorescence quantitative PCR was used to detect the differential expression of Lnc RNA PIK3CD-AS1 in normal kidney cells(HK-2)and various types of renal cancer cells(769-P,786-O,A498).PEGFP-N1-PIK3CD-AS1 recombinant plasmid was constructed.Then,the recombinant plasmid was transfected into renal carcinoma cells with the lowest expression of PIK3CD-AS1,and a cell line stably overexpressing PIK3CD-AS1 was selected by drugs.After that,proliferation and apoptosis of over-expressed cells were detected by proliferation and apoptosis assays to investigate the effect of PIK3CD-AS1 on the proliferation and apoptosis of renal cancer cells.The genes that interact with PIK3CD-AS1 on the proliferation or apoptosis of renal cancer cells were screened by cell proliferation or apoptosis-related gene expression profile microarrays.Western-blot was used to detect the changes of p AKT protein content in PIK3CD-AS1 high-expressing cells,and to lay a foundation for further study on the specific mechanism of PIK3CD-AS1.Results:Compared with normal renal cell HK-2,the expression of Lnc RNA PIK3CD-AS1 was significantly decreased in a variety of renal cancer cells(769-P,786-O,A498)(P<0.05),and the 769-P cells decreased most significantly.(P<0.01).After successfully constructing the PIK3CD-AS1 overexpression vector,it was transfected into 769-P cells and then screened with G418 drugs.PIK3CD-AS1 stably overexpressed 769-P cell line was successfully obtained and verified by real-time fluorescence quantitative PCR.The expression of PIK3CD-AS1 in these screened 769-P cells was significantly higher than that in negative control cells(P<0.05).Through proliferation assay,it was found that the PIK3CD-AS1 highly-expressingcells had a significantly decreased proliferative capacity compared with the negative control group(P<0.01),and the apoptosis rate was significantly higher than that of the negative control group(P<0.01).Apoptosis-related gene microarrays were used to screen for genes that were associated with PIK3CD-AS1's pro-apoptotic potential and heat maps were generated using the Morpheus webpage software.Three genes(DFFA,BCL2L1,and CASP9)were selected in combination with the relevant literature and verified several times.It was found that the expression levels of DFFA and BCL2L1 in PIK3CD-AS1 high-expressing cells were significantly lower than those in negative controls(P<0.01).There are many reports in the literature that DFFA and BCL2L1 can inhibit apoptosis.At the same time,the expression of gene CASP9 was significantly higher than that of the negative control group(P<0.01).It was reported that CASP9 could promote apoptosis.Western-blot results showed that after upregulation of PIK3CD-AS1 expression,the p AKT content in renal cancer cells decreased significantly.Conclusion:Through our research,it can be found that Lnc RNA PIK3CD-AS1 is likely to be a tumor suppressor factor,and its expression in a variety of renal cancer cells is generally reduced,and it plays a role in inhibiting the proliferation of renal cancer cells and promoting apoptosis.The mechanism may be that PIK3CD-AS1 inhibits the expression of p AKT protein and causes the change of PI3K-AKT-m TOR signaling pathway,which causes the abnormal expression of DFFA,BCL2L1 and CASP9 genes,so as to promote the apoptosis of renal cancer cells.At the same time,the alteration of PI3K-AKT-m TOR signaling pathway also has a certain effect on the proliferation of renal cancer cells.Therefore,Lnc RNA PIK3CD-AS1 as a tumor suppressor may be a new target for the treatment of renal cancer. |
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