Effects Of Tumor Extracellular Acidic Microenvironment On Invasion Ability And LncRNA Expression Profile In Renal Carcinoma Cell Line 769-p

Objective:To explore the effects of tumor extracellular acidic microenvironment on the invasion ability and LncRNA expression profile in renal carcinoma cell line 769-P and potential molecular mechanism.Methods:The tumor extracellular acidic microenvironment was simulated in vitro by using pH7.4 and pH6.6 cell culture medium.The renal carcinoma cell line 769-P was cultured in pH6.6 cell culture medium for 0h,0.5h,1h,2h,4h and 8h respectively.The real-time fluorescent quantitative PCR was used to examine the expressions of E-cadherin and its upstream gene Snail in renal carcinoma cell line 769-P.Transwell assay was used to detect the effects of acidic microenvironment on the invasion of renal carcinoma cell line 769-P.Western blot was used to detect the activation of pAKT/AKT and ERK signaling pathways in renal cancer 769-P cells cultured in acidic microenvironment.The changes of LncRNA expression profile in renal carcinoma cell line 769-P which was cultured in acidic cell culture medium was verified by using LncRNA expression profile chip.Finally,Lentivirus infection and CRISPR/Cpf1 gene editing techniques were used to construct renal cell carcinoma cell line 769-P which was knocked down LncRNA BC040587 stably.Results:The expression levels of E-cadherin gene was down-regulated significantly and its upstream gene Snail was up-regulated in renal carcinoma cell line 769-P treated with pH6.6 acidic cell culture medium for 4h compared with control group(both P<0.05).Transwell assay showed that the invasion ability of renal carcinoma cell line769-P treated with pH6.6 acidic cell culture medium for 4h was remarkably enhanced compared with control group(P < 0.05).Western blot results showed that the expression levels of pMEK1/2,p-P90 RSK,and pAKT in the renal carcinoma cell line769-P of experimental group were increased compared with control group,suggesting that the activation levels of pAKT/AKT and MEK/P90 RSK signal pathways wereincreased.The results of LncRNA expression profile chip screening showed that the LncRNA expression profile of renal carcinoma cell line 769-P treated with pH6.6acidic cell culture medium for 4h was significantly different from that of the control group.Compared with control group,the expression levels of BC040587,KCNQ1 DN and PCAT-43 were up-regulated while the expression levels of CTBP1-AS and MEG3 were down-regulated in renal carcinoma cell line 769-P treated with pH6.6 acidic cell culture medium for 4h(P<0.05).Exposure map after cutting with enzyme PacI/XhoI and gene sequencing results suggested that PY108/BC040587 plasmid was recombined sucessfully.It can be used to kncok down BC040587 by lentivirus infection.Compared to control group,the expression level of BC040587 in KD group was significantly decreased according to the RT-PCR results,suggesting that renal carcinoma cell line 769-P which was knocked down of BC040587 was constructed successfully.Conclusion:Tumor extracellular acidic microenvironment activates pAKT/ATK and MEK/P90 RSK signaling pathways and enhances the invasion ability of renal carcinoma cell line 769-P through the Snail/E-cadherin pathway.The expression levels of LncRNA BC040587,KCNQ1 DN,PCAT-43,CTBP1-AS,and MEG3 changed in the process.These LncRNAs may also be involved in the regulation of the invasion ability of renal carcinoma 769-P cell line mediated by tumor acidic microenvironment.This may provide a new direction for further research,diagnosis and treatment of renal carcinoma.