Study Of Function And Molecular Mechanism In LncRNA BC040587 Which Regulates Renal Cancer 769-P Cells In Acidic Microenvironment

Objective:This study aims to knock out long non-coding RNA BC040587(LncRNA BC040587)in kidney cancer 769-P cells via gene editing system CRISPR/Cpfl,and also tries to study the function and mechanism of this LncRNA,which regulates kidney cancer 769-P cells in acidic microenvironment.Methods:The gene editing technique CRISPR/Cpfl was used to knock out LncRNA BC040587 in renal cancer 769-P cells to obtain 769-PBC040587(-)cells.The knockout efficiency was detected by fluorescence quantitative PCR(qRT-PCR).In vitro tumor cell invasion test(transwell test)was used to detect the change of 769-PBC040587(-)cell invasion ability under acidic microenvironment;Snail and pAKT protein expression changes in acid microenvironment was observed in 769-PBC040587(-)cell by Western blot.In addition,the adjacent genes within 1M of the upstream and downstream of LncRNA BC040587 were also searched through the USUC database,and their expression in the control group 769-PVector cells and the experimental group 769-PBC040587(-)cells was detected by using qRT-PCR.Genes that may be related to the biological function of LncRNA BC040587 were screened out.SiRNA was then used to further decrease the expression of adjacent genes in 769-PBC040587(-)cells,and we observed whether related signal pathways were changed by Western blot.Results:The results of qRT-PCR showed that the expression of LncRNA BC040587 in the experimental group(renal cancer 769-PBC040587(-)cells)was significantly lower than that in the control group(renal cancer 769-Pvector cells)(P<0.05).Transwell experimental results showed that the invasion ability of kidney cancer 769-Pvector cells was enhanced under acidic microenvironment,while the invasion ability of kidney cancer 769-PBC040587(-)cells under acidic microenvironment did not change significantly.Western blot results showed that the expression levels of Snail and pAKT in kidney cancer 769-Pvector cells increased under acidic microenvironment(P<0.05),while the expression levels of Snail and pAKT in kidney cancer 769-PBC040587(-)cells under acidic microenvironment,there is no obvious difference.qRT-PCR results found that pseudogene RNU6-1200P and pseudogene RN7SL815P were stably and highly expressed in 769-PBC040587(-)cells(P<0.05).The expression of pseudogene RNU6-1200P and pseudogene RN7SL815P in 769-PBC040587(-)cells was down-regulated using siRNA.Western blot results showed that the experimental group(after the expression of pseudogene RNU6-1200P in 769-PBC040587(-)cells decreased,and the acid microenvironment for 1h)and the control group(769-PBC040587(-)cells in the acid microenvironment group 1h)increased pAKT expression(P<0.05);another experimental group(after the expression of pseudogene RN7SL815P in 769-PBC040587(-)cells decreased,group 1h under acidic microenvironment)and control group(769-PBC040587(-)cells under acidic microenvironment for 1h group)decreased pAKT expression(P<0.05).Conclusions:LncRNA BC040587 is a key gene that is involved in increasing invasion ability of renal cancer 769-P cell upon acidic microenvironment.This LncRNA exerts its biological role by regulating Snail and pAKT.The pseudogene RNU6-1200P is a downstream regulatory site of LncRNA BC040587 and plays an important role in the regulation of pAKT by LncRNA BC040587.There may be an acidic microenvironmental/LncRNA BC040587/pseudogene RNU6-1200P/pAKT pathway to regulate cell invasion in renal cancer 769-P cells.