Study On The Function And Molecular Mechanism Of Sunitinib Resistance Induced By LncRNA AFAP1-AS1 In Renal Cell Carcinoma

Objectives:To study the function and molecular mechanism of sunitinib resistance in renal cell carcinoma induced by LncRNA AFAP1-AS1.Methods:Using 241 kinds of LncRNA Sam library,which is closely related to the growth,metastasis and drug resistance of renal cancer,we used CRISPR/dCas9 method to construct lentivirus mediated up-regulation vector of Sam library gene.Through packaging virus,infection and drug screening of sunitinib,we obtained the up-regulated human renal cell carcinoma cells?RCC?of sunitinib resistant Sam library.RT-PCR was used to detect the expression of each gene in Sam library,and the AFAP1-AS1 related to sunitinib resistance was screened out.In RCC cell lines,CRISPR/dCas9 was used to up-regulate the expression of AFAP1-AS1 and to verify its drug resistance.RT-PCR was used to detect and compare the expression of the coding RNA within 1MB around AFAP1-AS1 before and after the upregulation of AFAP1-AS1,we also analyzed the mechanism of sunitinib resistance induced by AFAP1-AS1 in RCC.Western-blot was used to study the expression of related proteins before and after the upregulation of AFAP1-AS1 to find the possible molecular mechanism of the sunitinib resistance in RCC.Results:In this study,the 241 SAM library was used to infect 769-P renal cancer cells with lentivirus infection technology to obtain 769-P ^{SAM 241+}.769-P ^{SAM 241+}sunitinib resistant strains were screened and verified.RT-PCR was used to compare the relative expression of 241 LncRNA in the experimental group(769-P ^SAM241+sunitinib resistant strain)and the control group(769-P ^{SAM 241+}).Seven kinds of significantly up-regulated and stably expressed LncRNA were selected,which may be related to sunitinib resistance in 769-P renal cancer cells.The most one amony the all,which is LncRNA AFAP1-AS1,was chosen for further study,and then CCK8 was used for proliferation experiment to verify its drug resistance.In order to further elucidate the molecular mechanism of AFAP1-AS1-induced resistance of RCC to sunitinib,Western blot results suggested that AKT signaling pathway and cell protective autophagy played an important role in the process of AFAP1-AS1-induced resistance of RCC to sunitinib.SiRNA knockdown the expression of AKT in RCC cells,and the drug resistance of RCC cells to sunitinib was reversed after knockdown of AKT.In addition,we found a total of 1MB coding genes upstream and downstream of LncRNA AFAP1-AS1 through UCSC database.Using RT-PCR and Western blot technology,we found that HTRA3 and AFAP1 had obvious changes in the experimental group and the control group.HTRA3 may be the downstream mechanism of LncRNA AFAP1-AS1,which is worth further study.Conclusions:After up-regulation of AFAP1-AS1 in RCC cells,AKT signaling pathway was over-activated.It indicats that AKT signaling pathway mediates AFAP1-AS1-induced sunitinib resistance in RCC cells.Up-regulation of AFAP1-AS1 expression in RCC cells promoted the protective autophagy of RCC cells and the overexpression of HTRA3 protein,indicating that and HTRA3 may also be involved in AFAP1-AS1-induced sunitinib resistance in RCC cells.AFAP1-AS1 can induce drug resistance of RCC cells and play a role in promoting cancer.It is suggested that AFAP1-AS1 may be a factor to understand the prognosis of RCC and a new target to reverse sunitinib resistance.