| Background and purpose:The incidence of renal cell carcinoma(RCC)has been increasing rapidly in recent years.As one ofthe common malignant tumors,RCC seriously threatens human health.Almost all RCCs are resistant to traditional chemotherapy and radiotherapy.The rise of targeted therapy in recent years has opened a new way for the treatment of renal cell carcinoma.Long non-coding RNAs(LncRNAs)can be widely involved in the regulation of gene expression through a variety of mechanisms.A new,very conserved,long-chain non-coding RNA THOR recently discovered is associated with the development of malignant tumors.In this paper,the expression and biological function of THOR in renal cell carcinoma cells were studied,and its possible mechanism was explored in order to provide theoretical basis for finding new therapeutic targets.Methods:1,Real-time quantitative PCR(qPCR)was utilized to detect the expression of THOR in human renal cell carcinoma tissues,renal cell carcinoma cell lines(786-O,A498,ACHN and primary human renal cell carcinoma cells),and the expression of THOR in adjacent renal tissues,HK-2 and primary human renal epithelial cells.2.SiRNA was used to knockdown THOR expression,and CRISPR/Cas9 gene editing was used to knockout THOR in 786-0 cell lines cultured in vitro.After the above treatment,THOR was detected by qPCR;cell growth was detected by cell counting;cell survival was detected by MTT assay;cell proliferation was detected by clone formation test and BrdU ELISA method.For primary human renal cell carcinoma cells,HK-2 and primary human renal epithelial cells,the expression of THOR was knocked down by siRNA,and the changes of cell function were detected by the above methods.3.SiRNA was used to knockdown THOR expression,and CRISPR/Cas9 gene editing was used to knockout THOR in 786-0 cell lines cultured in vitro.After the above treatment,the expression of IGF2BP1-dependent gene IGF2,Glil,Myc mRNA and protein was detected by qPCR and Western blotting.Lentivirus expression vector was utilized to exogenous over-express THOR in 786-0 cell lines cultured in vitro.THOR was detected by qPCR,cell survival was detected by MTT assay;cell proliferation was detected by BrdU ELISA;IGF2BP1-dependent genes IGF2,Glil,Myc mRNA and protein were detected by qPCR and Western blotting.4.Transplantation tumor model of normal 786-0 cell in nude mice and transplantation tumor model of 786-0 cell after knocking out THOR by CRISPR/Cas9 gene editing were established to observe the growth of 786-0 cell line tumors in vivo;THOR of transplanted tumors were detected by qPCR;the expression of IGF2BP1 dependent genes IGF2,Glil,Myc mRNA and protein were detected by qPCR and Western blotting.Results:I.THOR was expressed in human renal cell carcinoma tissues,and was specifically expressed in all renal cell carcinoma cell lines,with the higher expression in 786-0 cell lines,But it was not detected in normal renal tissues,HK-2 and primary human renal epithelial cells.2.After being knocked down by targeted siRNA or knocked out by CRISPR/Cas9,THOR was significantly decreased in 786-0 cell lines(P<0.05),the number of cells decreased significantly(P<0.05),cell viability decreased significantly(P<0.05),cell proliferation decreased significantly(P<0.05).After being knocked down by siRNA,THOR in RCC1 and RCC2 cells decreased significantly(P<0.05),cell viability decreased significantly(P<0.05)and cell proliferation decreased significantly(P<0.05).HK-2 and primary human renal epithelial cells showed no significant changes in cell viability and proliferation after THOR knockdown.3.After being knocked down by targeted siRNA or knocked out by CRISPR/Cas9,IGF2BP1-dependent genes IGF2,Glil,Myc mRNA and protein expression were significantly down-regulated(P<0.05).After exogenous overexpression,THOR in 786-0 cell lines increased significantly(P<0.05),cell viability increased significantly(P<0.05),cell proliferation increased significantly(P<0.05),and the expression of IGF2BP1-dependent genes IGF2,Gli 1,Myc mRNA and protein increased significantly(P<0.05).4.In the subcutaneous implanted tumor experiment,compared with the control group,the tumors of 786-0 cell after THOR knockout decreased in size(P<0.05)、weight(P<0.05)、growth rate(P<0.05)、THOR expression(P<0.05)and IGF2BP1-dependent genes IGF2,Glil,Myc mRNA and protein expression(P<0.05).Conclusions:Long non-coding RNA THOR promotes RCC cell growth and proliferation in vitro and in vivo via regulating the expression of IGF2BP1-dependent genes,THOR could be a novel and important therapeutic target for human RCC. |
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