Lncrna Trerna1 Regulate The Proliferation And Migration Of The Hepatocellular Carcinoma Through PDS5B And KIF2C

Research purposes:Long non-coding RNA(lnc RNA)Tre TNA1 is also known as nc RNA-a7,which is an enhancer-like lnc RNA.Tre RNA1 involved in the occurrence and development of multiple cancers.The results of our previous studies showed that the expression of Tre RNA1 was obviously higher in the tumor tissue than the normal tissue of digestive tract tumor,such as gastric carcinoma,colonic carcinoma and hepatocellular carcinoma.And the most of it located in the cytoplasm.Overexpression of Tre RNA1 significantly improved the proliferation,migration and tube formation of hepatocellular carcinoma,and it significantly inhibited apoptosis of hepatocellular carcinoma.Meanwhile,the results of the microarray demonstrated that overexpression of Tre RNA1 caused the abnormal expression of many genes in the hepatocellular carcinoma.However,the tangible mechanism how Tre RNA1 improves the development of the hepatocellular carcinoma remains unclear.Methods:We used the stable isotope labeling with amino acids in cell culture(SILAC)to search the differential expressed proteins from the Hep G2-Tre RNA1 cells which overexpress Tre RNA1 stably and its control cells.We conducted conjoint analysis of the results of the microarray and SILAC.If the FC(fold change)of gene >2 in the results of the microarray and the FC of the gene>1.2 in the results of the SILAC,the gene was regarded as the downstream target gene of Tre RNA1.Then Real-Time PCR and Western Blot were conducted to verify the target genes that we had screened out.We formed the plasmids which overexpressed PDS5 B and KIF2 C and the corresponding control plasmids.Then we conducted the cell transient transfection using these plasmids to clarify if PDS5 B and KIF2 C could change the effection of Tre RNA1 on Hep G2 cells.Meanwhile,the cell scratch test was used to research whether PDS5 B and KIF2 C influenced migration of the hepatocellular carcinoma cells.The CCK8 test was utilized to study the impact of PDS5B and KIF2 C on proliferation of the hepatocellular carcinoma cells.We also conducted the tube formation assay test to explore the impact of PDS5 B and KIF2 C on the tube formation of hepatocellular carcinoma.To research how PDS5 B and KIF2 C function in vivo,we constructed the lentivirus which overexpressed PDS5 B and KIF2 C and conducted the cell transfection.The purinomycin and G418 were utilized to screen out the stable cell lines that overexpressed PDS5 B or KIF2 C stably.We constructed nude mouse models of the hepatocellular carcinoma proliferation in vivo through subcutaneous injection.We counted the rate of tumor formation and the weight of tumors to research the impact of PDS5 B and KIF2 C on the hepatocellular carcinoma proliferation in vivo.Results:The results of SILAC showed that the overexpression of Tre RNA1 caused abnormal expression of multiple genes in the hepatocellular carcinoma.We screened out 251 genes that differently expressed including 50 genes over-expressed and 201 genes down-expressed.Finally,we selected PDS5 B and KIF2 C as the downstream target genes of Tre RNA1 by conjoint analysis of the results of the microarray and SILAC.The results of the PCR showed that the expression level of PDS5 B m RNA of the Hep G2-Tre RNA1 cell which overexpressed tre RNA1 stably was obviously lower than the control group cells.However,the expression level of KIF2 C m RNA didn’t have obvious difference between Hep G2-Tre RNA1 cells and the control group cells.The results of the Western Blot demonstrated that the expression level of PDS5 B protein of the Hep G2-Tre RNA1 cells was obviously lower than the control group cells and the expression level of KIF2 C protein of the Hep G2-Tre RNA cells also obviously reduced compared to the control group cells.These results were in keeping with the results of the microarray and SILAC.To validate the effect of PDS5 B and KIF2 C,we transfected the plasmids which over expressed PDS5 B or KIF2 C and the control plasmids.Then we conducted real-time PCR and Western Blot.The results of the PCR showed that the expression level of PDS5 B m RNA of the Over-expression of PDS5 B or Over-expression of KIF2 C cells was obviously higher than the control group cells.The results of the Western Blot shown that the expression level of PDS5 B protein of the Over-expression of PDS5 B or Over-expression of KIF2 C cells was obviously higher than the control group cells.The results of the cell scratch test showed that the migration area of the Over-expression of PDS5 B cells was obviously less than the control group cells after 24 h and 48 h.Meanwhile,the migration area of the Over-expression of KIF2 C cells was also obviously less than the control group cells after 24h and 48 h.The results of the CCK8 test showed that the cell viability of the Over-expression of PDS5 B or Over-expression of KIF2 C cells was obviously inferior to the control group cell.The results of the tube formation assay test showed that the ability to form vessels of the Over-expression of PDS5 B or Over-expression of KIF2 C cells was obviously inferior to the control group cells.We structured the stable cell line which over-expressed PDS5 B or KIF2 C by lentiviral transfection.And we used PCR and Western Blot to validate the expression of PDS5 B and KIF2 C.The results of the hepatocellular carcinoma proliferation in vivo showed that the weight of cancers in over-expression of PDS5 B group was obviously less than the control group.Meanwhile,the weight of cancers in over-expression of KIF2 C group was obviously less than the control group.Conclusion:The results of the microarray and SILAC showed that the overexpression of TreRNA1 caused abnormal expression of many genes in the hepatocellular carcinoma.These genes may involve in tumorigenesis and the development of the hepatocellular carcinoma.We selected PDS5 B and KIF2 C as the downstream target genes of Tre RNA1.The results of the cell function test demonstrated that PDS5 B and KIF2 C could inhibit the migration,proliferation and tube formation of the hepatocellular carcinoma.And,the results were verified in vivo.Therefore,we can draw a conclusion that Tre RNA1 can promote the development of the hepatocellular carcinoma through down-regulating the expression of PDS5 B and KIF2 C.Our study proposes a new mechanism of Tre RNA1,which may be a new therapeutic target of treatment of the hepatocellular carcinoma.