ABSTRACT
Objective To investigate the effects of RNA interference targeting EphA7 gene on the growth of SMMC-7721 cell xenograft in nude mice.Methods Recombinant plasmid of EphA7 gene-targeting siRNA was transfected into hepatic cancer SMMC-7721 cells by LipofectamineTM2000,comparing with the empty vector transfected group,untransfected group and control group.The nude mice tumor model was established by subcutaneous injection of hepatic cancer cells in the left upper limb of the mice.Control group was injected with PBS as blank.Real-time PCR,immunohistochemistry and Western blot were employed to detect the mRNA and protein expressions of EphA7 in tumor tissues.The tumor formation time,tumor mass and weight of tumor were also considered in the analysis.Results About 9 ～ 12 days after the injection of tumor cells,the xenograft tumor formation can be observed around the injection site except the control group.35 days after tumor formation,there were obvious decreases in the tumor growth rate,tumor mass,as well as tumor weight in transfected group,comparing with empty vector transfected group and untransfected group (P ＜0.05).Transfection of RNA interference can inhibit the growth of xenograft tumor by 55％.Immunohistochemistry tests showed that there were less cells with positive staining of EPHA7 protein in transfected group,and the staining was lighter as pale yellow,in contrast with the untransfected group and the empty vector transfected group.Real-time PCR and Western blot revealed that the expression of EphA7 mRNA and EPHA7 protein of transfected group were significantly lower than those of untransfected group and empty vector trausfected group with statistically significance (P ＜ 0.05).Conclusion Silencing EphA7 gene with RNA interference can effectively inhibit the growth of SMMC-7721 cell in nude mice,which is expected to become a new target for gene therapy of hepatic cancer.
ABSTRACT
Objective:Ephs have been found to be over-expressed in numerous human tumors,and relate to the development and prognosis of cancers.The expression of EphA7 in colon cancer cell lines and primary colorectal cancers was detected.The role of EphA7 in the development of colorectal cancer was investigated. Methods:Quantitative reverse transcription-polymerase chain reaction was used to detect the expression of EphA7.Methylation specific PCR and bisulfite sequencing were used to examine the methylation status of EphA7.pEGFP-EphA7 plasmid was constructed and was transfected into HCT116 which lost the expression of EphA7.Gene chip was analyzed for the expression of genes between transfectant and mock cell. Results:Down-regulation of EphA7 was observed in cell lines of primary colorectal cancers,and the evidence of aberrant methylation was found in CpG island of EphA7 promoter.The methylation status of EphA7 gene was related to sex,differentiation and location of colorectal cancers.Some genes were up or down regulated in pEGFP-EphA7 transfectant. Conclusion: EphA7 gene may be involved in the carcinogenesis of colorectal cancer.The hypermethylation of CpG island is an important mechanism leading to the down regulation of EphA7 in colorectal cancer.