RESUMO
X-box binding protein1 (XBP1) is an important transcription factor, which participates in many signal transduction procession.To investigate the biological function of XBP1, yeast two-hybrid system to screen proteins interacting with XBP1 in hepatocytes was performed. The XBP1 coding sequence was amplified by polymerase chain reaction (PCR) method, and was cloned in pGEM-T vector. After the target region was sequenced, it was subcloned into the bait plasmid pGBKT7, then was transformed into yeast AH109(a type). After the expression of bait plasmid pGBKT7-XBP1 in AH109 yeast strains were proved by Western blot. The transformed yeast AH109 was mated with yeast Y187(α type) containing hepatocyte cDNA library plasmids pACT2 in 2 ×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medilium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After sequencing and verifying ORF of positive colonies,7 different kinds of proteins were obtained. In order to further testify the interaction between the screened proteins and XBP1, one of positive colonies MT1E was cloned. The interaction between MT1E and XBP1 in vitro/in vivo were examined successfully with GST pulldown and coimmunoprecipitations. It was shown that MT1E would be a new regulatory protein of XBP1. These screened proteins by yeast two-hybird system were closely correlated with liver fundamental metabolism,protein synthesis and transport,cell proliferation and apoptosis. The results mentioned above contributed to reveal the XBP 1 biological function, and brought some new clues for further exploration of the expressing and regulating mechanism of XBP1.
RESUMO
Objective:To silence the expression of HPV16 E6 gene in Ca Ski cell line with RNA interference technique.In order to understand if the HPV16 E6 siRNA could inhibit the expression of E6 gene specifically,recombinant eukaryotic vectors were constructed and transfected into Ca Ski cell line.The transfection efficiency was observed.Methods: According to the sequence of HPV16 E6 gene,the E6 siRNA was designed and cloned into the EGFP reporter plasmid pGenesil-1,and then transfected into CaSki cell line.The fluorescence expression was observed and the transfection efficiency was identified.Results: Two siRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into Ca Ski cell successfully,and the transfection efficiency achieved about 50%.Conclusion: The construction of recombinant E6 siRNA eukaryotic vectors and transfection of them into Ca Ski cell line successfully established a favourable foundation for further study.
RESUMO
Objective To learn the effect on carcinoma xenograft in nude mice by inhibiting human papillomavirus 16(HPV16) E6 gene expression in CaSki cell. Methods The recombinant plasmids expressing HPV16 E6 small interference RNA (siRNA) were transfected into CaSki cell. The cells expressing recombinant plasmid was screened out with G418. The expression of E6 mRNA was determined by RT-PCR. The cells were inoculated into BALB/c nude mice subcutaneouly and the growth of the xenograft carcinoma was observed. After the pGensil-CH2 recombinant was injected into the carcinoma, the growth of carcinoma and pathological changes of carcinoma were observed. Results The CaSki cell expressing E6 siRNA was obtained, and HPV16 E6 mRNA expression in CaSki cell was down-regulated. The oncogenicity of the CaSki cell expressing E6 siRNA was degraded, the inhibition rate was up to 71.4% as compared with that of control group. The growth of tumor in nude mice was inhibited after the E6 siRNA plasmids were injected into the nude mice. The volume and weight of the tumor treated by siRNA were smaller than that of control group significantly. More necrotic area and less cell division phase were observed under light microscope in the E6 siRNA treated tumor. Conclusion The oncogenicity of the CaSki cell was degraded after silencing HPV16 E6 gene in CaSki cell by E6 siRNA.
