The Expression And Function Of Germ Cell Nuclear Factor (GCNF) In Cervical Cancer Cells

Objective: To observe the expression of GCNF gene in normal cervix, cervical intraepithelial neoplasia, cervical cancer and cervical cancer cell line (HeLa cells) in vitro. Constructed the pcDNA3.1-GCNF eukaryotic expression vector, Observe the effect of GCNF genes on proliferation,apoptosis,adhesion and invasion of HeLa cells.Methods:(1) Collecting and picking up normal cervix, cervical intraepithelial neoplasia, cervical cancer paraffin blocks by the pathology department of the first auxiliary hospital of the Shihezi university, HeLa cells cultured in vitro, using immunohistochemical SP observed GCNF gene in these tissues and cells;(2) using PCR method amplified full-length GCNF cDNA from pMD18-T-GCNF vector, PCR products and pcDNA3.1 (-) plasmid were digested by restriction enzyme, recovered digestion products and using DNA directional connection technology construction of pcDNA3.1-GCNF recombinant plasmid, ligation products was transformed into E. coli DH5α, and colony PCR. The amplified DNA extracted for PCR identification of recombinant plasmid and selection sequence glycerol bacteria sent to the company; (3) liposome-mediated transient transfection of recombinant plasmids to cervical cancer HeLa cells, RT-PCR and indirect immunofluorescence observe transfection results; (4) inverted microscope observe morphology changes before and after transfection cell; MTT assay observa the proliferation of transfection cells; AnnexinⅤ-PI double staining flow cytometry analysis of apoptosis before and after transfection; matrix adhesion experimental observe adhesion of transfection cell; Transwell invasion assay observe cell invasion after transfection.Results:(1) Normal cervix was strongly positive expression of GCNF expression; CINⅠwas positive expression of GCNF; CINⅡonly individual cells expression of GCNF; CINⅢwas not expressed of GCNF; GCNF not expressed in cervical carcinoma. HeLa cells do not expression GCNF;(2) PCR products by gel electrophoresis can be seen the target band around 1450bp,by double-digested and connections obtain pcDNA3.1-GCNF recombinant plasmid, transformed into E.coli DH5αstrains were screened by monoclonal technology, plasmid DNA was extracted and identified by plasmid PCR sequencing and gene bank in the human GCNF login sequence comparison, the rate of 99.99%;(3)liposome positive recombinant plasmid was transected into HeLa cells, RT-PCR products gel electrophoresis, After gene transefection can be seen in the vicinity of a bright target band at 200bp, the intensity ratio (98.82±0.79)% was higher than that transfected with empty vector and untransfected group (10.29±0.59,9.67±0.29)%, difference statistically significant (P<0.05); indirect immunofluorescence showed that, after gene transfection, most of HeLa cells positive expression GCNF, transfected with empty vector and untransfected HeLa cells group no GCNF expression;(4)cell morphology no significant changes after transfection; MTT results showed that after transfected GCNF gene of HeLa cells absorbance maximum (0.239±0.011), higher than the untransfected group and empty vector transfected group (0.203±0.009,0.196±0.016) (P<0.05); flow cytometry apoptotic cells, indicating the purpose of gene transfection apoptosis rate as low as 11.46%(P <0.05), empty vector and untransfected group, the apoptosis rates were 16.5% and 15.34%; matrix adhesion assay with the cell migration rate, and the transwell invasion assay observe cell invasion using the same intervention condition, found no significant difference between the groups.Conclusion:(1) GCNF gene high expression in normal cervical tissues with the normal differentiation of epithelial cell;(2) First observed GCNF gene expression from strong to weak in CINⅠ,CINⅡ,CINⅢuntil in invasive cervical carcinoma to disappear, suggesting that GCNF gene may be not related with the abnormal differentiation or malignant differentiation of cervical epithelial cells;(3) GCNF gene not expression in cervical carcinoma and HeLa cells may suggest GCNF as an early diagnostic marker of cervical cancer;(4) pcDNA3.1-GCNF eukaryotic expression vector was successfully constructed, and obtain the transiently transfected cervical cancer HeLa cells; (5) GCNF gene overexpression can promoted the proliferation and inhibit apoptosis of cervical cancer HeLa cells in vitro,but did not affect the adhesion and invasion of HeLa cells;(6) The results to provide reference of early diagnosis of cervical cancer and cervical cancer pathogenesis.