Construction Of GSDML Gene Eukaryotic Expression Vectors And Its Prelimilary Functional Study

IntroductionSince gastric carcinoma ranks first both in incidence and mortality rate a-mong malignant tumors in our country, it is especially important and urgent to explore the molecular mechanism of carcinogenesis of gastric carcinoma. The pathogenesis of gastric carcinoma is a complex process involving the loss of function of tumor suppressor genes (TSGs) and overexpression of proto - oncogenes. Proto - oncogenes and TSGs are often clustered around recombination hotspots or fragile sites. The PPP1R1B - ERBB2 - GRB7 amplicon, which is frequently amplified in human gastric cancer and breast cancer, is located at the oncog-enomic recombination hotspot of human chromosome 17ql2, which is also in fragile site. Maybe there are the potential tumor suppressor genes linked to this site. Recently, the newly cloned gene gasdermin( GSDM) and the related gene gasdennin - like ( GSDML) are located at the evolutionary recombination hotspot of human chromosome 17q21. These two genes are tandemly linked and tightly clustered with the oncogenomic recombination hotspot of human chromosome 17q12 around the fragile sites. The expression of mouse GSDM gene is restricted to gastrointestinal tract, while human GSDM gene is only expressed in stomach. Moreover, their expressions are both significantly suppressed in human gastric cancer cells. It suggests that GSDM gene and GSDML gene may be the potential tumor suppressor genes. GSDML gene is generated due to local duplication of the GSDM gene.In order to verify the above hypothesis, we cloned the cDNA of GSDMIgene and constructed eukaryotic expression vectors pIRES - GSDML,pEGFP -C3 - GSDML,pFLAG - CMV -2 - GSDML. Then, we investigated the subcellular localization of GSDML protein and the effect of GSDML gene overexpression on the colony - formation ability of cells.Materials and Methods1. The cDNA of GSDML gene was cloned and inserted into eukaryotic expression vectors, constructed pIRES - GSDML,pEGFP - C3 - GSDML, pFLAG-CMV-2-GSDML.The total RNA of HeLa cell was extracted, the first strand of cDNA synthesized by reverse transcriptase. The full - length cDNA sequence of GSDML was PCR amplified . The resulting GSDML gene fragment was cleaved with BamH I and Hind M and inserted into BamH I and Hind III site of pFLAG - CMV -2. Then the GSDML gene was subcloned into the pEGFP - C3 and pIRESneo.2. Subcellular localization of GSDML proteinThe pEGFP - C3 - GSDML was transfected into HeLa and SGC7901 cells and the subcellular localization of GSDML protein was recorded by using fluorescent microscope.3. Effect of GSDML gene overexpression on the colony - formation ability of cells in vitropIRES - GSDML and pIRESneo were transfected into HeLa and SGC7901 cells respetively. Then cells were incubated for 2 weeks in selective medium containing G418. Giemsa staining was used to evaluate the number of colonies.Results1. We cloned a new cDNA transcript of GSDML gene and constructed eukaryotic expression vectors pEGFP - C3 - GSDML,pIRES - GSDML, pFLAG -CMV-2-GSDML.2. After pEGFP - C3 - GSDML was transfected into HeLa and SGC7901 cells, the green fluorescence was dispersed in the whole cell.