An In Vitro Study: Overexpression Of Cystatin M On Proliferation And Migration Of Gastric Cancer Cell Line

Background:Gastric cancer, the motality rate of which is currently the world’s second highest,continues to hold the first in digestive tract cancer in China. Maximum inhibition oftumor invasion and metastasis has always been a hot issue in oncology research.Cystatin M is a secreted cysteine protease inhibitor. Cysteine proteases whichcan break down the extracellular matrix is involved in the invasion and metastasis ofcancer cells, so the cysteine protease is closely related with invasion and metastasis oftumor cells, cystatin M can inhibit cysteine protease activity, so cystatin M was earlyon attention. Cystatin M was first discovered low expression in metastatic breastcancer cells. Cystatin M overexpression in vitro can inhibit invasion and metastasis ofbreast cancer cells. Such as William B. Coleman et al found that56%of primary and85%of breast cancer lymph node metastasis exists cystatin M low expression inbreast cancer cells. Later ovarian cancer, pancreatic cancer, prostate cancer so havelow expression of cystatin M. Our previous work was mainly around the DNA codingsequence of cystatin M gene, we forecasted cystatin M gene transcription initiationupstream sequence using bioinformatics online program,found that there is a highcontent of CpG islands in the gene transcriptional regulatory region, speculated thatthe hypermethylation in promoter region of the gene is a major cause of gene silencing, subsequent experiments have proved the correctness of our speculation andcystatin M promoter hypermethylation is closely related to its low expression ingastric cancer cells.When the cells were treated with methyltransferase inhibitor,cystatin M re-expressed.However, the role of cystatin M in the development of gastric cancer is unclear,the impact of gastric cancer cells on the biological behavior, gastric cancer treatmentand other aspects have not yet been reported. In the following experiments, we willconstruct cystatin M overexpression plasmid and transfected it into gastric cancercells and observe its effect on the proliferation and migration abilities.ObjectivesSynthetic CST6gene is used to construct overexpression vector. We transfectedit into gastric cancer cells and observe its effect on the proliferation and migrationabilities of the cell line,which will provide a theoretical basis for further study on themechanism of its anti-gastric.MethodsThe target sequence was synthesized according to the mRNA sequence of CST6（GenBank:NM₀01323.3） and the structure of vector pcDNA3.1（+）, HindIIIrestriction sites and a Kozak sequence （GCCACC）was added in upstream, BamHIrestriction site was added in downstream.It combined with pcDNA3.1（+） to constructthe recombinant eukaryotic expression vectors. The vector was identified by PCR,restriction enzyme digestion and DNA sequencing, then transfected byelectroporation to gastric SGC-7901cell line in logarithmic growth phase. G418wasused to screen positive clones. Stably transfected cell lines were named SGC-7901/CST6cells, SGC-7901/pcDNA3.1（+） cells. There were three groups: pcDNA3.1（+）-CST6group, pcDNA3.1（+） group and untransfected group. RT-PCR and WesternBlot were used to detect the mRNA and cystatin M expression of the three groups.MTT assay experiments and cell wound healing assay were used to test cellproliferation and migration ability repectively. Experimental data were analyzed by SPSS17.0software, ANOVA was used to analyze the differences in multiple groups,LSD-t method for comparison between groups, P<0.05means that the differencestatistically significant.Results1pcDNA3.1（+）-CST6eukaryotic expression vector was constructedsuccessfully.2Stably transfected cell lines which expressed cystatin M was successfullyscreened out.3In MTT experiments, the proliferation of pcDNA3.1（+）-CST6group cells wereinhibited compared with non-transfected group, the difference was statisticallysignificant, but the difference between pcDNA3.1（+） group and non-transfectedgroup was of no significance.4In cell wound healing assay, pcDNA3.1（+）-CST6group cells migrate into thescratch area relative less distance than non-transfected and pcDNA3.1（+） cells, thedifference was of significance （P<0.05）,while the difference between pcDNA3.1（+）group and non-transfected group was not significant.ConclusionCystatinM expression could inhibit the proliferation and migration of gastriccancer cells, revealing that it can be as a candidate gene of inhibiting the metastasis ofgastric cancer, and hopefully become a new target for gene therapy of gastric cancer.