The Influence Of Leucine Zipper Tumor Suppress 2(LSTZ2)on The Proliferation And Migration Of Ovarian Cancer Cells

LZTS2 is a tumor suppress gene,which is high expression in normal ovary,prostate,and testis,but it has low expression or expression deletion in cancerous ovary,prostate,testis and some other tissues.Studies on lung cancer and colon cancer at home and abroad indicate that LZTS2 can affect the proliferation and migration of tumor cells through the Wnt/β-catenin signaling pathway,and beta-catenin is a key factor in the Wnt pathway.It is found that high expression of LZTS2 can promote the degradation of beta-catenin,resulting in the loss of Wnt/β-catenin signaling pathway,thereby affecting the regulation of downstream target genes(c-myc cylinD1,etc.)on cell proliferation and migration.Beta-catenin has high expression in ovarian cancer.Whether the mechanism of LZTS2/β-catenin is present in ovarian cancer cells must be further confirmed.Objective: To investigate the effect of leucine zipper tumor suppressor 2(LZTS2)on proliferation and migration of ovarian cancer cells and its possible mechanism.Methods:1.Extracted the plasmid according to the steps of the Plasmid Extraction Kit,examined the extracted pcDNA3.1-3xFlag-C-LZTS2 plasmid and pcDN A3.1-3xFlag-C plasmid by means of agarose gel electrophoresis,and detected the plasmid containing the target gene by double enzyme digestion.2.Divided the cells into blank control group(SKOV3),experimental group(SKOV3-GDF15)and negative control group(SKOV3-N);respectively untransfected and transfected pc DNA3.1-3xFlag-C-LZTS2 and pcDNA3.1-3x Flag-C by Lipofectam-ine? 2000,and then observed the transfection rate through real time quantitative PCR 24 h after the transfection.3.Divided the cells into the experimental group(SKOV3-LZTS2),theblank control group(SKOV3)and the negative control group(SKOV3-N),and employed real time quantitative PCR to detect the expression level of LZTS2 and β-catenin mRNA in each group.4.Got cells transiently transfected with pcDNA3.1-3x Flag-C-LZTS2 or pcDNA3.1-3xFlag-C and SKOV3 cells.24 h after the transfection,trypsinization was done and cells were collected.Later the concentration was adjusted and planking was re-carried out.The MTS analysis was performed2 hs later when cell adherence occured.Set the initial adding time of MTS as0 h,and added 10μl/well MTS respectively at 0,24,48,72 h of the cell cultivation.Then detected the absorbance value at the wave-length of 490 nm by enzyme mark instrument after celles were cultured for 1-4h.5.Performed the cell wound scratch assay on cells transfected with pcDNA3.1-3xFlag-C-LZTS2 or pcDNA3.1-3 xFlag-C and cells of the blank control group 24 h after the transfection;then tested the effect of LZTS2 on SKOV3 cells migration.Results:1.The extracted plasmid containing the LZTS2 target gene underwent BamH /EcoR double enzyme and displayed two bands in agarose gel electrophoresis.The lower segment of the third band from left to right is near2000 bp,and it’s of similar size as the exon of the LZTS2 gene.The plasmid containing the LZTS2 target gene which didn’t undergo double enzyme digestion and the empty vector plasmid showed only one band.The second band from from left to right was empty vector plasmid and the fourth band was plasmid containing LZTS2 target gene.2.Real-time quantitative PCR was used to detect the transfection effect.The results showed that the LZTS2 mRNA expression quantity,2-Delta Delta CT value,of the transfected 2ug plasmid and 4ul liposome group was more than 1,suggesting that the transfection group was successfully transfected.3.Real-time quantitative PCR was also applied to test the expression of LZTS2 and β-catenin mRNA.The results showed that the expression of LZTS2 m RNA in SKOV3-LZTS2 group was significantly higher than that inSKOV3-N and SKOV3.The comparative difference among different groups has statistic significance(p<0.05).As for the expression quantity of β-catenin mRNA,the SKOV3-LZTS2 group is significantly lower than that in SKOV3-N and SKOV3 group.The comparative difference among different groups also has statistic significance(p<0.05).4.The test result of MTS showed compared to SKOV3-N and SKOV3 group,SKOV3-LZTS2 group’s OD value is significantly lower in 24,48,72 hours.And the comparative difference among different groups also has statistic significance(p<0.05).5.The results of scratch test showed that the migration ability of ovarian cancer cells in SKOV3-LZTS2 group was significantly lower than that in SKOV3-N and SKOV3 group,and the difference was statistically significant(p < 0.05).Conclusions:1.The overexpression of LZTS2 can suppress the proliferation and migration of ovarian cancer cells.2.The increase of LZTS2 expression in ovarian cancer cells can reduce the expression of beta-catenin,suggesting that the regulation mechanism of LZTS2/ beta-catenin may exist in ovarian cancer cells.