Restoration Of Klotho Gene Expression Induces Apoptosis And Autophagy In Gastric Cancer Cells

Gastric cancer is one of the most common malignant tumors in the world. It is the second death in malignant tumor.The incidence of gastric cancer is different from erery country. About170000people die in China every year, nearly occupy25percentage deaths in cancer.Because of a lack of specific symptoms, the majority of patients with gastric cancer are diagnosed at an advance disease stage. It is poor outcomes. Klotho gene is a new tumor suppressor gene which discovered recently. Early studies found that Klotho is closely associated with senescence gene. The early animal experiments demonstrated that Klotho gene deficient mice would appear a series of similar human senescence phenotype,whereas overexpression could prolong the lifespan of mice. It is found that the Klotho gene is closely related to the inhibition of insulin signaling. Recently it is found that Klotho gene is closely related with a variety of cancer, act as a tumor suppressor gene inactivation,which lead to the occurrence and development of tumor. Although it had been reported that Klotho may act as a tumor suppressor gene loss in gastric cancer, but the function of Klotho in gastric carcinoma is unclear.It has been shown that the Klotho gene can inhibite the insulin signal way which is anti-senescence and prolong life,so we propose Klotho whether in gastric cancer act as a tumor suppressor gene?it would inhibite insulin signal axis in gastric cancer? Therefore, we design the experimental as follows:1. We study the expression of Klotho gene in human gastric cancer cell lines MNK-45, AGS, GC-7901and human gastric mucosal cells, and analyze the causes of low Klotho Gene expression.whether it is closely related with promoter methylation.2. We give5-Aza-2-deoxycytidine (5-Aza) intervention GC-7901, Klotho can be recovered in gastric cancer cell lines,the restore of Klotho gene increase gastric cancer cell apoptosis,autophagy and insulin signal pathway is inhibited3. Construction of eukaryotic expression vector Klotho and transfected GC-7901cell line which is construction of high expression in GC-7901cell line. 4. The overexpression of Klotho in human gastric cancer cell line GC-7901whether influence cell proliferation, cell cycle, apoptosis, autophagy and related signal pathway in gastric cancer.Part Ⅰ The expression of Klotho in gastric cancer cell linesObjective:We study the Klotho gene and protein in gastric cancer cell lines MNK-45, AGS, GC-7901and GES-1and analyze the reason of Klotho Gene deletion.Method:The total RNA is abstracted from the cells, PCR was used to detect the Klotho Gene in human gastric cancer cell lines MNK-45, AGS, GC-7901and normal gastric cell line GES-1. Western blot detect the protein level of Klotho in all cell.Promoter methylation of Klotho Gene is founded in four cell lines by methylation specific PCR (Methylation-Specific, PCR, MS-PCR).Result:The mRNA expression of Klotho gene was detected by RT-PCR and obviously lower Klotho expression was observed in MNK-45, AGS, and GC-7901gastric cancercells than in the GES-1normal gastric epithelial cells Western blot also showed lower Klotho protein level in tumor cells than in normal cells.The bisulfate-based PCR method was applied to examine the CpG methylation of Klotho gene promoter,the tested CpG site exhibited almost full methylation in GC-7901cells, partial methylation in MNK-45and AGS cells, but almost no methylation in GES-1normal gastric epithelial cells.Conclusion:Klotho Gene and protein in gastric cancer cell lines MNK-45, AGS and GC-7901is significantly lower than in the GES-1cell line.the expression of Klotho in GC-7901is the lowest.Methylation of Klotho gene promoter and expression of the Klotho gene were detected in GC cells. GC-7901cell line is full Methylation. PartⅡ Restoration of Klotho gene expression induces apoptosis and autophagy in gastric cancer cells by5-AzaObjective:Restoration of Klotho gene expression was established by applying a demethylating agent5-Aza-2-deoxycytidine(5-Aza). The effects of Klotho Gene influence apoptosis,autophagy and insulin signal pathway into GC-7901cell line.Method:GC-7901cells were treated with5,10, and20μmol/L of5-Aza for three days, the expression of Klotho protein and autophagy related protein LC3-Ⅱ and LC3-Ⅰ detected by Western blot after72hours.We use10μmol/L5-Aza intervention into GC-7901and divided into three groups.Control group,5-Aza group, and the application of autophagy inhibitor3-MA group.Klotho and insulin signaling axis associated proteins IGF-1R, p-IGF-1R expression detected by Westernblot. We check IGF-1R downstream signaling pathway related proteins PI3K, p-PI3K, mTOR, p-mTOR expression and autophagy related protein LC3-II, LC3-I by Western blot.Result:5-Aza dose-dependently decreased LC3-Ⅰ level and increased LC3-Ⅱ level with a significant increase in LC3-Ⅱ/LC3-Ⅰ ratio, suggesting that a demethylating agent could significantly increase autophagy in gastric cancer cells. DNA demethylating agent (5-Aza) increased Klotho protein level in a dose-dependent manner.5-Aza can restore the expression of GC-7901Klotho gene,GC-7901cells could be induced autophagy and apoptosis.and the expression of phosphorylated insulin signaling pathway related protein such as p-IGF-1R,p-PI3K,p-mTOR significantly decreased.But it had no effect into total protein IGF-1R,PI3Kand mTOR. Conclusion:1.DNA demethylating agent5-Aza-2-deoxycytidine (5-Aza) increased Klotho protein level in a dose-dependent manner.2.5-Aza can restore the expression of GC-7901Klotho gene which induced autophagy and apoptosis in GC-7901cell line.3. Restoration of Klotho expression significantly decreased the levels of phospho-IGF-1R,phospho-PI3K and phospho-mTOR proteins, but had no effect on the total protein levels.3-MA significantly blocked the effect of Klotho gene expression. Part III Construction a vector overexpressing Klotho gene and delivered it intoGC-7901cellsObjective:we constructed a vector overexpressing Klotho gene and delivered it into GC-7901cells.Method:In this study, firstly we construct eukaryotic expression vector Klotho gene which is inserted into the eukaryotic expression vector form pZsGreenl-Cl-Klotho recombinant expression vector. Cut with restriction enzymes EcoR Ⅰ, Hind Ⅲ, Bgl Ⅱ, Hind Ⅲ restriction endonuclease.the amplified Klotho ORF was then cloned into pZsGreenl-C1vector at Bgl Ⅱ/Bam HI sites.To establish high expression Klotho into GC-7901cell lines.RT-PCR and Western-blot analysis the expression of Klotho gene and protein.Result:Western blot showed that GC-7901cells transfected with Klotho gene expression vector exhibited over4-fold increase in Klotho protein expression compared to cells transfected with blank vector-Conclusion:We successfully constructed a vector overexpressing Klotho gene and delivered it into GC-7901cells. Part Ⅳ The biological behavior of over expression of Klotho in gastric cancer cellsObjective:We study over expression of Klotho in GC-7901gastric cancer cells influence proliferation, apoptosis, period, autophagy and related signaling pathways.Method:It is divided into four groups:empty vector (blank vector, blank-V), the Klotho expression vector transfected GC-7901cell(Klotho expression vector, Klotho-V),the Klotho expression vector transfected GC-7901cell was treated with autophagy inhibitor3-MA.(Klotho vector plus3-MA, kV+3-MA), the Klotho expression vector transfected GC-7901cell was treated with apoptosis inhibitor Z-VAD-PMK (Z-VAD-PMK, kV+ZVP).CCK experimental curves each group of cell proliferation plot and comprehensively evaluate over expression of Klotho of gastric cancer cell lines GC-7901influence cell proliferation.Annexin V-PI flow cytometry to detect apoptotic cells in each group and the cell cycle, the evaluation of the impact on the GC-7901Klotho apoptosis.Western blot detection of the expression of IRS-1,IGF-1R,PI3K AKT,mTOR,p-IRS-1,p-IGF-1R,p-PI3K,P-AKT,p-mTOR,analysis Klotho impact axis of the insulin signaling pathway.Western blot assay and confocal autophagy-related proteins the expression of LC3-I and LC3-II, Overexpression of Klotho expression studies on the regulation of autophagy.Result:Relative cell viability in GC-7901cell transfected with blank vector (blank-V), Klotho expression vector (Klotho-V), or Klotho vector plus3-MA(k-V+3-MA),or Z-VAD-PMK(k-V+ZVP) incubation. The CCK-8assay showed that restoration of Klotho gene expression significantly decreased cell viability in GC-7901cell.Western blot showed that GC-7901cells transfected withKlothogene expression vector exhibited over4-fold increase in Klotho protein expression compared to cells transfected with blank vector.3-MA significantly blocked the effect of Klotho gene expression.Restoration of Klotho expression significantly decreased the levels of phospho-IGF-1R, phospho-IRS-1, phospho-PI3K, phospho-Aktl, and phospho-mTOR proteins, but had no effect on the total protein levels.Restoration of Klotho gene expression significantly inhibited cell proliferation, induced cell apoptosis.Flow cytometry of cell apoptosis in GC-7901cells transfected with blank vector (blank-Ⅴ) or Klotho expression vector (Klotho-V), transfected with Klotho expression vector plus3-MA (K-V+3-MA), or Z-VAD-PMK (k-V+ZVP) incubation. Percentage of apoptotic cells Klotho-V vs.blank-V p<0.001,Percentage of sub-G0/G1cells p<0.001Klotho-V vs.blank-V.To identify the location and expression of LC3-Ⅱ protein, we performed immunofluorescent staining in GC-7901cells,we detected a low rate of LC3-II/LC3-I expression in the GC cells and restoration of Klotho expression significantly increased the ratio. In addition, the autophagy inhibitor3-MA blocked Klotho-induced increases in LC3-II/LC3-I expression, the apoptosis inhibitor cannot completely block Klotho-induced authophagy and the same applies to the autophagy inhibitor. This implicates that Klotho-induced apoptosis and autophagy have different death pathways.Conclusion:Klotho was identified a tumor suppressor,which inhibited tumor cell proliferation, induced cell apoptosis and autophagy in GC.The tumor suppressive role of Klotho may be initiated by downregulation of IGF-1receptor phosphorylation, and subsequent decreases in IRS-1, PI3K, Akt, and mTOR phosphorylation.