The Expression Of Klotho In Colon Cancer Cell And Its Potential Effect On Cell Proliferation

Aim:To determine the expression and methylation of Klotho gene in colon cancer cell lines and to investigate its effect on colon cancer cell proliferation, thus to explore the potential relationship between Klotho and colon cancer.Materials and methods:RT-PCR was used to determine the expression of Klotho gene in colon cancer cell lines HCT116, HT29, DLD1, LS180, SW480 and SW620, and the methylation status was detected by methylation specific PCR (MSP). Colon cancer cell HCT116,HT29,LS180 and SW620 were treated with demethylation reagent 5-Aza-dC and then the expression of Klotho gene was detected. Clone forming analysis as well as MTS test were performed to check the effect on cell proliferation in HCT116 and HT29 cell lines after transfected with pCDNA3.1-Klotho plasmid or empty vector respectively.Result:Klotho gene was silenced in colon cancer cell HCT116 and HT29, and lower expressed in cell DLD1, LS180 and SW620 when compared to normal colon tissue. MSP indicated that the DNA sequence in the promoter of Klotho was fully methylated or partially methylated in DLD1, HCT116, HT29, LS180, and SW620 cells. Treatment with 5-Aza-dC could either restored or significantly increased the expression of Klotho gene in HCT116, HT29, LS180 and SW620 cells, indicating that hypermethylation in the promoter of Klotho took role on the down regulation of Klotho expression in colon cancer cells. Clone forming analysis showed that over expression with Klotho gene could inhibit the clone formation of HCT116 and HT29 cells. MTS results illustrated that, when the original cell dense was 2000/well in 96 well plate, the absorbance (OD value) of pcDNA3.1 empty vector cells on day 1,3 and 5 were 0.6680±0.0234. 1.439410.0597 and 1.6968±0.0251 respectively, when compared to that of 0.5814±0.0118,0.9760±0.0622, and 1.6264±0.0205 in HT29-Klotho cells, which had significantly lower on each time course (p<0.01). While the cell dense increased to 4000/well, the absorbance (OD value) of pcDNA3.1-Klotho tranfecant cells showed marked decreasing in day 1 and day3 (p<0.05), except for day 5, when compared with control empty vector tranfactants.Conclusion:Klotho gene was silenced or significantly down-regulated in series of colon cancer cell lines, which was closely related with hypermethylation in the promoter of Klotho gene. In vitro experiment demonstrated that Klotho could inhibit the clone formation and proliferation in colon cancer cells. In conclusion, our findings indicated that Klotho gene may function as a tumor suppressor in colon cancer whose expression was regulated by epigenetic mechanism of DNA sequence hypermethylation in promoter.