Study On The Effect Of GnRH And Its Analogue On Oral Cancer Cells Via GnRHR

Objective: Now we have known that type I Gn RH receptor(Gn RHR-I)was found to be expressed in a variety of cancer cells,but there is no report about the expression of Gn RHR in oral cancer cells or tissues.Many researchers believe that the signal transduction mechanism of Gn RHR on the surface of tumor cells is different from that in the hypothalamus pituitary gonad axis.Therefore,oral cancer cell line KB was seleted to verify the expression of Gn RHR-I gene and protein.Using cell proliferation inhibition assay,Real-Time PCR,Western Blot,i TRAQ and other experimental methods to explain how hormone levels in vivo affect growth of tumor cells and find the function of Gn RHR-I of oral cancer.Methods: 1.RT-PCR method was used for detection of gonadotropin releasing hormone receptor(Gn RHR-I)m RNA in KB cell line.Western blot was employed to detect the expression of Gn RH receptor I on KB cells.2.KB cells were treated with different concentrations(10,20,40,80 ?g/ml)of Gn RH-I or its analogue Triptorelin for 48 hours,respectively.And then OD450 was detected using CCK-8 assay to calculate the cell viability.3.Gn RHR-I m RNA expression level was detected in three oral cancer cell lines CAL-27,SCC-4 and KB using Real Time PCR.4.Using Real Time PCR to detect the variation of expression level of Gn RHR-I m RNA of KB cells after treated with 40 ?g/ml Gn RH-I or its analogue Triptorelin for 48 hours,respectively,and after treated with 40 ?g/ml Triptorelin for 0,12,24,36,48 hours.5.To further elucidate cellular mechanism and molecular function,we performed i TRAQ-based proteomics analysis to assess the protein expression profiles in KB cells with Triptorelin treatment.Results: 1.The results of agarose gel electrophoresis showed that the Gn RHR-I gene of KB cells were amplified correctly.Western Blotting has showed that the protein samples of KB cells have specific reaction at approximately 60 k Da.2.The absorbances of KB cells treated with 0,10,20,40,80 ?g/ml Gn RH-I are 1.475?0.017,1.455?0.013,1.437?0.017,1.418?0.016,1.392?0.019.Cell viability are 100.00?0.013%,98.537?0.009%,97.186?0.012%,95.784?0.012%,93.860?0.014%.There is no difference between 10 ?g/ml Gn RH-I treatment and control group(p=0.171).20 ?g/ml Gn RH-I treatment can decrease cell viability compared with control group(p=0.018).Cell viability of 40 and 80 ?g/ml treatment group compared with the control group,the cell viability was significantly different(p=0.002,p=0.0001).The absorbances of KB cells treated with 0,10,20,40,80 ?g/ml Triptorelin are 1.475?0.017,1.444?0.019,1.429?0.023,1.401? 0.018,1.332?0.032.Cell viability are 100.00?0.013%,97.701?0.014%,96.431? 0.017%,94.507?0.013%,89.485?0.024%.There is no difference between 10 ?g/ml Triptorelin treatment and control group(p=0.118).20 ?g/ml Triptorelin treatment can decrease cell viability compared with control group(p=0.018).Cell viability of 40 and 80 ?g/ml Triptorelin treatment group compared with the control group,the cell viability was significantly different(p=0.002,p=0.0001).Comparison between the same concentration of Gn RH-I and Triptorelin showed the inhibition on the proliferation of KB cells was not significantly different.3.The analysis of relative expression level of three oral cancer cells(CAL-27,SCC-4,KB)showed the 2-??Ct value of SCC-4,CAL-27 and KB are 1.012?0.196,3.563?0.066,1.810?0.049.4.Real Time PCR results showed that the expression level of Gn RHR-I m RNA in KB cells treated with Gn RH-I or Triptorelin was downregulated.The 2-??Ct value of control,Gn RH-I group,Triptorelin group are 1.01?0.15,0.26?0.02,0.12?0.01.2-??Ct value of KB cells treated with Triptorelin for 0,12,24,36,48 hours are 1.00? 0.05,0.93?0.02,0.56?0.02,0.25?0.02,0.18?0.03.5.i TRAQ results showed proteasome subunit were significantly downregulated.Conclusion: 1.KB cell line has the gene and protein expression of Gn RH receptor.2.Gn RH-I and its analogue Triptorelin have inhibitory effect on proliferation of KB cells.3.CAL-27 showed the highest expression level of Gn RHR-I m RNA in three oral cancer cell lines SCC-4,CAL-27 and KB,and SCC-4 was the lowest.4.The expression level of Gn RHR-I m RNA in KB cells treated with Gn RH-I or Triptorelin was downregulated.The m RNA expression of Gn RHR-I was downregulated by Triptorelin in a time dependent manner.5.The inhibitory mechanism of on the proliferation of KB cells is the down-regulation of key proteins such as proteasome.It shows that Triptorelin inhibits cell proliferation as a proteasome inhibitor.