Study on Expression of Beclin1and HLA in Ovarian Cancer Cells Line SKOV3
|School||Hebei Medical University|
|Course||Pathology and Pathophysiology|
|Keywords||ovarian cancer SKOV3cell line Beclin1 HLA-Ⅰ HLA-Ⅱ autophagy immune escape|
Objective: Examined the detection of Beclin1and HLA in human ovariancancer cell line SKOV3through this experiment, used the vector of Beclin1expression by DDK tagged to transfect into SKOV3cell line.Explored therelationship between expression of gene and protein of Beclin1and HLA inthe clinical pathology of ovarian cancer and explored the effect of Beclin1onhuman ovarian cancer cell SKOV3in the expression situation, further clarifiedthe expression of Beclin1in the progression of ovarian cancer and the role ofthis aspect on immunity affected by autophagy, the research results mayprovide a theoretical foundation for the study on the etiology and clinicalimmunity treatment of ovarian cancer.Methods:1Used the experiment method of cell gene transfection technique、RealTime-PCR、RT-PCR and Western blot to study the effect of the antigenexpression of HLA after over expression of exogenous Beclin1transfectedinto ovarian cancer cell line SKOV3.2Used the method of MTT to study the effect on the proliferation afterover expressing of exogenous Beclin1transfected into ovarian cancer cell lineSKOV3.3Autophagy phenomenon was observed under fluorescence microscope.Results:1At first,constructed the expression vector of pCMV6-Entry-Beclin1and pCMV6-Entry, then transfected into SKOV3cells line. The results ofRT-PCR showed that: the gene expression of Beclin1increased significantlyafter transfected the vector of pCMV6-Entry-Beclin1, compared with thecontrol blank group. It suggested that expression vector could be used for the subsequent experiments.2Studied the biological behavior of Beclin1and HLA in ovarian cancer,this experiment use the technique of transient transfection. Firstly transfectedpCMV6-Entry-Beclin1and pCMV6-Entry into SKOV3cell line, then studiedexpression changes of mRNA and protein of HLA-Ⅰ and HLA-Ⅱand cellproliferation.2.1Firstly transfected pCMV6-Entry-Beclin1into SKOV3cells line(Beclin1transfection group), the expression level of mRNA and protein onBeclin1, HLA-Ⅰ a nd HLA-Ⅱ were higher than that of empty transfectiongroup (SKOV3-Entry group) and blank group (SKOV3group).2.2The results of MTT showed that, the growth of the Beclin1transfect-ion group significantly be inhibited, compared with the empty transfectiongroup and the blank control group. After the transfection of24h,48h,72h and96h, the growth inhibition rate of the Beclin1transfection group were42.60%,37.87%,24.35%,14.81%. These results showed that it could inhibit the prolif-eration of SKOV3cell line after transfected pCMV6-Entry-Beclin1.2.3The results showed that detected the expression of Beclin1and HLA-Ⅰalleles and HLA-Ⅱalleles in the blank group, the Beclin1transfectiongroup and the empty transfection group through RT-PCR. The gene expressionof Beclin1in these cells groups were0.6609±0.0457,1.5973±0.1491,0.6661±0.0316.The HLA-Ⅰalleles expression of HLA-A, HLA-B, HLA-Cwere0.8223±0.0704,1.6227±0.2427,0.7835±0.0842;0.6785±0.1527,1.4491±0.2190,0.5992±0.1705;0.6626±0.0803,1.2284±0.1742,0.6820±0.0591inabove groups.The HLA-Ⅱalleles expression of HLA-DP, HLA-DQ and HLA-DR were0.5043±0.1030,1.6283±0.1698,0.4844±0.1731;0.2551±0.1128,1.1219±0.2517,0.3212±0.1598;0.5670±0.0642,1.5132±0.2356,0.5014±0.2524in above groups. The Cells groups were detected by0ne-way ANOVAand nonparametric test were statistically significant (P <0.01).2.4The results showed that, detected the expression of Beclin1and HLA-Ⅰ alleles and HLA-Ⅱalleles in the blank group,the Beclin1transfectiongroup and the empty transfection group through Real Time RT-PCR. The gene expression level of Beclin1in these groups were1.3861±0.0066,9.2922±0.2715,1.8350±0.1015;The HLA-Ⅰalleles expression of HLA-A, HLA-B,HLA-C in each cells group were5.9848±0.0783,14.5475±0.0545,6.0894±0.0140;1.7167±0.0580,2.7984±0.1515,1.7973±0.0192；3.8161±0.0449，12.1393±0.0463，4.6966±0.1527；The HLA-Ⅱalleles expression of HLA-DP,HLA-DQ,HLA-DR in each cells group were6.8787±0.1833,16.7675±0.4850,7.6501±0.4856;2.9097±0.2281,25.5259±0.7104，3.8230±0.1273；6.1550±0.0511，19.1355±0.6053，6.3781±0.1222. The cells groups weredetected by one-way ANOVA and nonparametric test were statisticallysignificant (P <0.01).2.5The detection of the protein expression of Beclin1,HLA-ⅠandHLA-Ⅱ in the blank group, the Beclin1transfection group and the emptytransfection group through Western blot. The protein expression of Beclin1were5.6656±0.8535,11.6792±2.8931,5.1258±0.6148in these cellsgroups.The protein expression of HLA-Ⅰ and HLA-Ⅱin each cells groupwas0.2131±0.0421,0.4208±0.0989,0.2208±0.0564;0.2143±0.0544,0.3503±0.0778,0.2143±0.0543. The cells groups were detected by one-wayANOVA were statist-ically significant (P <0.01).2.6The Beclin1transfection group produced a significantly higher MDCfluorescent intensity than control group in its cytoplasm appeared obviouslygreen fluorescence.Conclusions:1Over expression of autophagy gene Beclin1could inhibit the proliferat-ion of cells line SKOV3.The gene therapy of autophagy gene Beclin1forovarian cancer may have a certain feasibility.2Down regulated expression of HLA may increase the immune escapeon ovarian cancer cells, and autophagy may promote immune responses totumor cells, through autophagy enhance immunity may become a newapproach to immunotherapy.