| Objectives To study the expressions(including the positive rate and the amount of relative expression)of ZNF703 in papillary thyroid carcinoma and the relationship of the positive rate and the related clinical characteristics,and redece the expression of ZNF703 by si RNA in order to observing the expression of P27,E2F1 and CCNE1 in the papillary thyroid carcinoma cell line K1,explore the mechanism of ZNF703 in the occurrence and development of papillary thyroid carcinoma.Methods 1 Immunohistochemistrical PV9000 method was used to detect the positive expression rate of ZNF703 in organize chips(including 36 cases papillary thyroid carcinomas,14 cases Para-Carcinoma tissues),and analyze the relationship of positive expression rates of ZNF703 in papillary thyroid carcinoms and clinical characteristics(including age,gender,tumor size,lymph node metastasis,clinical stage).Additionally collect 20 cases papillary thyroid carcinomas and para-Carcinoma tissues to detect the relative expressions of ZNF703 by Western blot and rt-pcr.2 Devise the micro-RNA sequences(Si-ZNF703-1,si-ZNF703-2,si-ZNF703-3)embaded into lentivirus carrier,to infect these sequece into papillary thyroid cell K1.Set MOI values to 30,50,70,90 respectively and observe infected cells under a fluorescence microscope to choise the best MOI.The K1 cells respectively transfected by si-ZNF703-1,si-ZNF703-2 and siZNF703-3 were used as experimental group 1,2,3.The cells without being transfected was used as control group,and the cells transfected with empty vector was used as negative control group.PCR was used to detect the relative expression of m RNA in order to select the best interference sequence.Western blot was used to detect the expression of ZNF703 in blank control group,negative control group and the group transfectde bset sequece to check up the effect of interferation,next detect the expression of P27,CCNE1 and E2F1 in all kinds of cells.Results 1 Immunohistochemistry Results:ZNF703 is mainly expressed in the nucleus.the positive expression rates of ZNF703 in papillary thyroid carcinoma(66.7%)were obviously higher than those(21.4%)in Para-Carcinoma tissues,the differences were statistically significantit(P < 0.05).The positive rates of the papillary thyroid carcinomas were correlated with tumor size,lymph node etastasis,clinical stage(P<0.05),but had no correlations with the age,gender.2 The results of RT-PCR and Westernblot :The relative expression of ZNF703 protein [(0.893 ± 0.113)vs(0.510 ±0.072)]and the relative expression of ZNF703 m RNA [(6.399 ±2.096 vs 1.285 ±0.354)]in 20 cases papillary thyroid carcinomas were higher than those in mached ParaCarcinoma tissues,the differences were statistically significantit(P < 0.05).3Optimization of transfection efficiency and si RNA screening: The best transfection efficiency(> 90%)was achieved when MOI was 70.The result of PCR shows that the relative expression of m RNA in experimental group 1,experimental group 2 and experimental group 3(0.142 ± 0.013,0.492 ± 0.008,0.550 ± 0.007 vs 0.982 ±0.032,P <0.05)was lower than that in blank control group,and down rate of experimental group 1 was 85.5±0.594%(P<0.05),which indicates its silent interference is the best,and there was no significant difference between the blan control group and negative control group.4 Western-blot analysis showed that the relative expression of ZNF703 protein in experimental group 1 was significantly lower than that in blank control group(P <0.05),the relative expression of CCNE1 and E2F1 protein in experimental group 1was significantly lower than that in blank control group(P <0.05).The expression of P27 protein in experimental group 1 was significantly higher than that in blank control group(P <0.05),there was no significant difference between blank control group and negative control group.Conclusions 1 The expression of ZNF703 in papillary thyroid carcinoma is higher than that in Para-Carcinoma tissues,which may play a role as a carcinogenic factor and is related to clinical prognosis.2 ZNF703 may regulate cell growth cycle change to promote tumor development through the P27-E2F1-CCNE1 molecular pathway... |
PDF Full Text Request