Study On Correlativity Between Progesterone Receptor Gene Polymorphism And Endometrial Cancer

Objective: To establish a technology platform for the detection of progesterone receptor gene(PGR)+331G/A and G1978 T polymorphisms for genotyping endometrial cancer patients.To study the influence of PGR promoter +331G/A polymorphisms caused to PRA,PRB m RNA and protein expression.Therefore,to explore the relationship between +331G/A polymorphism and endometrial cancer.Methods: DNA from normal human blood was extracted as a template,and two-point site-directed mutagenesis primers designated as +331G/A and G1978 T were designed.According to the principle of splicing by overlapping extension PCR(SOE-PCR)site-directed mutagenesis,PGR fragment containing mutant +331A,1978 T were obtained.The mutation of the gene fusion fragments were verified by DNA sequencing.The wild-type detection primers and mutant-type detection primers of +331G/A and G1978 T were designed with 3? terminal phosphorothioate modification using the constructed recombinant gene fragment as a template respectively.Designing an orthogonal design scheme,from the amount of primers,template volume,annealing temperature and the number of cycles,the optimum conditions for the detection of two-point polymorphisms by molecular switches were determined.Then the genotypes of PGR +331G/A and G1978 T polymorphisms in whole blood DNA of normal human and endometrial cancer patients were genotyped under the best detection conditions.Next,the total RNA of endometrial tissues of normal human and endometrial cancer patients was extracted,and the expression of m RNA was detected by qRT-PCR.The protein was extracted and the protein expression was detected by Western Blot.Finally,statistics were performed to analyze the association between the polymorphisms of the PGR gene and the occurrence,development,and treatment of endometrial cancer.Results:1.Two sites mutant type gene fragment templates of PGR gene were successfully established by site-directed mutagenesis using overlapping PCR,respectively.The optimal conditions for the molecular switch technology genotyping the PGR gene +331G/A and G1978 T were successfully established.Under the optimal reaction conditions,the molecular switch phenomenon was obvious.When the wild type/mutant type detection primer and the DNA template are completely matched,a corresponding band appears on the gel electrophoresis,that is,there is a PCR product;otherwise,if there is an incomplete match,the gel electrophoresis does not have a band,i.e there is no PCR product.2.The molecular switch was used to genotype two spots of PGR gene in whole blood DNA of 66 normal human and 62 endometrial cancer patients.The gene frequencies of the +331 G/A polymorphism were GG(93.55%)and GA+AA(6.45%)respectively,and there was no significant difference(P>0.05)compared with the normal human group GG(90.91%),GA+AA(9.09%).The frequencies of the G allele in the endometrial cancer group and the normal human group were 94.70% and 95.97%,and the frequencies of the A allele in the two groups were 5.30% and 4.03%,respectively.There was no significant difference of the gene frequency distribution(P>0.05).3.As for the frequency difference of genotype distribution of the G1978 T polymorphic between patients GG(100.00%),GT+TT(0.00%)and the normal human GG(96.97%),GT+TT(3.03%),there was no statistically significant(P>0.05).The frequencies of the G allele in the endometrial cancer group and the normal human group were 97.73% and 100.00%,and the frequencies of the T allele in the two groups were 2.27% and 0.00%,respectively.There was no significant difference of the gene frequency distribution(P>0.05).4.The results of qRT-PCR showed that compared with the control group(normal endometrium tissue),the m RNA levels of PRB,PR,and PRA(0.396,0.306,and 0.237)in the patients tissue group were significantly lower(P<0.05).The difference in PRA/PRB level between the two groups was not statistically significant(P>0.05).Further analysis showed that the relative expression of PRB and PR m RNA in GA+AA group and GG were not statistically significant(P>0.05).But there was a significant increase compared to GA+AA group and GG group of PRA m RNA relative expression and PRA/PRB value(P<0.05).5.Western blot results showed that the expression of PRA and PRB protein in endometrial cancer tissues was significantly lower than that in the control group(P<0.05).Moreover,the level of PRB protein between the GG group compared to the GA+AA group with endometrial cancer was significantly higher(P<0.05),while the PRA difference between the two groups was not statistically significant(P>0.05).Regarding the PRA/PRB levels,it in the GG group and the GA+AA group were significantly higher(P<0.05)than those in the control group,and there was a significant decrease between the two groups(P<0.05).Conclusion:1.The technology platform were successfully established to genotype the polymorphisms of PGR +331G/A and G1978 T.2.The incidence of endometrial cancer in the Chinese population and PGR +331G/A,G1978 T two polymorphism may not be relevant.3.The occurrence and development of endometrial cancer may relate to the decreased expression of PR,PRA,PRB m RNA,decreased expression of PRA,PRB protein,and increased the value of PRA/PRB.4.The polymorphism of PGR promoter +331G/A may not affect the expression of PR and PRB m RNA,but may affect the expression of PRB protein and the value of PRA/PRB.