Overexpression Of Progesterone Receptor B Enhance The Sensitivity Of Endometrial Cancer Cells To Medroxyprogesterone Acetate

Objective: To study overexpression of progesterone receptor B can enhance the sensitivity of endometrial cancer cells Ishikawa and KLE to the progestin drug medroxyprogesterone acetate,and provide a theoretical basis for the conservative treatment of endometrial cancer progesterone.Methods: RNA from Ishikawa cell was extracted as a template,and reverse transcription as templates for cDNA.Then design PRB CDS area length of specific primers and amplification of the target fragment.The product purpose fragment cloning into pcDNA3.1(+)in the plasmid vector,and transformed into Escherichia E Coli.DH5? competent cells and then cultured on the Luria-Bertani(LB)-agar plates containing ampicillin.Following randomly picking up positive clones,the double enzyme digestion and sequencing identification,and pcDNA3.1(+)-PRB recombinant plasmid vector was constructed.The endometrial cancer cells Ishikawa and KLE were cultured in vitro,and transfected pcDNA3.1(+)-PRB recombinant plasmid vector into Ishikawa and KLE cells.The expressions of PRB mRNA and protein in Ishikawa and KLE cells were detected by Real-time PCR and Western blot,to determine whether the cell lines with overexpression of PRB were formed.Then medroxyprogesterone acetate was used to treat endometrial cancer cells Ishikawa and KLE.The cck-8 method,scratch test and monoclonal formation test were used to detected cell proliferation activity and invasion ability.Finally,the effect of PRB expression on the sensitivity of endometrial cancer cells treated with medroxyprogesterone acetate was analyzed statistically.Results:1.The CDS of PRB which was amplified by PCR was coloned into pcDNA3.1(+)-PRB plasmid successfully.2.The results of Real-time PCR showed that Isk-pcDNA3.1(+)-NC group(transfected with empty vector)and Isk-pcDNA3.1(+)-PRB group(transfected with pcDNA3.1(+)-PRB recombined Plasmid vector)PRB mRNA levels were(1.56 � 0.74)and(40.38 � 6.72),the Isk-pcDNA3.1(+)-PRB group mRNA expression of PRB was increased,which was statistically significant compared to the KLE-pcDNA3.1(+)-NC group(p<0.0001).The KLE-pcDNA3.1(+)-NC group(transfected empty vector)and KLE-pcDNA3.1(+)-PRB group(transfected pcDNA3.1(+)-PRB recombinant plasmid vector)PRB mRNA levels were(1.89 � 0.52)and(51.27 � 11.38).The KLE-pcDNA3.1(+)-PRB group mRNA expression of PRB was increased,which was statistically significant compared to the KLE-pcDNA3.1(+)-NC group(p<0.0001).3.The Western blot results showed that the PRB protein expression levels of the Isk-pcDNA3.1(+)-NC group and the Isk-pcDNA3.1(+)-PRB group were(0.71 � 0.05)and(1.05 � 0.07),respectively.The comparation of Isk-pcDNA3.1(+)-PRB group and Isk-pcDNA3.1(+)-NC group,the difference was statistically significant(p<0.05).The expression levels of PRB protein in KLE-pcDNA3.1(+)-NC group and KLE-pcDNA3.1(+)-PRB group were(0.18 � 0.01)and(0.70 � 0.03).The difference between KLE-pcDNA3.1(+)-PRB group and KLE-pcDNA3.1(+)-NC group was statistically significant(p <0.01).4.The CCK-8 results showed that the Isk-pcDNA3.1(+)-PRB group(64.12�1.37)compared to the Isk-pcDNA3.1(+)-NC group(42.21�1.67),the cell inhibition was increased(p<0.01);KLE-pcDNA3.1(+)-PRB group(39.88 � 2.22)compared to KLE-pcDNA3.1(+)-NC group(15.04 � 1.34),the cell inhibition rate was increased(p<0.001).The results of the monoclonal formation experiment showed that after the treatment of medroxyprogesterone acetate,the number of monoclonal cells in the Ishikawa-pcDNA3.1(+)-PRB group and the KLE-pcDNA3.1(+)-PRB group was less than the empty vector group.The scratch experiment showed that the cell migration rate of the Ishikawa-pcDNA3.1(+)-PRB group was reduced compared with the Ishikawa-pcDNA3.1(+)-NC group(13.81 � 0.60 vs 22.06 � 0.84,p <0.01);compared with KLE-pcDNA3.1(+)-NC group,the cell migration rate of KLE-pcDNA3.1(+)-PRB was reduced(15.66 � 1.07 vs 24.60 � 2.31,p<0.01).Conclusion:1.The recombinant plasmid vector of pcDNA3.1(+)-PRB was successfully constructed which can enhance the expression of progesterone receptor B in endometrial cancer Ishikawa cells and KLE cells.2.The overexpression of progesterone receptor B can enhance the sensitivity of endometrial cancer cells Ishikawa and KLE to medroxyprogesterone acetate.