Nuclear Progesterone Receptor B Subtype Gene Promoter Region +331G/A Polymorphism Enhances The Sensitivity Of Endometrial Cancer Cells Ishikawa To Mapetate By Promoting Prb Expression

The literature shows that the nuclear progesterone receptor gene promoter+331G→A mutation will increase the incidence of endometrial cancer,and +331A will significantly increase the expression of PRB(progesterone receptor B,PRB).Accordingly,this project puts forward the scientific hypothesis that "nuclear progesterone receptor gene promoter region +331G/A polymorphism enhances the expression of PRB to enhance the drug sensitivity of endometrial cancer Ishikawa cells to medroxyprogesterone acetate",in order to To verify this hypothesis,this study was divided into two parts:(1)The effect of +331G/A mutation on transcriptional activity and PRB expression was detected by constructing PRB promoter region +331G and +331A reporter gene vectors;(2)Nuclear progesterone The effect of medroxyprogesterone acetate(MPA)on the malignant behavior of endometrial cancer cell line Ishikawa after changes in the expression of receptor B subtype protein;Objective: In order to explore the effect of mutations at +331G/A on transcriptional activity and PRB expression,we constructed the nuclear progesterone receptor B subtype gene promoter +331G and +331A luciferase reporter gene vector and combined it with the previous laboratory The constructed PRB overexpressing Ishikawa cell line(Isk-PRB-OE)and the Ishikawa cell line with significantly reduced PRB protein expression(Isk-PRB-KD)were constructed in this study to investigate whether changes in PRB protein expression affect endometrial cancer Sensitivity of the cell line Ishikawa to medroxyprogesterone acetate(MPA).Methods: Frist,search the promoter region of PRB gene(2000bp before the transcription start site)on NCBI,use Primer Premier 6.0software to design primers for amplifying G wild-type promoter region,and then design overlapping PCR primers to amplify A mutant promoter Zone primers.Insert the amplified fragment into the pGL-4.10 plasmid,transform it into DH5α E.coli,extract the plasmid and Enzyme digestion was performed to verify that the target fragment was well connected to the plasmid.pGL-4.1-hPgR-331G+ p RL-TK,pGL-4.1-hPgR-331A+p RL-TK,pGL-4.1-Basic+ p RL-TK was transfected into 293 T cells to detect the activity of the luciferase reporter gene.Then insert the PRB CDS region sequence into the back end of the PRB promoter in pGL-4.10-hPgR-331 G,pGL-4.10-hPgR-331 A and pGL4.10-Basic to construct plasmid pGL-4.10-CDS,pGL-4.10-hPgR-331 GCDS,pGL-4.10-hPgR-331A-CDS.The plasmid pGL-4.10-CDS,pGL-4.10-hPgR-331 G,pGL-4.10-hPgR-331 A,pGL-4.10,pc DNA3.1-PRB,pGL-4.10-hPgR-331G-CDS,pGL-4.10-hPgR-331A-CDS,was transfected into KLE cells,and Western-blot technology was used to test the effect of mutation at position +331 in the Pg R promoter region on the expression of PRB protein.Last of all,the constructed PRB overexpression cell line was used as a control,four PRB sh RNA sequences were designed,transfected plasmids into the Ishikawa cell line using lipo3000,Using real-time quantitative PCR(RT-PCR)test designed sh RNA.The sh RNA with the lowest expression of PRB was detected.Construct a PRB knockdown cell line(Isk-PRB-KD)and construct a cell line stably expressing its negative control(Isk-sh NC).A series of cell malignant biological activity experiments were carried out.This experimental design is divided into 4groups,PRB expression silencing were negative control group sh-NC;PRB group decreased expression Isk-PRB-KD;empty plasmid vector overexpressed protein PRB group Isk-pc DNA3.1;overexpression PRB group Isk-PRB-OE.Use the proliferation of CCK-8 experiments and experimental test PRB monoclonal protein expression mediated MPA on Ishikawa cells affect cell scratch test PRB protein-mediated migration of MPA on Ishikawa cells,the use of flow cytometry PRB expression were mediated apoptosis with MPA on Ishikawa cases in each group.Results:1.The Friefly/Renilla value of pGL-4.1-hPgR-331A+ p RL-TK is significantly higher than that of pGL-4.1-h PRB-331G+ p RL-TK(P<0.5).2.Western-Blot test showed that the mutation of +331A can increase the expression of PRB in KLE cells,and the expression of PRB protein in KLE cells transfected with pGL-4.10-hPgR-331A-CDS plasmid was significantly higher than that of transfected pGL-4.10-hPgR-331G-CDS KLE cells(P<0.05).3.Plasmid vector using a fluorescence inverted microscope after transient transfection taken to observe four sh RNA sequences transfection efficiency,48 h after transfection efficiency reaches nearly 80% extraction of total RNA in each group,after reverse-transcribed into c DNA template was detected by RT-PCR.m RNA expression.PR-PCR results show that,Isk-sh-NC group and the group Isk-sh RNA-1747,Isk-sh RNA-920 group,Isk-PRB-OE(+)m RNA expression levels were PRB group (1±0.00),(0.17±0.02),(0.29±0.07),(44.43±4.11).Compared with Isk-sh NC group sh RNA-1747 and sh RNA-920 group the PRB m RNA expression was significantly decreased,withstatistical significance(P <0.05)..4.Western-Blot results showed that the expression of PRB protein in the Isk-PRB-KD(the plasmid sh RNA-1747 with the best knockdown efficiency)group(0.26±0.01)was significantly lower than that of Isk-sh NC.The results showed that Isk-PRB-KD(-)could induce PRB protein expression significantly(P <0.05),while Isk-PRB-OE(+)could decrease PRB protein expression significantly(P <0.001).5.CCK-8 results showed that there was a significant difference in inhibition rate between Isk-PRB-OE and Isk-pc DNA3.1-NC(P <0.5),after MPA treatment for 48 h,and the cell inhibition rate of Isk-PRB-KD was significantly lower than that of Isk-sh NC(P <0.01).6.The results of monoclonal formation showed that the number of monoclonal in Isk-PRB-KD group was significantly more than that in Isk-sh NC group,and the number of monoclonal in Isk-PRB-OE group was significantly less than that in Isk-pc DNA3.1-NC group.At the same time,there was no difference in the number of monoclonal between the two negative control groups,while the number of monoclonal in Isk-PRB-OE group was significantly less than that in Isk-PRB-KD group.7.The results of scratch test showed that after being treated with MPA for 24 hours,the healing rate of cells in Isk-PRB-KD group was the highest(228.97 ±0.69),and that in Isk-PRB-OE group was the lowest(65.26 ±4.74).There was significant difference between the two groups.8.The results of flow cytometry showed that the average apoptosis rate of PRB overexpression Isk-PRB-OE group treated with MPA was as high as 48%,which was significantly different from that of empty plasmid vector Isk-pc DNA3.1-NC group treated with MPA(P < 0.05).The results of flow cytometry showed that the expression of PRB in Ishikawa cells increased the apoptosis rate of Ishikawa cells,while the decrease of PRB protein expression could inhibit the apoptosis rate of Ishikawa cells.Conclusion:(1)The +331G→A mutation in the promoter region of progesterone receptor B subtype gene promotes the protein expression of PRB,which may be related to the increase of gene transcriptional activity.(2)PRB reduced expression will diminish Ishikawa endometrial cancer cell sensitivity to the MPA.