The Study Of PD-L1 Expression Regulation Mechanism In Cervical Cancer Patients And Its Prognostic Value For HPV Infection

Objective:PD-L1 as an important component of PD-1/PD-L1 immunosuppression pathway,which play an important role in clinical applications of PD-1/PD-L1immunosuppression pathways checkpoint antibody drugs.cervical cancer as the potential clinical applicators of these antibody drug,detecting the PD-L1 expression in the surface of tumor cell,as well as the regulation mechanism of PD-L1 expression has very important clinical significance.Which could provide the corresponding detecting targets for PD-1/PD-L1 antibody drugs in the clinical treatment of cervical cancer patients,it also provide help for the treatment and prognostic judgement of HPV infection.Methods:1)66 tissue samples from patients with squamous cell carcinoma were collected,data of their clinical characteristics were also gathered.Based on these samples,the expression levels of MLH1,MSH2,and PD-L1 in cancer cells were tested by immunohistochemical assay(IHC).Moreover,PD-1/PD-L1 expressions in tumor invading lymphocytes(TILs)were detected by IHC as well.Fluorescence Amplified restriction fragment polymorphism were used to test the Microsatellite instability.According to the expression of MLH1/MSH2 and MSI test,all 66 cases were divided into dMMR or pMMR groups.The comparisons of PD-L1 in cancer cells and PD-1/PD-L1 in TILs were compared in dMMR and pMMR subgroups.Finally through RNA interference to silence MLH1 protein expression in HeLa cells,to analog form dMMR expression of cell lines,by RT-PCR and Western blot techniques respectively detected PD-L1 mRNA and protein expression before and after MLH1 inhibit in HeLa cells;2)Collected 20 cases of HPV infection cervical cancer patients with frozen samples,first using the Agilentsureselect liquid enrichment combined chips and the second generation sequencing technology from Illumina to test the integration of the HPV and host cell genome.First analyzed the gene integration situation in the 9p24.1 and 3'-UTR of CD274,then synthetic integration of HPV gene segment and CD274gene 3'-UTR recombinant plasmid vector transfection the HeLa cells,meanwhile,other synthetic micRNA-384 artificial simulation and recombinant plasmid transfected the HeLa cells together.Respectively using the qRT-PCR and Western blot detected PD-L1mRNA and protein expression in HeLa and two transfected cells;3)Cervical exfoliated cells samples of 54 premalignant cervical lesion patients with HPV16 infection were collected;meanwhile the cervical exfoliated cells samples from 20 healthy women without HPV infection and 20 patients with only HPV16 infection but cervical cytology normal were collected as 2 control groups.The PD-L1 expression and DNA ploidy analysis was tested by Flow cytometry analysis,the methylation-sensitive high-resolution melting(MS-HRM)was used to test the HPV16 L1 gene methylation.The 54 premalignant cervical lesion patients were followed up in 18 months by three months apart,to evalute the influence of HPV clean time from the PD-L1 on the surface of cervical exfoliated cells,L1gene methylation and HPV subtypes infection state by Kaplan-Meier method and multivariate Cox risk assessment model.Results:1)25.8%of patient samples were associated with dMMR.PD-L1 in cancer cells,PD-L1 in TILs,and PD-1 in TILs took up59.1%,47.0%,and 60.6%,respectively.Statistic difference of PD-L1 in cancer cells could be observed between dMMR and pMMR subgroups.In dMMR group,the PD-L1 in cancer cells and PD-1 in TILs had no correlation(r_s=0.161,P=0.537),but in pMMR group,they had good correlation(r_s=0.645,P<0.001).RNA interference make MLH1protein expression level of HeLa cells reduce to 2%of normal levels,also can be thought a stable state of dMMR cell lines to simulate form,dMMR state of cell lines compared with normal HeLa cell lines,the PD-L1 mRNA level increased to 1.50 times,while PD-L1protein expression increased to 1.27 times;2)There were HPV host cell genome integration phenomenon in 20 cervical cancer patients,there were 8 cases exist integration phenomenon on chromosome 9p24.1 region,which involving 10genes in the region,including CD274(PD-L1)gene,the specimens of 8 cases also had high expression of PD-L1.For the HPV integrated into CD274 specimen,and its integration sites was in CD274gene 3-'UTR region,and the integration sites also was in the region of micRNA-384 to combine CD274gene at the same time.Through synthetic consolidated CD274 gene 3'-UTR gene into HeLa cell line and found that the PD-L1 mRNA and protein expression level increased 23.68 times and 5.65 times to compare with the normal HeLa cells respectively.While to import micRNA-384 artificial simulation and consolidated CD274gene 3'UTR-gene into He La cells,PD-L1 mRNA and protein expression level only increased 14.29 times and 3.42 times respectively than the normal HeLa cell line;3)The PD-L1 positive cell rate and mean fluorescence intensity of PD-L1positive cell in premalignant cervical lesion patients with HPV16 infection were higher than 2 control groups.Mean fluorescence intensity of PD-L1 positive cell were increased in 54 cases when existing multiple HPV status,DNA heteroploid and high HPV16-L1gene methylation(L1gene methylation more than 50%).High PD-L1 expression(PD-L1positive cell rate more than 10%),high HPV16-L1gene methylation,and multiple HPV infection status could prolong the time to clean HPV infection by Kaplan-Meier analysis.Multivariate Cox proportional hazards analysis also showed that all of high PD-L1expression,high HPV-L1 methylation,and multiple HPV infection status should increase the risk of HPV unclearance in premalignant cervical lesion patients;the hazard ratio(HR)was 2.043(CI:1.050-3.973),2.797(CI:1.277-6.122),and 3.050(CI:1.406-6.615).Conclusion:PD-L1 was common in cervical cancer cell surface expression,dMMR state can enhance the expression of PD-L1 on the surface of cervical cancer cells;HPV integrate into the specific areas of host cell genomecan also could increase the expression of PD-L1,in certain integrate situations could also inhibit micRNA physiological functions,leading to the high expression of PD-L1;the high PD-L1 expression on cervical exfoliated cells could enhance the difficulty of HPV infection treatment in premalignant cervical lesion patients.