| Background:Pancreatic cancer is an aggressive malignancy,and traditional radiotherapy and chemotherapy are often difficult to work,so developing new treatment strategies(such as immunotherapy)is very important.The PD-1 / PD-L1 signaling pathway can induce and maintain the immune tolerance of peripheral tissues in order to prevent excessive inflammatory response and autoimmunity in tissues.The PD-1 / PD-1 signaling pathway can induce and maintain the immune tolerance of peripheral tissues.And tumor can utilize the PD-1 / PD-L1 signaling pathway to inhibit the immune system,and we found that miR142-5p can regulate the expression of PD-L1 to block this immunosuppressive signaling pathway and enhance anti-tumor immunity to suppress tumor.Aim:To investigate whether miR-142-5p can regulate the expression of PD-L1 and the effect of miR-142-5p on anti-tumor immunity.Research methods and content:First,we predicted the mi R-142-5p target gene by the bioinformatics method through the Targetscan database and the PicTar database.It was found that PD-L1 was the target gene of miR-142-5p in both databases.The relationship between the expression of PD-L1 and the expression of miR-142-5p was analyzed from the data derived from the TCGA database,and the correlation between the expression of PD-L1 and the expression of miR-142-5p was further confirmed.The results were analyzed by statistical analysis.The expression of miR-142-5p was negatively correlated with the expression of PD-L1 gene and it is statistically significant.Panc02 cells were mouse pancreatic cancer cells.Panc02 cells were transfected with miR-NC mimics or miR-142-5p mimics respectively.Then the cells were used to conduct luciferase reporter assay,RT-PCR and Western Blot to determine whether PD-L1 is regulated by miR-142-5p.And then mouse pancreatic cancer cell line Panc02 cells were transfected with with miR-142-5p overexpressing lentivirus and control lentivirus.The stable cell lines were screened and then analyzed by luciferase reporter assay,RT-PCR and Western Blot to determine whether PD-L1 is regulated by miR-142-5p.Then C57BL/6 mice were challenged with stable miR-142-5p or vector control(miR-NC)overexpression Panc02 cells to construct the model of mouse pancreatic cancer.Mice were sacrificed two weeks after implantation,and tumors were isolated,measured,weighed and imaged.The changes of tumor growth between two groups were observed and the flow cytometry was carried out to detect the changes of tumor immune microenvironment.The miR-142-5p overexpression Panc02 cells or miR-NC overexpression Panc02 cells were injected into the tail vein of mice respectively to test whether miR-142-5p can influence the metastasis of pancreatic cancer.And cell proliferation experiments,cell migration experiments were conducted to observe whether the miR-142-5p can directly promote proliferation or promote migration.Results:1)Firstly,we analyzed date from two miRNA target gene prediction websites(Targetscan and PicTar)and found that PD-L1 is the potential target gene of miR-142-5p in both human and house mouse.Human and house mouse have exactly same mature miR-142-5p sequence.Secondly,we use the TCGA database composed of 48 large B cell lymphoma samples,122 thymoma samples and 181 pancreatic cancer samples to further evaluate the relevance of PD-L1 and miR-142-5p.From these data,we found that miR-142-5p expression is inversely correlated with PD-L1.2)The overexpression of miR-142-5p by transfecting mimics inhibited wild-type but not mutant luciferase reporter activity in Panc02 cells.The overexpression of miR-142-5p by transfecting mimics decreased the mRNA and protein level of PD-L1.The stable overexpression of miR-142-5p by lentivirus transfection decreased the mRNA and protein level of PD-L1.3)Mice were challenged with stable miR-142-5p or vector control(miR-NC)overexpression Panc02 pancreatic cancer cells.mi R-142-5p overexpression reduced tumor size and tumor weight.In the metastasis model,miR-142-5p overexpression significantly reduced tumor metastasis.Cell proliferation experiments including cell count experiment and CCK-8 cell proliferation experiment,the results show that there is no significant difference between miR-142-5p overexpression group and miR-NC group.In the Transwell experiment,miR-142-5p overexpression Panc02 cells significantly migrated less than control.4)Mononuclear cells in mouse tumor were extracted and analyzed by flow cytometry.It was found that miR-142-5p increased the infiltration of T lymphocytes,including CD4+ T lymphocytes and CD8+ T lymphocytes,while reducing PD-1+ T lymphocytes.There was no significant change in Treg,macrophage and dendritic cells infiltration after miR-142-5p overexpression.The expression of IFN-γ and TNF-α was increased and the expression of IL-10 was decreased after high expression of mi R-142-5p.Conclusion:1)miR-142-5p regulates PD-L1 expression by binding to the 3’UTR of PD-L1.2)miR-142-5p inhibited cancer growth and suppressed cancer metastasis.3)miR-142-5p overexpression inhibited the migration of cancer cells,but didn’t have significant influence on cancer cell proliferation.4)miR-142-5p enhances anti-tumor immunity. |
PDF Full Text Request