microRNA-375在胃癌PD-L1过表达中的作用和机制研究

Our previous studies found that PD-L1 was overexpression in gastric cancer. Moreover the overexpression of PD-L1 was associated with poor prognosis. Till now the mechanism of PD-L1 overexpression in gastric cancer remains unclear. We used The Cancer Genome Atlas (TCGA) gastric cancer database to analyze the difference of miRNA expression between PD-L1 mRNA high expression and low expression patients. We found that the expression of miR-375 was significantly lower in the PD-L1 high expression patients than in the PD-L1 low expression patients. Quantitative real-time PCR in gastric cancer samples from our cancer center further verified that miR-375 expression was significantly downregulated in PD-L1 positive patients compared with PD-L1 negative patients. Meanwhile, we found a significant inverse correlation between miR-375 and PD-L1 mRNA expression in gastric cancer cell lines in vitro. When the cells were transfected with miR-375 precursor, the expression of PD-L1 significantly decreased. We employed miRTarBase and TargetScan websites to search for putative human gene targets of miR-375 and found that JAK2 was a putative target. The protein level of JAK2 can be inhibited by transfecting miR-375 precursor. In vitro, we found that JAK2 inhibitor can also inhibit the expression of PD-L1. We therefore hypothesized that miR-375 could regulate the expression of PD-L1 through the JAK2/STATs pathway. In this study, we will further verify the target gene of miR-375 and explore the role of miR-375/JAK2/STATs pathway in the regulation of PD-L1 overexpression in gastric cancer. Moreover, we would also like to explore the effect of miR-375/JAK2/STATs/PD-L1 pathway on the activation of T cells. This study will provide evidences for us to understand the mechanism of PD-L1 overexpression in gastric cancer and promote the development of precision treatment in gastric cancer.

PD-L1在胃癌中高表达，且与不良预后相关，但其过表达的机制不详。我们使用TCGA胃癌数据库分析PD-L1 mRNA高表达和低表达患者的miRNA差异，发现miR-375在PD-L1高表达患者中显著下调，在本中心胃癌患者组织中也证实了这一发现；我们还发现胃癌细胞株中miR-375表达与PD-L1负相关，转染miR-375前体后PD-L1表达降低；通过miRTarBase和TargetScan平台筛选发现JAK2可能是miR-375的下游靶基因，抑制JAK2可降低PD-L1的表达。我们猜想miR-375可能通过JAK2/STATs通路调节PD-L1的表达。本课题将验证miR-375的靶基因；探索miR-375/JAK2/STATs通路对PD-L1表达的调控作用；阐述该通路对T细胞激活的影响。探索 PD-L1 高表达的机制有助于挑选合适的患者进行免疫治疗，推动胃癌精准治疗的发展。

PD-L1在胃癌中高表达，且与不良预后相关，但其过表达的机制不详。我们使用TCGA胃癌数据库分析PD-L1 mRNA高表达和低表达患者的miRNA差异，发现miR-375在PD-L1高表达患者中显著下调，在本中心胃癌患者组织中也证实了这一发现；我们还发现胃癌细胞株中miR-375表达与PD-L1负相关，转染miR-375前体后PD-L1表达降低；通过miRTarBase和TargetScan平台筛选发现JAK2可能是miR-375的下游靶基因，抑制JAK2可降低PD-L1的表达。通过构建荧光素酶报告基因质粒，发现miR-375对JAK2具有负调控作用，在NUGC3和MKN7胃癌细胞株中过表达mir375均可减少PD-L1、JAK2和STAT3的mRNA表达，划痕实验显示在NUGC3和MNK7胃癌细胞株中过表达mir375，细胞的侵袭能力降低，Transwell实验提示在NUGC3和MNK7胃癌细胞株中过表达mir375，细胞的侵袭能力降低。细胞克隆形成实验提示在MKN7和NUGC3胃癌细胞株中过表达mir375可抑制细胞的增殖，体内成瘤实验发现过表达mir375可以抑制胃癌动物模型的生长。本研究通过扎实的前期研究结果，阐明miR-375/JAK2/STATs通路对PD-L1表达的调控作用；阐述该通路对胃癌增殖、侵袭转移和凋亡的影响。