Linc-POU3F3 Promotes Cell Proliferation In Gastric Cancer Via Increasing T-reg Distribution

Background and aimsGastric cancer is a sort of common malignant tumor,it is characteristic of high morbidity,mortality rate.According to the statistics in the world,gastric cancer is is the fifth most common malignancy,and the third leading cause of cancer death.In China,gastric cancer is the second malignant tumor,and the mortality rate is the third highest.So we will study the mechanism and metastasis of gastric cancer,explore the new interventions to improve the therapeutic effect of gastric cancer,in order to increase the survival rate and quality of life.Tumor mediated immunosuppression is considered to be one of the key factor for tumor immune evasion,which is closely related to the occurrence and development of tumor.T-reg cell is an important medium for tumor immune escape mechanism.CD4+CD25+Fox3+Treg cell is a special subset of T-reg cell,which has two characteristic:immune suppression and immune incompetent.It shows high expression of malignant tumor tissues and peripheral blood.It may inhibit tumor cell responses in specific sites of tumors.At the same time,with the widely used of high-throughput sequencing technology,the function of protein coding genes has been studied.Long chain non-coding RNA has been shown to be involved in the formation and development of gastrointestinal tumors,and may be involved in gene transcription and post transcriptional regulation.It also has the potential to be a biomarker for early cancer.The previous studies were mainly concentrated in the abnormal expression of lncRNAs,and rarely involve the mechanism of tumor immune cells.In this study,we will detect the abnormal distribution of T cells in the peripheral blood of patient with gastric cancer by comparing with the healthy control group.Through high-throughput microarray screening technology,we will explore the abnormal expression of IncRNA in T cells.Linc-POU3F3 has high expression and stable expression level,so to explore the relationship between linc-POU3F3 and T-reg cells.In addition,studies have found that T-reg cells take effect through TGF-beta signaling pathway.And some researchers suggest that linc-POU3F3 can directly activate TGF-beta signal pathway,but the specific mechanism is not clear.At last,we intend to reveal the regulation of linc-POU3F3 and the interaction with T-reg cells by immunoprecipitation.MethodIn this study,we collected peripheral blood from gastric cancer patients and healthy control group.We extracted T cells from peripheral blood lymphocytes by isolation and culture technique,then distribution of T-reg was detected by flow cytometry method.At the same time,screen lncRNAs through high throughput screening to choose the target lncRNA.We analyses the relation between the clinical information of patients and lncRNAs.The RT-PCR was used to detect the expression of IncRNA in the peripheral blood of gastric cancer.In the second part,we mainly study the function of linc-POU3F3 in immune cells and tumor cells.In order to further explore the effect of linc-POU3F3 on T cells,especially the differentiation of T-reg cells,we combined T cells with linc-POU3F3 by by lentivirus packaging and transfection.Immunofluorescence and RT-PCR technology were used to detected the expression of linc-POU3F3.T cells were co-cultured with linc-POU3F3 cells and cultured in Transwell culture system to observe the proliferation of tumor cells.In the last part,we aimed to explore the regulation mechanism of Linc-POU3F3.According to the study,linc-POU3F3 can directly activate the TGF-beta signaling pathway,the differentiation of T-reg cells is also closely related to the TGF-beta signaling pathway.So we detected the interaction between linc-POU3F3 and TGF-beta by radioimmunoassay and Western blot.ResultPeripheral blood T-reg cells were significantly up-regulated in plasma samples of gastric cancer patients.Between gastric cancer patients and controls,IncRNA microarray detection indicated an aberrant expression profiling of IncRNAs in T-reg cells,in which linc-POU3F3 was selected as a potential biomarker with the highest fold change value as well as the most stable expression level in each group.Combined with the clinical information of patients with gastric cancer,it is suggested that linc-POU3F3 is closely related to the growth of tumor,TNM stage and metastasis of gastric cancer.In addition,over-expression of linc-POU3F3 elevated Treg distribution in vitro and promoted tumor cell proliferation in the co-culture system.In vitro experiments,Linc-POU3F3 can be directly combined with TGF-beta and up-regulated TGF-beta.We further found that linc-POU3F3 could recruit TGF-beta which increased the phosphorylation of SMAD2/3,which is important in the occurrence and development of gastric carcinoma.ConclusionWe found that linc-POU3F3 could promote the distribution of T-regs in peripheral blood T cell which caused an enhanced cell proliferation of gastric cancer cells by recruiting TGF-beta as well as activating TGF-beta signal pathway.This finding may provide a theoretical basis for the further exploration of IncRNAs function in immune cell cells of gastric cancer.