The Expression Mechanism And Screening Of Differentially Expression Proteins In Microenvironment Of Gastric Cancer

Objectives: Gastric cancer is one of the most frequently occurrence malignancies in China. This research aimed to discover proteins in microenvironment of gastric cancer and explore possible mechanisms of how microenvironment effecting the occurrence and development process of gastric cancer.Methods: 1. The stromas of normal gastric tissue mucosa(NGM)and gastric cancer(GAC) was respectively purified from fresh NGM and GAC tissues by laser capture microdissection(LCM), and obtained high purity stromas of NGM and GAC. Then, the differentially expression proteins between NGM and GAC tissues were identified by i TRAQ-tagging combined with 2D LC-MS/MS in microenvironment of gastric cancer. Then, the cross-talk between proteins in microenvironment of gastric cancer was analyzed by bioinformatics. The expression of FN1,MCL1, EMILIN1 and COL1A1 in gastric cancer microenvironment was detected by Western-blot and immunohistochemistry staining to understand the clinical pathological significance of their expression in NGM and GAC tissues. 2. According to the results of quantitative proteomics research, the miRNA regulating the expression of protein in gastric cancer microenvironment was predicted by bioinformatics software Targetscan 6.2. The miR-125 b regulating multiple protein expression was in-depth analyzed. Firstly, the expression of miR-125 b was detected by q RT-PCR and in situ hybridization in NGM and GAC tissues, and the relationship between the expression of miR-125 b and the clinical and pathological data and prognosis of clinical pathology was evaluated. 3. Then, the miR-125 b was transfected in gastric cancer MGC-803 cells. The effects of over-expression of miR-125 b to the proliferation, invasion and apoptosis of gastric cancer MGC-803 cells were observed by the growth curve, Transwell invasion assay, flow cytometry, Western-blot and tumor formation in nude mice. Secondly, the target gene regulated by miR-125 b in gastric cancer cells was predicted by Targetscan6.2. The target gene MCL1 gene regulated by miR-125 b was analyzed by luciferase activity, q RT-PCR and Western-blot. Lastly,the expression of MCL1 protein in nude mice tumor tissue of miR-125 b over-expression was observed by immunohistochemistry staining. At the same time, the interference vector of MCL1 was constructed. The effects of interference expression of MCL1 to the proliferation, invasion and apoptosis of gastric cancer MGC-803 cells were observed by the growth curve, Transwell invasion assay, flow cytometry and Western-blot.Results: 1. 123 differentially expressed proteins were identified from microenvironment of gastric cancer, 56 proteins were up-regulated, 67 were down-regulated in GC tissue. These proteins were involved in cancer-associated signaling pathways such as MAPK, VEF, cell cycle,apoptosis and p53 signaling pathways. These proteins in microenvironment of gastric cancer was associated with allelotaxis,cellular growth, cellular proliferation, metabolism, apoptosis, response to biotic stimulus, translation, metabolism, protein transport, localization and humoral immune response. They were the nodes in the STRING network and between mutual relations. The results of immunohistochemical staining showed that the EMILIN1 and COL1A1 proteins were over-expressed and the FN1 and MCL1 proteins were low-expressed in GC tissue. The over-expression of FN1 and MCL1 proteins in gastric cancer was positively correlated with the stage of tumor differentiation, tumor size, lymph node metastasis and TNM staging, which the expression of EMILIN1 and COL1A1 proteins was negatively correlated(P<0.01).2. The mic RNAs regulating gastric cancer microenvironment proteins were predicted by Argetscan 6.2, which miR-125 b regulated multiple proteins. The low-expression of miR-125 b in gastric carcinoma was positively associated with gastric cancer tissue differentiation(P<0.01). The positive rate of miR-125 b expression in TNM stage I-II patients was higher than that of III-IV patients, which patients of gastric cancer with TNM staging was negatively correlated(P<0.01). Similarly,the expression of miR-125 b was negatively correlated with the metastasis of lymph node(P<0.01). The Kaplan-meier survival curve was display that the survival rate of patients with low-expression of miR-125 b was lower than that of the miR-125 b over-expression(P<0.01). The disease free survival rate of miR-125 b over-expression in patients was higher than that of miR-125 b low expression(P<0.01). When the expression of miR-125 b in gastric cancer was low the prognosis was worse, and its expression was positively correlated with the prognosis of the patients(P<0.01).3. The growth rate and invasion ability of MGC-803 cells were decreased, cell apoptosis rate increased, and cleaved caspase-3 and cleaved PARP cells were increased when the miR-125 b was over-expression(P<0.01). The gastric cancer xenografts in nude mice were smaller and lighter when the miR-125 b was over-expression(P<0.01). MCL1 is the target gene of miR-125 b that miR-125 b binds2613-2620 nucleotides of MCL1 3’UTR. When the expression of miR-125 b was decreased, the luciferase reporter activity including of MCL1 3’UTR sequence were reduced. The expression of m RNA and MCL1 protein was decreased in MGC-803 cells after the over-expression of miR-125b(P<0.01). In addition, the growth rate and cell invasion ability were decreased also, the rate of apoptosis, cleaved cells and caspase-3 cleaved PARP increased after the expression of MCL1 interference in MGC-803 cells(P<0.01). The expression of MCL1 protein was reduced in tumor tissues of nude mouse after the over-expression of miR-125b(P<0.01).Conclusions: 1. The research identified 123 differentially expressed proteins in gastric cancer microenvironment by laser capture microdissection combined with quantitative proteomics, of which 56 proteins were up-regulated and 67 proteins were down-regulated.2. MiR-125 b is involved in regulating multiple proteins in gastric carcinoma microenvironment; its low expression in gastric cancer tissues was closely related with the differentiation degree of gastric carcinoma,TNM staging, lymph node metastasis and prognosis of patients.3. MiR-125 b inhibited the expression of MCL1 gene and activated Caspase-3 signaling pathway to reduce the invasion and proliferation of MGC803 cells.