| Background and purpose:Pancreatic cancer is one of the common digestive tract malignant tumors, while the percent of pancreatic cancer in total digestive tract malignant tumors is8%～10%. Pancreatic cancer is characterized by rapid growth and metastasis in the early stages, while the resection rate is still low. and recurrence and distant metastases often occur, as one of the ten malignant tumors caused deaths in China. Clarifying the pathogenesis of pancreatic cancer, exploring the methods for early diagnosis of pancreatic cancer, and looking for value drug targets for cancer treatment are major tasks for biomedical scientists, because of the great harmfulness of pancreatic cancer on human health.B7-H4(also known as B7x or B7S1) is a newly discovered member of the B7family that inhibits both the adaptive immune response, by reducing the proliferation, activation, and cytokine production of T cells. It is reported that B7-H4is expressed either in the membrane or in the cytoplasm of cells, with a diffuse pattern in many tumor cells such as pancreatic cancer, and correlated with clinical stage and grade. B7-H4inhibits T cell mediated immune response, and therefore enables tumors to avoid immune detection. However, the role of B7-H4role in the pathogenesis of cancer in addition to inhibiting immune responses has no clear conclusion, such as whether B7-H4expression in tumors can directly affect the biological activity of tumor cells, and whether B7-H4regulate tumor cell growth and survival resulting in contributing to the progress of tumor by influencing cancer cell signaling pathways. This research will focus on the pathology mechanism of B7-H4mediated malignant development of pancreatic cancer cells though a variety of methods and technology including cytobiology, molecular biology, histopathology, and gene target to expression regulation, in order to discuss whether B7-H4can directly affect biology activity of tumor cell, and participate in pathology process of pancreatic catheter epithelial cell malignant transformation and pancreatic cancer occurrence and development. Thus, we expect to clarity the role of B7-H4in pathology of pancreatic cancer, and provide theoretical and experimental basis for clinical targeting therapy of pancreatic cancer.Methods:1. We prepared mouse anti-B7-H4monoclonal antibodies though hybridoma technology, and tested the function and usage of these monoclonal antibodies.2. The expression characteristics of B7-H4in pancreatic cancer tissue and cells were detected using these mouse anti-B7-H4monoclonal antibodies through Western blotting, flow cytometry and immunohistochemical (IHC).3. siRNAs specific for B7-H4were synthesized, and their ability to knockdown B7-H4mRNA and protein expression in pancreatic cancer cells was evaluated though RT-PCR and Western blotting.4. In vitro experiment:the effect of B7-H4gene silencing in L3.6pl pancreatic cancer cells was determined though a number of functional assays. Morphology changes were observed with an optical microscope. Changes of surface molecules (stem cell markers and the epithelial or mesenchymal phenotype markers) were observed though flow cytometry and western blotting. Cell proliferation was determined though CellTiter96AQueous One Solution Cell Proliferation Assay kit (MTS). Clone formation and migration of L3.6p1cells were observed after B7-H4gene silencing. Apoptosis assays were performed using an annexin V-FITC apoptosis detection kit. Cell signal molecules such as Erkl/2and AKT were detected through western blotting assay.5. In vivo experiment:for inoculations, approximately1×106L3.6p1cells in log phase were injected subcutaneously into the dorsal region near the neck of the mice. The tumor volume was measured after treated with B7-H4siRNA. Tissue sections were then analyzed using an In Situ Cell Death Detection Kit. The tissue pathological changes were observed through H&E staining, and cell signal molecules were analyzed using IHC staining.6. Several distinctive miRNAs which were closely related to B7-H4were preliminarily selected in pancreatic cancer cells using miRNA microarray combined with siRNA technology. And the potential role and possible target molecules of these miRNAs in mediating pathological mechanisms of pancreatic carcinoma were analysed by bioinformatics methods.Results:1. Obtain mouse monoclonal antibodies specific to B7-H4. The mouse anti-B7-H4mAbs3E8specifically recognized B7-H4through ELISA, IP, western blotting, flow cytometry and IHC staining analyses.2. B7-H4expression was significantly higher in the pancreatic cancer tissues than in normal pancreatic tissues. Further, the B7-H4expression was related to the stage of the tumors and lymph node metastasis.3. B7-H4siRNAs actively suppressed the mRNA and protein expression of B7-H4in L3.6p1pancreatic cancer cells.4. The results from in vitro experiment indicated that B7-H4could directly affect biology activity of tumor cell. ①Inhibition of B7-H4increased cell-cell adhesion, and decreased refraction and pseudopodia. The expression of the epithelial phenotype marker E-cadherin was increased, and the expression of mesenchymal phenotype marker Vimentin and pancreatic cancer stem cell marker CD44was decreased. The results indicated that B7-H4could induce morphological change of L3.6pl pancreas cancer cells.②B7-H4siRNA inhibited cell proliferation, colony formation, and migration of L3.6pl pancreatic cancer cells.③B7-H4downregulation led to a significant increase in apoptosis. Increased levels of the pro-apoptotic protein Bax, reduced expression of the anti-apoptotic Bcl-2protein following B7-H4silencing, and activation (cleavage) of caspase9and caspase3relative to untransfected and non-target siRNA controls were observed. And the expression of phosphorylated Erkl/2(Thr202/Tyr204) was markedly reduced.5. The results from in vivo experiment indicated that B7-H4siRNA inhibited the L3.6p1tumor formation.①Tumors treated with B7-H4siRNA were significantly smaller throughout the time course compared with controls.②Tumors were excised and DAB-coupled TUNEL assays in tumor sections revealed advanced-stage apoptosis in TUNEL-positive B7-H4siRNA-treated cells that were concentrated in large areas near regions of cell death, while chromatin concentration and marginalization, nuclear membrane lysis, and chromatin splitting into massive (apoptosis corpuscles). There was a significantly higher apoptotic index for B7-H4siRNA-treated cells than controls.③Average protein staining measurements in tumor tissue from B7-H4siRNA-injected mice confirmed a significant increase in signal intensity for Bax, cleaved caspase3, and cleaved caspase9and a significant decrease in signal intensity for Bcl-2and phospho-Erkl/2compared with controls, which were consistent with the results of in vitro experiments. 6. The expression of hsa-let-7c, hsa-miR-186-5p, and hsa-miR-335-5p were significantly decreased in B7-H4gene silencing pancreatic cancer cells, while the expression of hsa-miR-299-5p and hsa-miR-483-3p were significantly increased. These miRNA involved in the regulation of mRNA surveillance pathway, MAPK signaling pathway and other cell signaling pathways influenced biological function of pancreatic cancer cells.Conclusions:Different expression levels of B7-H4in the tumors indicated that B7-H4might be involved in tumor formation and development. Loss of B7-H4function induced the activation of caspase9and caspase3and inhibited the Erkl/2signaling pathway, and then promoted apoptosis of L3.6p1pancreatic cancer cells and prevented tumor growth, indicating that B7-H4might be a pancreatic cancer promoter. This research suggested that abnormal expression of B7-H4might promote pancreatic cancer growth. Targeted inhibition of B7-H4could effectively inhibit pancreatic cancer pathology progress, and B7-H4may become a potential target for pancreatic cancer treatment. |
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