| Colorectal cancer(CRC) is a serious threat to human health. And metastasis is the key factor of poor prognosis. More than 1/3 of patients with colorectal cancer will eventually develop to metastasis, which will bring great difficulties to clinical treatment, seriously affect the efficacy and living quality of patients. Tumour metastasis remains one of the major challenges in management for CRC patients. Therefore, we must find a molecular target that can effectively interfere with the metastasis of CRC. And this depends on the breakthrough of the mechanism of metastasis.However, the mechanisms of tumor metastasis have not been completely elucidated, and progression of CRC is a process that involves multiple genetic changes, multi-factor, multi-step process. Because of obvious heterogeneity, not all tumor cells can metastasize. Only those tumor cells with the abilities of migration and invasion can break the barriers to form the metastases in the distant tissues. Recent evidences have suggested that epithelial-mesenchymal transition(EMT) is an important cause of invasion and metastasis in tumor cells. And it was a crucial step in triggering tumor metastasis. The EMT program enables cancer cells to disseminate from a primary tumor also promotes their self-renewal capability. Indeed, just such a connection between metastasis and stem-cell state has previously been proposed in a speculative voice as a means of enabling the formation of macroscopic metastases.In recent years, researches have found that the negative B7 family molecules PD-L1(B7-H1) /PD-1, B7-H3, B7-H4, CTLA-4 and Tim-3 play an important role in tumor immunity. On one hand, these molecules express on a series of tumor tissue or immune cells to participate in immune escape, invasion and metastasis process of tumor. On the other hand, they are related with the clinical pathology and prognosis of patients. Especially, the monoclonal antibody CTLA-4, PD-1 and anti PD-L1 have an outstanding curative effect in curing many tumors. So in tumor immunotherapy the attention is unprecedentedly paid to negative B7 family molecules.B7-H3 was cloned from the human DC c DNA library in 2001. The human B7-H3 gene, located in chromosome 15q24, belongs to I type transmembrane protein of the Ig super family. So far, the protein of B7-H3 has been found in many tumor tissues, including gastric cancer, lung cancer, prostate cancer and so on. Besides, B7-H3 abnormal expressed in human tumor tissues and took part in the occurrence and development of tumors, but the mechanism is not completely clear.So far, we have focused on the research of PD-L1, B7-H3, B7-H4, CTLA-4 and Tim-3 in tumor immunity. Recently, we have found a protein c-Met which can be combined with B7-H3 by mass spectrometry analysis. This work has laid a good foundation for further investigation. c-Met was highly expressed in a variety of tumors. Once c-Met was abnormal activated, it would play a key role in the occurrence, development, invasion and metastasis of tumor. In general, we take the human colorectal cancer as the research object, the B7-H3 and c-Met interaction as the main line, and make a profound study in several aspects of CRC such as clinical specimens, in vivo and in vitro experiments, to further explore the mechanism and clinical significance of B7-H3/c-Met regulation of EMT in CRC.Part ⅠThe expression and clinical significance of negativecostimulatory molecule B7-H3 in CRCObjective: To investigate the expression of costimulatory molecule B7-H3 in CRC cells, fresh tumor tissues and paraffin-embedded tissues, and then to analysis the correlation to pathological parameters and prognosis of patients, at last to explore the relationship between abnormal expression of B7-H3 and tumor metastasis in CRC.Methods: The m RNA and protein of B7-H3 in 6 CRC cell lines were analyzed by Real-time PCR, Flow cytometry(FCM), and Western Blot. At the same time, the soluble B7-H3 from CRC cells’ culture supernatant was detected by ELISA. Furthermore, the m RNA and protein of B7-H3 were analyzed in 10 matched pair cases(fresh CRC tissues and normal intestinal tissues). Finally, 197 cases of CRC tissues with complete pathological data were collected as well, and then B7-H3 expression was detected by immunohistochemistry(IHC), and the correlation was analyzed with statistic software.Results: The 6 CRC cells expressed different degrees of membrane B7-H3, and secreted soluble B7-H3. Compared with the normal tissue, B7-H3 highly expressed in the fresh surgical resection of CRC tissues. The IHC staining showed that low B7-H3 expression was found in 73 cases of tissues, and high B7-H3 expression was found in 124 cases of tissues. Furthermore, we found that B7-H3 expression level is closely related with patients’ postoperative overall survival and disease-free survival time(P < 0.01). In other words, high expression of B7-H3 greatly reduced the patients’ overall survival and disease-free survival. B7-H3 expression was uncorrelated with the patients’ gender, age, sizes of tumor, stages of tumor and lymphatic metastasis, while correlations were found between B7-H3 expression and patients’ Duke’s stages. In addition, there is a rising tendency of expression of B7-H3 along with the rising level of distant metastasis in CRC.Conclusion: Costimulatory molecule B7-H3 was abnormally expressed in CRC cells and tissues, correlating to CRC Duke’s stages and patients’ prognosis. It is suggested that B7-H3 plays an important role in the development of CRC.Part Ⅱ The function of B7-H3 in regulating EMT andmaintaining stemness in CRC cellsObjective: To investigate the effect of B7-H3 on the characteristics of EMT and stemness in CRC cells, and then to explore the regulation of B7-H3 in CRC cells’ migration, invasion and stemness maintenance.Methods: After blocking B7-H3 by Small interfering RNA(Si RNA) in CRC cells(HCT116 and SW480), Real-time PCR was performed to detect the m RNA expression of B7-H3, EMT related transcription factors(TF) and stem genes. And then Western Blot and Immunofluorescence were used to analyze the protein expression of B7-H3, EMT related makers and TF on CRC cells with or without treated with Si RNA. Furthermore, the migration, invasion and wound healing assay were used to observe the capabilities of migration and invasion of tumor cells which blocking B7-H3 by Si RNA or over-expression of B7-H3 by lentivirus. Finally, cell spheroids formation, chemo-resistant assay and tumor animal model under subcutaneously inoculated were performed to explore the role of B7-H3 on stemness maintenance.Results: Silencing of B7-H3 caused E-cadherin enhanced expression and vimentin and N-cadherin reduced expression, together with reduced expression of EMT-promoting TF(Snail, Slug, Zeb1 and Twist) in HCT116 as well as SW480. And moreover, the expression of stem genes(CD133, Nanog, Bmi1, Lgr5, ABCB1, ABCG2, Oct4) were down regulated after knockdown B7-H3. Furthermore, knockdown B7-H3 in HCT116 and SW480 inhibited their abilities of migration and invasion. However, the abilities of migration and invasion in CRC cells enhanced after over-expression of B7-H3. Besides, cell spheroids formation assay showed that the ability of forming cell spheroids was inhibited in CRC cells after treated with Si RNA. Chemo-resistant assay further revealed that CRC cells down-regulated expression of B7-H3 were more sensitive to chemotherapy drugs. At last, the tumor animal model confirmed that blocking B7-H3 by short-hairpin RNAs in CRC cells reduced the tumor forming ability in vivo.Conclusion: Knockdown the expression of costimulatory molecule B7-H3, EMT in CRC was blocked, and tumor cells’ migration and invasion ability was inhibited in vitro. At the same time, the expression of stem cell markers was also significantly reduced, and then the abilities to forming cell spheroids and resisting chemotherapy drugs were weaken in vitro. In addition, the ability to forming tumor was also significantly reduced in vivo.Part Ⅲ The co-expression and clinical significanceof B7-H3, E-cadherin and c-Met in CRCObjective: Based on the discovery of interaction between B7-H3 and proto-oncogene encoding protein c-Met, and clarifying the regulation of B7-H3 in EMT, to investigate the expression of costimulatory molecule B7-H3, epithelial marker E-cadherin and proto-oncogene encoding protein c-Met in CRC paraffin-embedded tissues, and then to analysis the correlation to pathological parameters and prognosis of patients, at last to explore the relationship between abnormal expression of B7-H3, E-cadherin, c-Met and development or prognosis in CRC.Methods: 197 cases of CRC tissues with complete pathological data were collected as well, and then B7-H3, E-cadherin and c-Met expression were detected by IHC, and the correlation was analyzed with statistic software.Results: We assessed the expression of E-cadherin in these specimens by IHC staining. In the tumor tissues, 31.5%(62/197) had membranous staining of E-cadherin, which was considered as normal, and 68.5%(135/197) had aberrant E-cadherin expression with cytoplasmic staining or negative staining. The IHC staining also showed that abnormal c-Met expression was found in 71.1% of tissues, and no c-Met expression was found in 28.9% of tissues. Furthermore, we found that there was low expression of membrane E-cadherin in B7-H3 high expressed specimens. In contrast, there was high expression of membrane E-cadherin in B7-H3 low expressed specimens. And the overall survival and disease-free survival time of patients who expressed high degree of B7-H3 and cytoplasm E-cadherin were significantly decreased. In addition, the statistical analysis revealed a strong positive correlation between B7-H3 expression and c-Met expression.Conclusion: Costimulatory molecule B7-H3, cytoplasm E-cadherin and c-Met were abnormally expressed in CRC tissues. The analysis revealed a negative correlation between B7-H3 expression and E-cadherin membranous expression, correlating to CRC patients’ prognosis. And there also was a strong positive correlation between B7-H3 expression and c-Met expression. In a word, these results suggested that the combination of B7-H3 and c-Met was involved in the regulation of EMT in CRC.Part Ⅳ The mechanism of B7-H3 in regulatingEMT in CRC cells by c-Met signalingObjective: To investigate the mechanism of interaction between B7-H3 and c-Met in regulating EMT in CRC cell lines, and then to provide theoretical basis for B7-H3 to promote the progression and metastasis of human colorectal cancer.Mothods: After over-expression of B7-H3 by lentivirus in SW480, Co-immunoprecipitation and Western Blot were performed to observe the physical binding of B7-H3 and c-Met in CRC cells. And then double immunofluorescent staining were used to reveal co-localization of c-Met and B7-H3 proteins in SW480/LVCD276 cells. After co-transfection of RKO cells with lentivirus-mediated overexpression of B7-H3 and Si RNA- mediated knockdown of c-Met, migration and wound healing assay were used to analyze the effect of B7-H3/c-Met complex on the invasion ability of tumor cells, stem gene detection and cell spheroids formation were applied to explore the effect of B7-H3/c-Met complex on the maintenance of stemness. Furthmore, Western Blot were performed to observe the changes of p-Met and c-Met after addition of human recombinant B7-H3 protein. In addition, The phosphorylation expression of p Akt which was a crucial downstream signaling molecule of B7-H3/c-Met was observed by Western Blot in HCT116 treated with Si RNA, and the expression of EMT TF Snail was also detected.Results: We verified that B7-H3 could bind to c-Met by IP and Western Blot in SW480/LVCD276 cells. And by immunofluorescence double-labeling experiments confirmed the existence of two co-localized phenomen. Compared with control group, the expression of stem cells makers CD133 and the abilities of migration, wound healing and cell spheroids forming significantly enhanced in B7-H3 over-expressed group. However, tumor cells in B7-H3 over-expressed and c-Met silenced group showed lower expression of CD133 and weaker abilities of migration, wound healing and cell spheroids forming than the B7-H3 over-expressed group. Furthermore, exogenous addition of human recombinant protein B7-H3 could up-regulate the expression level of p-Met. In addition, blocking B7-H3 by Si RNA in CRC cells significantly reduced the expression of p Akt, p Erk and Snail.Conclusion: The costimulatory molecule B7-H3 could be combined with c-Met, and then affected the EMT of CRC cells by affecting the expression of p-Met, and then through the downstream signal axis Akt/Snail.In summary, we verified that the costimulatory molecule B7-H3 were closely related to the clinical pathology and progression of CRC through the clinical specimen analysis, in vivo and in vitro experiments. And then we found that B7-H3 could accelerate the mesenchymal transition of CRC cells and promote the progression and metastasis of tumor. So the molecule B7-H3 is expected to become a valuable biomarker for monitoring metastasis of colorectal cancer. More important, we have found the interaction between B7-H3 and proto-oncogene encoding protein c-Met in CRC cells for the first time. And then it would promote c-met signaling and corresponding biological function. In general, the achievement of this program will improve the regulation mechanism research of EMT and provide the new target for clinical tumor immunotherapy. |
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