The Role And Mechanism Of SEMA3F And Its Receptor NRP2 In The Progression Of Colorectal Carcinoma

IntroductionColorectal cancer (CRC) is one of the most common malignant tumors in human alimentary tract, which has very high morbidity with modest mortality among common cancers worldwide. Despite of substantial improvement in the treatment of this tumor, the outcome of CRC after regular therapy still remains poor. Accumulating evidence suggest that the initiation and progression of CRC is a complex process of which multiple genetic aberrations and other factors are involved. Metastasis remains the most important adverse prognostic factor for the patients with CRC. Thus, identification of more specific gene(s) related to metastasis and prognostic markers is of prime importance so that the mechanisms underlying tumor progression can be revealed, and novel and effective therapeutic strategies to treat CRC can be explored.Axon guidance molecule Semaphorin 3F (SEMA3F) and its receptor Neuropilin 2 (NRP2) are initially identified in repulsing superior cervical ganglion and regulating development of the nervous system. However, it has become apparent that SEMA3F and NRP2 express in a wide variety of human tumors including colon, breast, ovary, and lung cancers, implicating that they may play important roles in tumor biology. Growing evidence indicates that NRP2, as an obligate co-receptor, is involved in other signal pathways independent of SEMA3F, which results in totally different biological effects. The molecular crosstalk between SEMA3F and NRP2, and the clinical significance are of considerable interest. I hypothesize that the ratio of SEMA3F and NRP2 expression levels may be an important determinant of tumor development. However, absent as the studies on large samples of human malignant tumors are, the expressions of SEMA3F and NRP2 in CRC and complicated relationship of SEMA3F/NRP2 pathway as well as pathologic significance, especially their values in predicting prognosis, remain to be elucidated. Furthermore, the roles and underlying molecular mechanism of SEMA3F on CRC require further clarification.Therefore, in this study, the first section was focused on the investigation of the expressions of both SEMA3F and NRP2 on a large sample of surgical specimens of human CRC for revealing pathologic significance. In the second section, I specifically silenced SEMA3F expression in colon cancer cells through lentivirus interference, and I attempted to explore the molecular mechanism of SEMA3F underlying CRC by analyzing the different gene expression profiling based on DNA microarray.The experiment data and main conclusions are reported as following:1.NRP2 expression exhibits a double-humped pattern in the tumorigenesis and progression of CRCIn comparison to its lower expression in the epithelium of normal colorectal tissues, immunohistochemistry analysis of tumor specimens showed that NRP2 expression was found higher in the cytoplasm of tumor cells with a corresponding increase in the tissues adjacent to tumors. NRP2 expression in the tissue adjacent to tumors was correlated to the dysplasia, which gradually elevated from the lowest expression in normal epithelium, to modest one in mild dysplasia and moderate dysplasia, to the highest one in sever dysplasia. Additionally, the expression of NRP2 in the adenomas nearby tumors exhibited similar pattern as found in dysplasia. At early Dukes'stages, NRP2 was down-regulated in the tumors with a lower level expression than that in the adjacent mucosa. At advanced Dukes'stages, NRP2 expression in the tumors was significantly up-regulated, which was close to or even stronger than that in their adjacent tissues. NRP2 expression was positively associated with Dukes'stage, lymph node metastasis and distant metastasis as well as recurrence,but was negatively correlated to differentiation grade. NRP2 expression enhanced significantly in the paired lymph node metastases compared with that in primary CRCs. These results implicate that up-regulation of NRP2 occurs in the prophase of carcinomatous change of colon. Thus, NRP2 expression may be correlated to the tumorigenesis, differentiation, invasion and metastasis of CRC.2.SEMA3F expression is up-regulated in the tumorigenesis but down-regulated in the progression of CRCSEMA3F was found in the cytoplasm of both normal epithelium and tumor cells. Similar to NRP2 expression, SEMA3F expression was lower in the normal colorectal tissues than that in the tissues adjacent to the tumors. The levels of SEMA3F expression elevated from mild dysplasia, to moderate dysplasia, and reached to plateau in severe dysplasia. Similar expression pattern was found in adenomas. Expression of SEMA3F was attenuated sharply in the carcinoma tissues. SEMA3F in tumor tissues and their adjacent tissues was found significantly decreased at both limited Dukes'stage and advanced Dukes'stage. Furthermore, SEMA3F expression was negatively correlated to lymph node metastasis, advanced Dukes'stage and recurrence of CRC. SEMA3F expressions were found significantly changed in the paired lymph node metastases compared with that in primary CRCs. These results suggest that up-regulation of SEMA3F expression may be an early event in human colorectal carcinogenesis. Down-regulation of SEMA3F is closely correlated to CRC progression, which may promotes lymph node metastasis and recurrence of CRC.3. Expressions of SEMA3F and NRP2 are correlated to lymphangiogenesis of CRCCancer lymphangiogenesis was heterogeneous not only in distribution and morphology but also in the quantity of lymphovasculature. The microlymphatic densities (MLDs) of both tumor tissues and their adjacent tissues were positively correlated to metastasis. SEMA3F expression was negatively correlated to tumorous MLD. On the contrary, NRP2 expression was positively correlated to tumorous MLD.4. Expressions of SEMA3F and NRP2 have an impact on the prognosis of CRCA clear survival advantage was identified from the patients with negative NRP2 expression as compared to those with positive NRP2. Patients with a high ratio of NRP2 expressions in tumor tissues to adjacent tissues (T/A,≥2) were poorer in survival than those with a low ratio (T/A<2). Patients with positive SEMA3F expression had longer survival time than the patients with negative SEMA3F expression. The poorest prognosis was found in the patients with N>S, while the patients with N≤S showed the best prognosis and the patients with negative expression of both NRP2 and SEMA3F had a moderate survival. Multivariate Cox analysis identified that metastasis, recurrence, SEMA3F expression and N/S were independent prognosticators of CRC.5. Down-regulation of SEMA3F regulated expression of multiple genesFollowing SEMA3F gene silenced by RNAi, DNA array analysis showed that in shSEMA3F group, 709 differentially expressed genes (DEGs) were found up-regulated whereas 759 DEGs were down-regulated. The Gene Ontology (GO) comparison results demonstrated that the total 1468 genes could be grouped into 63 GO categories. Among these 63 GO categories, the GOs in biological process (BP) were primarily concentrated on cell proliferation, cell motion, actin filament-based process, and regulation of developmental process, while the GOs in cellular component (CC) were significantly concentrated on cell leading edge. These results implicate that SEMA3F gene is generally involved in the regulation of cellular process to cast several significant biologic effects.6. SEMA3F may be involved in the changes of multiple signaling pathways An analysis of KEGG pathways showed that SEMA3F was involved in the multiple pathways related to cancer, cell cycle, and regulation of actin cytoskeleton, and etc. Thus, the changes of pathways on cell proliferation, cell motility and metastasis should be a part of the molecular mechanism of which SEMA3F inhibits the tumorigenesis and development of CRC.In conclusion, the above data demonstrate that1.The up-regulation of NRP2 and SEMA3F may be an early event in the tumorigenesis of CRC, implicating that they might be involved in tumorigenesis;2.The up-regulation NRP2 is positively correlated to the malignant features including tumor differentiation, metastasis and recurrence, as well as lymphangiogenesis, indicating that NRP2 may act as a pro-metastatic factor for the clinical CRC progression.3.The expression of SEMA3F in CRC is negatively related to lymph node metastasis, recurrence and lymphangiogenesis. The changes of pathways on cell proliferation, cell motility and metastasis should be a part of the molecular mechanism of which SEMA3F inhibits the tumorigenesis and development of CRC, suggesting that SEMA3F might act as a tumor suppressor in CRC to play an anti-tumor role.4. SEMA3F, T/A ratio of NRP2 and the balance of NRP2 and SEMA3F may be the key markers for CRC prognosis.