| Background and objective:Colorectal cancer (CRC) is the third most prevalent cancer in the world, accounting for approximately1.2million new cases and more than600,000cancer-related deaths each year worldwide. In China, CRC ranks fourth in cancer mortality, and in recent years, the morbidity and mortality of CRC in China shows an obvious upward trend. The occurrence and development of CRC is a complex process involving multi-gene, multi-step, and multi-stage. More than80%of CRC begins with adenomatous polyp, further advances to adenoma with high grade dysplasia and finally progresses to an invasive cancer. This process mainly involves three types of genetic changes in oncogenes, tumor suppressor genes, and mismatch repair genes. The defect of tumor suppressor genes plays a key role in the development of CRC, which could cause cells to grow out of control and tumor progression. Therefore, it is necessary to find the tumor suppressor genes involved in the development of CRC, to explore their roles in tumor development, and to further clarify the pathogenesis at the molecular level in the etiology study, which could provide an important theoretical basis for early detection, diagnosis, and treatment of CRC.In previous study, protein dynamic expression patterns in different stages of CRC were established using two-dimensional gel electrophoresis. The results showed that SDH exhibited potential tumor suppressor function in the pathogenesis of CRC. SDH (also known as mitochondrial complex Ⅱ) is a heterotetrameric protein consists of A, B, C and D subunits encoded by corresponding nuclear genes, localized on the inner mitochondrial membrane which catalyse the oxidative dehydrogenation of succinate and, as part of complex Ⅱ of the electron transport chain, couple this to the reduction of ubiquinone (succinate-ubiquinone oxidoreductase). As a tumor suppressor gene, genetic defects or abnormal expression in SDH are associated with a diverse collection of diseases, including paragangliomas (PGL), pheochromocytoma, renal cancer, gastrointestinal stromal cell tumor, and Leigh syndrome. Initially, the SDH expression was detected by immunohistochemistry using tissue microarray (TMA) sections which containing103cases of CRC tissue specimens. Expression of SDHB protein was found to be significantly lower in CRC tissues compared to the normal mucosa. And the expression of SDHB was negatively correlated with the differentiation status of CRC tissues.The role of SDH in CRC development has not been reported previously. Based on preliminary work, we detect SDHB expression in poorly differentiated CRC tissues with expanded sample to further confirm the correlation between SDHB expression and differentiation status of CRC in the present study. In order to further investigate the potential SDH tumor suppressor functions in CRC, we stably expressed SDHB protein in human colon cancer cells to determine the effects of SDHB expression on tumor cell growth and tumorigenesis. And differential expression of genes induced by ectopic SDHB expression in cancer cells was screened by microarray analysis. Moreover, we investigated the mechanism for downregulated expression of SDHB in CRC in genetic and epigenetic aspects, such as gene mutations and DNA methylation.Methods:In this study, we examined SDHB protein expression in a TMA containing32cases of poorly differentiated colon cancer tissues by immunohistochemistry. Then SDHB was transfected into colon cancer cell line (SW620) by lipofectin and a stable cell line of pIRES-SDHB overexpressing SDHB gene was established. The effect of SDHB on malignant behavior of SW620was assessed by growth curves of cells, colony formation, and tumor formation in nude mice. The effect of SDHB on cell cycle progression in SW620was investigated by flow cytometry.The expression of cell cycle related proteins cyclin D1and PCNA was detected by western blot and immunocytochemistry. In order to identify genes modulated by SDHB in CRC, the expression profile of SDHB expressing colon cancer cells and controls were investigated. Moreover, genomic DNA was extracted in forty CRC and paired normal mucosa tissues. On the one hand, SDHB gene exons were amplified by PCR and all products were purified and sequenced from both directions to detect gene mutations. On the other hand, genomic DNA was treated by sodium bisulfite modification and then amplified by methylation-specific PCR to assess the SDHB promoter methylation status.Results:(1) Protein expression of SDHB in CRC①Immunohistochemical results showed that expression of SDHB protein was found to be significantly lower in CRC tissues compared to the normal mucosa. And the expression of SDHB in poorly differentiated CRC was significantly lower compared to that in moderately and well differentiated CRC.②Western blot showed the same results which were consistent with that of immunohistochemistry.(2) Effects of SDHB expression on tumor cell growth and tumorigenesis①Cell growth curves showed the restoration of SDHB significantly inhibited the growth of SW620cells.②Colony formation showed the ectopic expression of SDHB substantially inhibited tumor cell colony formation.③Xenograft tumor formation assay showed that SDHB inhibited tumorigenicity in SW620cells.④Flow cytometry results of the cell cycle distribution showed that SDHB induced cell cycle arrest at the G1phase.⑤Western blot and immunocytochemistry showed that SDHB downregulated expression of cyclin D1and PCNA.(3) Effects of SDHB expression on the gene expression profile of colon cancer cells①cDNA microarray analysis showed total of1,653differentially expressed genes with more than a twofold change (p<0.01), of which1,382gene expressions were upregulated and271gene expressions were downregulated in SW620-SDHB cells compared with vector control cells.②The differentially expressed genes were validated by real-time RT-PCR.③Most of the differentially expressed genes were grouped in GO with regard to "cell cycle","cellular process","transcriptional regulation" or "cell signaling".④The KEGG database analysis showed that the differentially expressed genes were mainly involved in the p53, MAPK, Toll-like receptors, ErbB, Jak-STAT, T cell receptor, Notch, Wnt, TGF-β, VEGF, cytokines interaction signal transduction pathway.(4) Mechanism for downregulated expression of SDHB in CRC①Forty cases of CRC and paired normal mucosa tissues contained a single nueleotide polymorphism in SDHB exonl which located in the18th base pair, C substituted by A, making the6th codon changed from GCC to GCA. However, both GCC and GCA are encode for alamine. And no pathogenic mutation was found. The results suggested that the exon mutation in SDHB gene may not be the cause of CRC.②Methylation-specific PCR failed to detect SDHB promoter methylation in all these CRC and paired normal mucosa tissues, which suggested that the mechanism for downregulated expression of SDHB in CRC may not be caused by promoter methylation.Conclusion:(1) Expression of SDHB protein was significantly lower in CRC tissues compared to the normal mucosa, and its expression was negatively correlated with the differentiation status of CRC tissues.(2) Restoration of SDHB could inhibit tumor cell growth, colony formation, tumorigenicity, and induce cell cycle arrest at the G1phase.(3) The mechanism for downregulated expression of SDHB in CRC may not be due to exon mutation or promoter methylation. |
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