Proteomic Analysis Of RNA Interference HIF1-α Gene In Colon Cancer Cell

Colorectal cancer is one of the most common gastrointestinal tumors. According to statistics, new cases of colorectal cancer increased 3.9 percent annually and mortality rates of colorectal cancer added 4.0 percent annually in worldwide. Colorectal cancer is ranked as the fourth most frequently diagnosed cancer and the second leading cause of cancer death in the United States. In 2009, an estimated 106,100 new cases of colon cancer and approximately 40,870 new cases of rectal cancer occurred in the United States. During the same year, it is estimated that 49,920 people will have died from rectal and colon cancer. In China, colorectal cancer is ranked as the third most frequently diagnosed cancer and the fourth leading cause of cancer death, the number of incidence rate was twice the world average. Faced with this situation, it must be do more intensive studies on the incidence and development of colorectal cancer.With the penetrating of cancer research, many methods are confirmed that intermittent hypoxia is a common feature in most solid tumors such as colon cancer, rectal cancer. The adaptive response to hypoxia is adjusted by a family of transcription factors, the most important member in this family is hypoxia-inducible factor 1-α. The transcriptions of many genes that are critical for cellular function under hypoxic conditions are activated by HIF1-α. HIF1-αis considered as a master regulator favoring tumor cell survival, promotion invasion and metastasis and also resistance to chemotherapy and radiotherapy. Many scholars believe that HIF1-αis an ideal target for cancer therapy. However, it has been reported that more than 2% of all human genes are regulated by HIF1-αdirectly or indirectly. Therefore, it is very important to reach a comprehensive understanding of HIF1-αin cancer biology in intermittent hypoxic environment and its mechanism.The characteristics, quantity and function of proteins could be comprehensive analyzed by proteomics. Through the expression of different proteins, we could understand the role of specific factors on the tumor and its mechanism. Because of specific and high efficiency of RNA interference to silence target gene, it be also widely used in cancer research. In this study, we first detected the expression of HIF1-αin colorectal cancer and its effect in colorectal cancer cell SW480; Second, established electrophoretic technique, combined RNA interference and proteomics to identified proteins with differential expressions, in order to find proteins interacting with HIF1-α; At last, the expression of interested protein was detected in colorectal cancer tissues. PARTⅠThe Expression of HIF1-αin Colorectal Canerand its Effect in Colon Cancer CellObjective: To observe the expression of HIF1-αin colorectal neoplasm tissues and under intermittent hypoxic conditions. Investigate its effect in colon cancer cell SW480.Methods: We detected the expression of HIF1-αby immunohistochemistry in colorectal cancer tissues. Colon adenocarcinoma SW480 cells were cultured under normoxia and intermittent hypoxic conditions,the expression of HIF1-αwas detected by immunofluorescence, RT-PCR and Western blot. We also silenced HIF1-αby RNA interference in order to detect the change of cell morphology and cell apoptosis by electron microscope and flow cytometry. In vivo xenograft tumor model, the effect of HIF1-αon cancer development was evaluated.Results: The expression of HIF1-αprotein was 71.88% in colon cancer tissue samples. In colon cancer cell SW480, the expression of HIF1-αmRNA and protein was significantly increased under intermittent hypoxic condition than in normoxia conditions. The inhibition of HIF1-αby RNA interference led to the apoptosis of colon cancer cell SW480. Silenced HIF1-αcould inhibit tumor growth in vivo xenograft tumor model.Conclusion: In the colorectal carcinoma, HIF1-αprotein expressed at a high level. In colon cancer cell SW480, the expression of HIF1-αwas significantly increased under intermittent hypoxic condition. It played an important role in cell apoptosis and tumor growth. PARTⅡProtein Sample Preparation, 2-DE and Image Analysis for ProteomeObjective: To investigate the protein sample preparation and the conditions of two-dimensional electrophoresis in order to analysis the change of proteomics after HIF1-αwas silenced by RNA interference in SW480 cells.Methods: The lytic agent of test samples, the method for treat samples, quantity of sample for loading as well as condition for two-dimensional electrophoresis were optimized. The PDquest software was used to analysis the image after stained with silver nitrate. Results: Extracted protein by lytic agent containing thiourea, increased the reaction time of DNase and RNase, loaded at a quantity of by 200ug, prolonged the time for demineralization and focused for 50000Vh could obtain a good electrophoretic profile, while the number of protein spots was more than 1,200. In positive interference group, thirteen down-regulated proteins and ten up-regulated proteins were apparently different among negative interference group.Conclusion: The electrophoretic technique for RNA interfering colon cancer cell SW480 was successfully established with highly reproducible and well-resolved.PARTⅢThe Identification and verification of Differences ProteinsObjective: Before and after HIF1-αgene silenced, the differential protein spots were identified and verified, the functions of HIF1-αin colorectal cancer were analyzed.Methods: Twenty-three differential protein spots were analyzed by HPLC-CHIP-MS/MS and bioinformatics analysis. Western blot was used to check the interested proteins.Results: Nineteen proteins were identified. In positive interference group, PRDX2 and other nine proteins were significantly decreased, INPP5D and other eight proteins were significantly increased. These proteins might have a direct or indirect contact HIF1-α. They involved in cell division, proliferation, apoptosis, cell transformation, cell migration, energy metabolism, signal transduction and hypoxia adaptation. The expression of PLD2, ANXA2 and PRDX2 protein was detected by Western blot, the result was consistent with proteomic analysis.Conclusion: HIF1-αcould regulate a variety of cell factors then involved in tumorigenesis and development.PARTⅣThe expression of peroxiredoxins in colorectal neoplasms and the mechanism of PRDX2Objective: PRDX expressions were investigated in histological samples of colorectal neoplasm and the normal tissues. The mechanism of PRDX2 was analyzed.Methods: The expression of PRDX in histological samples of colorectal neoplasm and the normal tissues was detected by immunohistochemistry and Western blot. In colon cancer cell SW480, interacting proteins with PRDX2 was detected by immunoprecipitation.Results: Normal colorectal tissues were almost negative for PRDX except PRDX4 (46.88%, 15/32). In colorectal cancer tissues, the most prominent reactivity was observed with PRDX2 (71.88%) in 23/32 cases, the corresponding figures for others was PRDX1 (65.63%, 21/32), PRDX3 (56.25%, 18/32), PRDX5 (56.25%, 18/32), PRDX6 (56.25%, 18/32) and PRDX4 (25.00%, 8/32). PRDX1, PRDX2, and PRDX5 were the isoforms that showing significantly increased expression in colorectal cancer patients with stageⅢor lymphnodes metastasis-positive cases. There was a significant relationship between the expression of PRDX1 and PRDX2 and between PRDX3 and PRDX4. In SW480 cells, seventeen proteins were obtained from immunoprecipitation by using specific antibodies against PRDX2; nine of them have been reported to be regulated by c-Myc gene.Conclusion: PRDX played some roles in the development and metastasis of colorectal cancer, PRDX2 might be associated with c-Myc gene regulatory pathway.