Study On The Expression And Promotive Role Of Gene HCLOCK In Human Colorectal Carcinoma

Circadian gene is a system that sets and regulates circadian rhythm in organisms, as an oscillation with an approximate24-hour cycle. Gene hCLOCK acts as the core of the circadian gene family. The changes of hCLOCK gene’s expression and function result the body’s circadian rhythm disorder, which promote tumori genes is. Colorectal carcinoma is one of the most common malignancy affecting human health worldwide. Although big progress has been made in surgical resection, chemotherapy and radiotherapy, colorectal cancer mortality remains high. Looking for much more new key genes or specific molecular markers, which affect the progression of colorectal tumorigenesis, and explore their function, will be very significant for the diagnosis, prognostic monitoring, as well as the invention of new anticancer drugs of colorectal cancer. This study will be divided into four parts to explore the expression and promotive role of gene hCLOCK in colorectal cancer in vitro and in vivo. After that, we want provide some theoretical basises and hope hCLOCK gene could be a new target for the treatment of colorectal cancer.Part I The expression of hCLOCK and some of hCLOCK controlled genes in human colorectal cancerObjective1. Analysis the expression of hCLOCK in human colorectal cancers and matched normal tissues; then analysis its relationship with the pathology and clinical features of the patients;2. Detection the expression of hCLOCK controlled genes, including (1) circadian gene hPER2;(2) tumor angiogenesis-related genes (HIF-1a, ARNT, VEGF);(3) apoptosis-related genes (BAK, BAX, BID, TNFR I, TNFR II), and analysis with hCLOCK expression changes.Methods1. Analysis the expressions of protein hCLOCK and hPER2in cancers and paired non-cancerous tissues by immunohistochemistry; 2. Analysis the expressions of hCLOCK and some of hCLOCK controlled genes (HIF-1α、 ARNT、VEGF、BAK、BAX、BID.TNFR I.TNFR II) mRNA in cancers and paired non-cancerous tissues by real-time quantitative reverse transcription-polymerase chain reaction. Results1. Protein hCLOCK and hPER2were expressed in both cancerous and noncancerous colorectal tissues. Higher expressive level of protein hCLOCK while lower expression of protein hPER2were found in human colorectal carcinoma than paired non-cancerous tissues (P<0.01);2. Higher expression of hCLOCK protein in colorectal cancer is related to lymph node metastasis, and TNM stage. In the occurrence of metastasis of lymph nodes and vascular, higher expression of hCLOCK can always be found (P<0.05);3. Higher expression of hC10CK is related to lower expression of hPER2in colorectal cancer. The younger the patient is, the lower degree of differentiation, deeper depth of invasion, incidence of lymph node metastasis, late stage of TNM, all these facts induce the lower expression of hPER2;4. Higher expression of hCLOCK mRNA was found in human colorectal carcinoma than paired non-cancerous tissues (P<0.01), while no difference was found in expression of hPER2mRNA;5. Gene hCLOCK was significantly positively linear correlated with gene ARNT, HIF-1a and VEGF in human colorectal carcinoma (P<0.05), but not with BAK、BAX、BID、TNFRI and TNFR II. Conclusions1. The expression of circadian gene hCLOCK is higher in human colorectal carcinoma, and related with tumor progression, lymph nodes and vascular metastasis;2. The expression of circadian gene hPER2is deregulated in colorectal cancer, and related with patient’sage, tumor differentiation, depth of invasion, incidence of lymph node metastasis, and stage of TNM;3. Higher expression of hCLOCK is related to lower expression of hPER2in tumorous tissue; 4. Expression level of circadian gene hCLOCK was positively correlated with gene HIF-1α, ARNT and VEGF in human colorectal carcinoma.Part Ⅱ The expression of hCLOCK in colorectal carcinoma cell lines Objective1. Analysis the expression of hCLOCK in human colorectal carcinoma cell lines:HT29, LoVo, SW480and SW620;2. Analysis the expression of hCLOCK with different ability of cell activity, invasion, and metastasis;3. Choose one cell line which has high expression level of hCLOCK, choose another cell line which has low expression level of hCLOCK.Methods1. Analysis the expression of protein by immunohistochemistry;2. Analysis the expressions of mRNA by real-time quantitative reverse transcription-polymerase chain reaction.Results1. The expression of hCLOCK protein and mRNA is highest in cell SW620, which has high metastasis ability;2. The expression of hCLOCK is low in cell SW480, which has low metastasis ability. Conclusions1. The higher expression of hCLOCK is related with higher degree of tumor cells’ malignancy and metastasis ability;2. We choose SW620, which has high expression level of hCLOCK, and SW480, which has low expression level of hCLOCK, for further research.Part Ⅲ Construction and packaging of hCLOCK-lentivirus and hCLOCK-RNAi-lentivirusObjective1. Construct and package hCLOCK-lentivirus, which can express hCLOCK gene stably; 2. Construct and package hCLOCK-RNAi-lentivirus, which can interfere the expression of hCLOCK gene stably.Methods1. Construction of recombinant lentiviral vector plasmids;2. Package the lentivirus.Results1. Get hCLOCK/GV186lentiviral concentrate successfully, which can express gene hCLOCK stably;2. Get hCLOCK/GV113-RNAi lentiviral concentrate successfully, which can interfere the expression of gene hCLOCK stably.Part IV Study on the role and mechanism of hCLOCK in promoting colorectal carcinoma cells proliferation, invasion and metastasisObjective1. Obtain SW480-hCLOCK cells, which can express hCLOCK stably, and use SW480-Vec cells, which were infected with valid plasmid virus, as controlled group;2. Obtain SW620-shhCLOCK cells, which can interfere the expression of hCLOCK stably, and use SW620-Vec, which were infected with valid plasmid virus, as controlled group;3. Study on the inference of hCLOCK on the cell proliferation, invasion and metastasis;4. Analysis the expression of tumor angiogenesis related genes (VEGF. ARNT. HIF-1α), and apoptosis related genes (AKT\P-AKT. BID. BAX), to explore the mechanism of hCLOCK in promoting colorectal carcinoma progression and metastasis.Methods1. Infect cell SW480with hCLOCK/GV186lentiviral concentrate; test and verify the higher expression of hCLOCK in SW480-hCL0CK than SW480-Vec with fluorescence detection and Western Blot;2. Infect cell SW620with hCLOCK/GV113-RNAi lentiviral concentrate; test and verify the lower expression of hCLOCK in SW620-shhCLOCK than SW620-Vec is more than70%with fluorescence detection and Western Blot.Results1. Cell proliferation is higher in SW480-hCLOCK than SW480-Vec, and lower in SW620-shhCLOCK than SW620-Vec (P<0.01);2. Cell apoptosis is lower in SW480-hCLOCK than SW480-Vec, and higher in SW620-shhCLOCK than SW620-Vec (P<0.05);3. Cell invasion is stronger in SW480-hCLOCK than SW480-Vec, and weaker in SW620-shhCLOCK than SW620-Vec (P<0.01);4. Higher expression of VEGF、HIF-1a、ARNT protein was found in SW480-hCLOCK than SW480-Vec (P<0.01), while lower expression of them was found in SW620-shhCLOCK than SW620-Vec;5. Lower expression of apoptosis promotive genes BAX、BID protein was found in SW480-hCLOCK than SW480-Vec (P<0.05), while the expression of anti-apoptotic gene P-AKT protein was found increased in SW480-hCLOCK than SW480-Vec, and decreased in SW620-shhCLOCK than SW620-Vec (P<0.01)Conclusions1. hCLOCK inhibits colorectal carcinoma cells apoptosis, but promote the cancerous cells proliferation, invasion and metastasis;2. hCLOCK promotes the expression and activity of gene VEGF、HIF-1a and ARNT in colorectal cancer, through which it plays an positive role in tumor neovascularization, and promotes the progression such as infiltration, invasion and metastasis of colorectal carcinoma;3. hCLOCK promotes the expression and activity of anti-apoptosis gene P-AKT, and inhibits the expression and activity of apoptosis promotive genes BAX、BID, by which way it can inhibit cancerous cells apoptosis.