| Backgrounds:Colorectal cancer is one of the commonest malignant tumors of the digestive system, it has the second highest incidence rate from cancer of all types in the United States and other Western countries, and there was an increasing trend in developing countries. With the development of the economy and the change of people’s lifestyle, diet and habits, the the incidence of colorectal cancer in the world showed an increasing trend and the age of onset has been getting younger in recent two decades. In 2012, statistics of China Cancer Registry Annual Report showing that the incidence of colorectal cancer is in the third place and mortality rates is fifth in all incidence of tumor in 2009 in China, moreover, the incidence in Beijing, Shanghai and other big cities havs been close to or even higher than developed countries. Colorectal cancer has become a serious threat to people’s health, and brought heavy financial burden to society and family.The etiology and mechanisms of colorectal cancer is connected with environmental, genetics, diseases and other adverse factors, it is also involved with the activation of oncogenes and inactivation of tumor suppressor. Some early colorectal cancer patients can be discovered by early screening technology, survival rate of patients also increased and mortality reduced. Furthermore, a variety of new chemotherapy drugs and molecular targeted drugs are available continually to improve the clinical efficacy, however, advanced colorectal cancer patients still accounted for a higher ratio, poor curative effect,5-year survival rate is still low. Therefore, it has important clinical value and social benefits to find new markers for early diagnosis and new therapeutic targets, so that it can improve the efficacy and prognosis.Silent information regulator 2 (Sir2) is an anti-aging gene that was originally discovered in budding yeast, which encodes a small protein sirt2 with NAD+ dependent histone deacetylase activity. SIRT1 is a homology analogy of sirt2 in mammals, which involved in cell energy metabolism, proliferation, senescence, multiple inflammatory processes, neuroprotection, tumorigenesis and so on, by deacetylating multiple transcription factors.The implementation of SIRTl functionality is mainly by deacetylation of the key protein that regulate gene expression. SIRT1 can deacetylate specific lysine residue of many histone in yeast and mammals, such as H1 (K26), H3 (K9, K14), H4 (K16), etc., and some studies found that H4 (K16) is in the deacetylation state in some cancers, SIRT1 can also make some non-histone deacetylation. P53 was first discovered, the classical tumor suppressor protein P53 is a clear downstream protein of sirtl. SIRT1 can regulate the activities and expression of P53 by deacetylation, so that it participate in the P53-mediated cell proliferation and apoptosis. Some research has indicated P53 mutated in over 50% human tumors, mutational P53 in tumor suppressor genes change into tumor promoters.may plays an important role in the process of P53 mutation. Some other non-histone proteins can also be deacetylated by SIRT1, such as HIF-1α, HIF-2α,β-catenin, etc.. It has been reported that SIRT1 can inhibit the proliferation of multiple adenomatous polyps of colon by P-catenin deacetylation, SIRT1 may affect tumor development by regulating the activities of these proteins.There is still controversy about SIRT1 in tumor research, however less in the field of colorectal cancer research. SIRT1 plays a role in tumor promoters or tumor suppressor in colorectal cancer? SIRT1 is localized in the nucleus or cytoplasm in colorectal cancer? If the subcellular localization of SIRT1 is related to nuclear cytoplasm shuttling in colorectal cancer? Currently, these controversial studies about specific mechanisms of SIRT1 is not clear, it is necessary to discuss in depth.In addition, it has reported that phosphorylation can regulate deacetylation and nuclear cytoplasm shuttling of SIRT1.However, the researchs on phosphorylation in colorectal cancer are rarely reported, so that in the first part of this study we focused on the Phospho-SIRT1 in colorectal cancer and its subcellular localization, in order to reveal the role of SIRT1 phosphorylation in tumor formation.The expression and correlation of SIRT1, P53 and P-catenin in colorectal cancer and their relationship to clinicopathological features will be discussed and analyzed in the second part of this study.PART1The study on SIRT1 subcellular localization and its phosphorylation in colorectal cancerObjectives:1. To investigate the expression and subcellular localization of SIRT1 and Phospho-SIRT1 in colorectal cancer tissues and normal tissues.2. To investigate the expression of SIRT1 mRNA in colorectal cancer tissues and corresponding normal tissues.3. To investigate the possible correlations between SIRT1 or Phospho-SIRT1 expression.4. To investigate the possible correlations between Phospho-SIRT1 and SIRT1.5. To explore the role of phosphorylation in colorectal cancer.Methods:1. Immunohistochemical staining and Western blot were performed to detect the expressions and subcellular localization of SIRT1 and Phospho-SIRT1 in colorectal cancer tissues and corresponding normal tissues.2. Real-Time PCR was performed to detect the expression of SIRT1 mRNA in colorectal cancer tissues and corresponding normal tissues.Results:1. SIRT1 was completely localized in the nucleus and was highly increased in colorectal cancer tissues.2. Phospho-SIRT1 was completely localized in the nucleus and was highly increased in colorectal cancer tissues..3. Phospho-SIRT1 and SIRT1 expression in colorectal cancer was positively correlated. The ratio of Phospho-SIRT1 and SIRT1 was higher in cancer tissues than in normal tissues.4. SIRT1 mRNA was no significant difference in colorectal cancer tissues and normal tissues.Conclusions:1. SIRT1 and Phospho-SIRT1 was overexpressed in colorectal cancer tissues, indicating that both of them may be involved in tumor formation.2. Phospho-SIRT1 and SIRT1 expression in colorectal cancer was positively correlated. The ratio of Phospho-SIRT1 and SIRT1 was higher in cancer tissues, suggesting Phospho-SIRT1 may determine the role of SIRT1 in colorectal cancer.3. SIRT1 and Phospho-SIRT1 are both located in the nucleus, without cytoplasm localization. This research cannot demonstrate the nucleocytoplasmic shuttling of SIRT1.4. Changes in SIRT1 protein levels are not associated with changes in SIRT1 mRNA transcriptional levels, suggesting post-transcription may play an important role in SIRT1 regulation.PART2The expressions of SIRT1 P53 and p-catenin in colorectal cancer and their association with clinicopathological characteristics.Objective:Through the detect of the expression level of Silencing Information Regulator Type 1 (SIRT1) in colorectal cancer tissue and its adjacent normal mucosa, and the expressive correlation of SIRT1, P53 and P-catenin in colorectal cancer tissue, to explore the role of SIRT1 in the occurance and development of colorectal cancerMethod:Immunohistochemistry were performed to investigate the expressions of SIRT1、 P53 and β-catenin in 76 colorectal cancer tissues and expressions of SIRT1 in 30 adjacent normal tissues. The relationship between SIRT1 P53 and β-catenin and clini-copathological characteristics of colorectal cancer was analyzed.Results:1. The expression of SIRT1 localized in the nucleus both in colorectal cancer tissues and adjacent normal tissues, there was no expression in cytoplasm.2. SIRT1 expression in cancer tissues and normal tissues were 64.47%(49/76) and 36.67%(11/30) respectively. The average score of SIRT1 in cancer tissues and normal tissues were 4.667±0.374 and 2.650±0.425, respectively. The expressions of SIRT1 and Phospho-SIRT1 were significantly higher in colorectal cancer tissues than normal tissues (P<0.05).3. SIRT1 expression in cancer tissues was associated with tumor invasion depth, degree of differentiation, Lymph node metastasis and TNM stage. And the expression of SIRT1 was increasing with the deepen of tumor invasive, poorer of differentiation, combining with Lymph node metastasis and the rise of clinical grade. The overexpression of P53 was associated with tumor invasion depth Lymph node metastasis and TNM stage. The overexpression of β-catenin was involved with tumor invasion depth, degree of differentiation, Lymph node metastasis and TNM stage4. The expression of SIRT1 was positive correlation with expression of P53 (r=0.258,p<0.05), there was no association between expression of SIRT1 and expression of β-catenin (r=0.213, P>0.05).Conclusions:1. We found that SIRT1 was over-expressed of in colorectal cancer tissue, so it can be speculated that SIRT1 promotes the formation of colorectal cancer.2. The correlation between SIRT1, P53,β-catenin and the clinicopathological features of colorectal cancer suggests that the combined detection of the expression of SIRT1, P53 and β-catenin has a certain significance in the prognosis of patients.3. There is a positive correlation between the positive expression of SIRT1 and the expression of P53 in colorectal cancer tissue (R=0.287, P=0.009), therefore it is presumed that SIRT1 may be a promoting gene of colorectal cancer. The mechanism may lies in that the deacetylation of P53 could be used to regulate its activity and expression, which inhibits the apoptosis of cells mediated by P53 and participates in the progress of colorectal cancer.4. There is no correlation between the positive expression of SIRT1 and β-catenin in colorectal cancer tissue (R=0.213, P=0.058), which dose not suggests its role as an anti-oncogene through the deacetylation of β-catenin. This result may be related to the different effects caused by the different positions of β-catenin nucleus and cytoplasm. Further study on the coorelation between the subcellular localization of β-catenin and SIRT1 could be helpful to reveal their relationship. |
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