| Background and Objection:Although tremendous progress has been made about pathogenesis and gene therapy of tumor, cancer is still a major problem endangering human health. Because of the WHO’s advocating for cancer early prevention, early diagnosis and early treatment in recent years, the incidence and mortality of colorectal cancer began to show a downward trend. Even so, every year over1000000new cases were diagnosed, nearly half of the cases died. Colorectal cancer remains the third leading cause of cancer-related death. In our country one of the main characteristics of CRC patient is younger age trend. High risk groups of colorectal cancer were men over the age of40. In the treatment of colorectal cancer, the survival rate of patients are improved with traditional radical resection and constantly development of radiotherapy and chemotherapy, but the recurrence and metastasis of colorectal cancer is still the leading cause of cancer-related death.it is a serious threat to our people’s physical and mental health. Despite a large number of basic and clinical research for development and transferring mechanism of colorectal cancer were carried out by many scholars, the detailed mechanism is still not completely clear. Therefore, it is important to explore the invasion and proliferation ability and cell growth regulatory mechanism of CRC for early prevention and early diagnosis and improving survival rate at the level of gene and protein and molecular biology.RNA interference （RNAi） is found in genome research program of Caenorhabditis elegance by Fire and other scholars in1998.they firstly elaborate that dsRNA which named RNAi technology can express selective and specific inhibition of target gene. Since then, RNAi technology has been adopted and applied in the global scope. The researchers found the phenomenon of RNA interference existed both in eukaryotes and mammalian cells. RNAi is a new technology used for genetic research in recent years. In the field of cancer research about occurrence, development and metastasis, RNAi technology is used for any abnormal high expression of gene function. RNAi is a kind of specific transcription of gene silencing process induced by double-stranded RNA sequence.lt can lead to specific gene silencing and provides a powerful method for reverse genetics research to analyze gene function in vitro and in vivo. At present in the experimental study, a small interfering RNA of the nucleic acid sequence specific gene silencing molecule is the most widely used, known as siRNA, is usually made up of21-23base pairs of symmetric structures. This method can inhibit the expression of target gene specifically and effectively. Concept of gene therapy （genetherapy） is that the functional gene is transferred to correcting the defective gene of patient and enable patients to restore the original function of genes in order to achieving the purpose of clinical treatment. RNAi technology which is composed of double-stranded RNA promoter gene silencing specific sequence, by mean of cell transfection technique, can interfer series gene expression of tumor suppressor gene and proto-oncogene by importing DNA. RNAi has the following characteristics. Firstly, with a high degree of specificity, according to the principle of complementary base pairing, siRNA binding to homologous specific sequence of mRNA, leading to the degradation of homologous mRNA and silencing homologous gene expression, wheal other non homologous gene expression changes will not occur. Therefore, the feature of gene is used for therapy of disease. Secondly,another characteristics of RNAi is efficient. Several times to ten times concentration of mRNA is inhibited and degraded with small amounts of dsRNA. The reason of the phenomena is that when mRNA is degraded, siRNA can be degraded continuously. The second reason is function of RNA dependent RNA polymerase, i.e. dependent RNA polymerase in the presence of RNA, dsRNA began to copy, ultimately resulting in a large number of dsRNA leads to the degradation of mRNA. Furthermore, with a high degree of stability, chemical structure of siRNA molecules is very stable, especially the3’end dangling TT base of dsRNA. It is different from the antisense nucleic acid technology, without extensive chemical modification. For shRNA, because of inserting the loop ring structure, the efficiency and stability of shRNA is greatly improved. At present RNAi technology has been widely used in the identification of gene function and expression as well as post-transcriptional regulation and other popular research field. It can provide new ideas for the development of gene therapy for various diseases. The purpose of the study is silencing SLPI gene expression in colon cancer cells by using RNAi technology and studying the effects on morphology and function of Lovo colon cancer cell cultured in vitro. Secretory leukocyte protease inhibitor （SLPI） belonging to the WAP family is a serine protease inhibitor and inhibitor of inflammation and protease inhibitor. SLPI can regulate cell differentiation and proliferation and inhibit tumorigenesis and metastasis. At present domestic and foreign literatures of SLPI are closely correlated with tumor, but it’s levels of expression in different tumor is different. Some scholars found that the expression of SLPI protein and mRNA is increased in gastric carcinoma and the level of expression is closely related with tumor stage. the level of expression in advanced gastric cancer was significantly higher than that of in early gastric cancer. the level of expression with serosal invasion was significantly higher than that of without serosal invasion. Compared with normal prostate tissue, expression of SLPI is down-regulated in prostate cancer tissues.it is suggesting that SLPI may act as a tumor suppressor genes playing a role in the genesis and development of prostatic tumor.The mechanism may be that role of SLPI in different tissues is different, resulting in the level of protein expression.At present there are no relevant reports about SLPI gene and colorectal cancer research. Using tissue chip technique, a retrospective analysis of150cases of colon cancer tissues and adjacent normal tissues performed by general surgical department in NO.2hospitals in Xiamen city from2008January to2012December were used to investigate the correlation between expression of SLPI protein in colorectal cancer and the clinical pathological factors of the degree of tumor cell differentiation and clinical staging and TNM lymph node metastasis by using immunohistochemical method. Then, using RNA interference to inhibit the expression of SLPI protein and SLPI mRNA in colon cancer Lovo cells, the effect on tumor cell biological function and mechanism were observed. In order to provide evidence to revealing the role of SLPI in the development of colon cance and provide new ideas to studying early diagnosis and treatment of colon cancerMethods and materialsThe first chaptera retrospective analysis of150cases of colon cancer tissues and adjacent normal tissues performed by general surgical department in NO.2hospitals in Xiamen city from2008January to2012December were fixed with10%formalin and paraffin embedding. All specimens are in detail recorded with patient information including tumor TNM staging and differentiation degree and lymph node metastasis and distant metastasis. Immunohistochemical SP method was used to detect difference of the expression level of SLPI protein in different specimens. Mouse anti-human SLPI monoclonal antibody was purchased from American Santa Cluz Company. Experimental procedures were carried out According to kit. The results are as follows: the negative judgment standard is colorless, positive for cytoplasmic staining brown. According to the percentage of positive staining cells score:10representative view field were selected at high magnification. one hundred tumor cells were counted in each chip and each field. A total of one thousand cells were counted.score0:The positive cell number is less than or equal to5%; the number of10%-25%positive cells was score1; the number of25%-50%positive cells was score2; the number of positive cells was more than50%to score3; score0-2definite as low expression; score3definite as high expression.Tissue chips were used to investigate the correlation between expression of SLPI protein in colorectal cancer and the clinical pathological factors of age and gender and the degree of tumor cell differentiation and clinical staging and TNM lymph node metastasis by using immunohistochemical method.The second chapterWe use the RNAi technology to silence SLPI gene, respectively using qRT-PCR and Western blot to detecting silence of SLPI mRNA and SLPI protein. Effect of SLPI silencing on colon cancer Lovo cell proliferation was observed by cell counting. Effect of SLPI silencing on cell adhesion ability was observed by cell adhesion assay; the migration ability influenced by SLPI silencing on colon cancer cell line Lovo was evaluated by Transwell test.Statistical analysisChi-squared test and Fisher exact test were used in enumeration data, and Independent-Samples T test was used in quantitative data. When expected frequency of cells was more thanl and less than the expected frequency of1/5cell is less than5, Chi-squared test with continuity correction was used. A small sample of Student t was used to compare cell adhesion and migration and proliferation ability to experiment, test level a=0.05, Probability values<0.05were considered statistically significant.Results:The first chapterThe expression of SLPI in colon cancer was analyzed by using methods immunohistochemical. SLPI was not expressed in normal tissues. The positive expression of SLPI of tumor tissues was in the cytoplasm. Relationship between clinical features and expression of SLPI protein in patients was analyzed.（Fig.1-1） The positive expression of SLPI was positively correlated with the degree of tumor differentiation （P<0.05）. The positive expression rate of SLPI is high and staining cell is deep in low differentiated tumors. In addition, strength of the SLPI expression showed significant correlation between with lymph node metastasis and without lymph node metastasis. Strength of the SLPI expression in patients with lymph node metastasis was significantly higher than that without metastasis （P<0.05）. It is significant correlation between SLPI and TNM staging of tumor.the rate of positive expression in patients with stage Ⅲ and Ⅳ was significantly higher than that with stage Ⅰ and Ⅱ.（P<0.05） Moreover, the expression of SLPI protein in patients with distant metastasis is higher than that in patients without metastases （P<0.05）. In addition, the expression of SLPI was not correlated with age and sex （P=0.594, P=0.902）（table.1-1）.The second chapterExperimental study of SLPI gene silencing by RNAi technique was carried out to investigate its biological role in colon cancer Lovo cells. The suitable proportion transfection reagent is SiRNA75ng,1.5ul for transfecting colon cancer Lovo cells. 72hours after transfection to silence SLPImRNA about83%, HsSLPI5siRNA can silence SLPI gene89.9%. Silencing of SLPI gene results in decreasing cell proliferation and enhancing performance of adhesion and reducing ability of migration.it suggested that SLPI may play an important role in the adhesion and migration of colon cancer cells. Combined with the first part of the experimental results, the expression of SLPI protein in colorectal carcinoma tissues was significantly higher than that in adjacent cancer tissues. SLPI may play a key role in the process of invasion and metastasis in colon cancer cells. However, the detailed mechanism remains to be further studied.Conclusion1.The positive reaction of SLPI protein was mainly located in the cytoplasm and negative expression in normal colon mucosa.In colon cancer its high expression of SLPI correlated with low differentiation and tumor stage Ⅲ-Ⅳand regional lymph node metastasis and distant metastasis. The expression of SLPI strength increased with decreasing degree of differentiation. Expression level of SLPI in patients with stage III-IVwas significant higher than that of patients with stage Ⅰ-Ⅱ. Expression level of SLPI in patients with distant metastasis and lymph node metastasis was higher than that of patients without lymph node metastasis and distant metastasis. SLPI play an important role in the occurrence and development of colon cancer, it may become one of biological markers of early detection of colon cancer.2. By using siRNA silencing of SLPI gene in colon cancer Lovo cell line can decrease the expression of SLPI protein inhibiting the ability of proliferation and migration and enhancing the adhesion ability of colon cancer cells. |
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