TPM4Expression And Biological Effect In Colorectal Cancer

Colorectal cancer (Colorectal cancer, CRC) is one of today’s common malignancies, serious gastrointestinal cancer. Approximately1.2million patients worldwide are diagnosed with colorectal cancer each year, and there are more than600,000patients die of colorectal cancer directly or indirectly. In China, the incidence of colorectal cancer ranks the fourth most common cancer after lung cancer, stomach cancer and liver cancer.Colorectal cancer is one of the fastest rising incidence of cancer tumors. Due to the occurrence of colorectal cancer mechanism is not entirely-clear, it is still difficult in the diagnosis and treatment of colorectal cancer, morbidity, mortality remains high. Currently, surgery is the most effective treatment means of colorectal cancer, non-surgical treatment include chemical treatment, radiation therapy, biological therapy and targeted therapy, in the treatment strategies, individual treatment rapture to unprecedented heights. If patients is unable to surgery, they need to chemotherapy and radiotherapy. Disease staging of colorectal cancer is the most important prognostic factor. In our country, most patients are diagnosed when the disease has advanced, and more than a third of patients with recurrence, which seriously affect the quality of life of patients and shorten survival. So efficient screening, early diagnosis and early detection of recurrence is crucial to improve the efficacy and prognosis of colorectal cancer, to alleviate the suffering of patients and improve the quality of life and prolong the lives of patients.Studies have shown that early diagnosis and early treatment of colorectal cancer, early treatment is a chance to cure. Tumor markers, also known as tumor marker (tumor marker, TM). is present in malignant cells characteristically, or a substance produced by the abnormal malignant cells, or the host response to stimuli substances produced by the tumor. Tumor markers are present in the patient’s tumor tissue, body fluids and excreta. Detecting TM’s presence or quantitative change may be prompted and can reflect tumor development, understanding of tumor tissue, cell differentiation and cell function, as well as helping cancer diagnosis, classification, prognosis and treatment guidelines. Tumor markers are one of the main aid diagnosis of colorectal cancer. At present, the widespread use of colorectal cancers tumor markers are CEA, CA19-9, CA724, CA125, CA242, CA50, As the widely used serological marker for early diagnosis,but their sensitivity and specificity are not high. Because of the complexity of the tumor genes, it is very difficult to find a high sensitive and specific tumor marker. Studies have shown that a single tumor marker detection has a low accuracy rate, tumor markers combined can improve positive rate and accuracy.Medical researchers are actively trying to find a highly sensitive and highly specific tumor markers to help diagnose colon cancer, which if of great significance.Emergence of proteomics technology enables detection of tumor markers in faster, more accurate, saving time and effort. Comparative proteomics plays an important role in Disease research. It was first detected in a tumor tissue of all proteins, and by comparing the disease and control groups (normal group, and/or other diseases), and detection of disease-specific protein group, by differential proteomics analysis can be found the sensitivity and specificity of early tumor marker for early detection of cancer, that provides a great convenience for early diagnosis, early treatment. It has important implications for disease pathogenesis, diagnosis, treatment, research and development of new drugs.In this study, the proteins of colorectal cancer and adjacent tissues were analyzed by using two-dimensional chromatography and mass spectrometry,and the results showed that24proteins were found,of which15proteins are up-regulated and9are down-regulated.This result is consistent with the results of previous experiments in this laboratory.The laboratory will verify the24proteins by one by one.the study only for Tropomyosin4(TPM4) protein validation.Tropomyosin (TPM) is a thin filament with the actin-binding protein, a molecular weight of2×35KPa, length of41nm, a helical configuration consists of two parallel polypeptide chains, each tropomyosin inclusive phase forming a continuous chain with actin thin filaments combined. Tropomyosin is an important regulatory protein during muscle contraction, which in a large number of isomeric forms are widely distributed in a variety of eukaryotic cells. Mammals4TPM genes have been identified, i.e., TPM1, TPM2, TPM3, TPM4, express at least20kinds of TPM isomers. Previous studies have shown that different TPM mutations can cause different diseases. Current research on tropomyosin4(TPM4) less, mainly in its muscle-related diseases and cancer. Largely confined tumors and breast cancer metastasis in bladder cancer research and related studies. Is not yet TPM4and colorectal cancer occurred Correlation development and metastasis.。TPM4expression in colorectal cancer, and colorectal cancer occurrence, development and metastasis of research has been reported rarely. We intend to use the two-dimensional chromatography,mass spectrometry. immunohistochemistry, fluorescence quantitative real-time PCR (Real-Time PCR) and western blot, RNAi approach, to explore the relationship between TPM4expression changes and the incidence of colorectal cancer.Part Ⅰ:The differential proteomics of colorectal cancer and adjacent tissues by two-dimensional chromatography and mass spectrometry.Method:1Clinical samples drawn:23cases of TNM Ⅲ stage cancer patients, adjacent tissues and cancer tissues.2Samples of total protein extraction, protein concentration analysis.3Preparation proteolytic peptide mixtures.4Analysis of the two-dimensional chromatography and mass spectrometry and database retrieval.5Western blotting experiments.Results: 1By MS data collection and collation,846proteins identification information was obtained from Carcinoma:.535proteins identification was obtained from adjacent tissues.2Through differential proteomics,24proteins were found to be significantly different, in which the differential protein upregulation has15,and downregulation has9.3Western blot analysis showed TPM4protein in cancer tissues was significantly higher than that of the adjacent tissues.Summary:1Successful application of two-dimensional chromatography and mass spectrometry to complete colorectal cancer and adjacent tissues proteomic data identification.2Through differential proteomics,24proteins were found to be significantly different.of which15were up-regulated and9were down-regulated.The expression of TPM4protein in cancer tissues was significantly higher than that in adjacent tissues by Western blot.Part Ⅱ:Colorectal cancer tissues TPM4level expression and clinical significanceMethod:1Collecting of ciinical and pathological data.2Measureing colorectal cancer tumor tissue TPM4. by immunohistochemistryResults:1The expression of TPM4protein in cancer tissues was significantly higher than that in adjacent tissues.2TNM staging of colorectal cancer, pathological stage III and IV of TPM expression level is not significantly different with stage I and II (p>0.05).3TPM4expression in tissue of colorectal cancer with lymph node metastasis is is not significantly different with those without lymph node metastasis of colorectal cancer (p>0.05).4TPM4expression in tissue of colorectal cancer with distant metastases is not significantly different than those without distant metastasis of colorectal cancer (p>0.05).5TPM4expression in poorly differentiated adenocarcinoma is significantly higher than those in moderately differentiated and well differentiated adenocarcinoma (p<0.01).6TPM4expression in tissue of Colon cancer is not significantly different with Rectum cancer (p>0.05).7TPM4expression was unrelated to tumor size (p>0.05)Summary:1TPM4expression is significantly increased in colorectal cancer tissues.2There was a significant association between positivity for TPM4and the degree of differentiation. In contrast, TPM4expression was unrelated to TNM stage, lymph node metastasis and distant metastasis, tumor size, location, gender, age.Part III:Selection of colorectal cancer cell lines, Construction of Expression Vector and identification of expression vectorMethod:1TPM4protein expression in various cancer cell lines and normal colon cell lines.2TPM4siRNA expression vector construction and plasmid amplification.3TPM4siRNA vector transfection efficiency measurementResults:1SW480cell lines TPM4protein expression is highest.2The siRNA TPM4expression vector was successfully constructed and the plasmid concentration was normal. 3The transfection efficientcy of siRNA TPM4vector was in accordance with the requirements.Summary:1Successfully screened out TPM4high expression cell line SW480from colon cancer cell lines.2This part of the test required TPM4siRNA vector completed by the santa cruz company, the successful use of liposome-mediated by the recombinant plasmid TPM4siRNA was transfected into SW480.TPM siRNA expression vectors can efficiently transfect SW480cell..Part Ⅳ:The effect of TPM4siRNA transfection on the biological behavior of human colon cancer cellsMethod:1TPM4siRNA transfecting on SW480cells effect TPM4mRNA transcription.2TPM4siRNA vector effect on SW480cell activity by CCK-8detection.3After TPM4siRNA vector transfection.changing SW480cell" morphological by light microscopy and changing cell’ submicroscopic structures, by electron microscopy.4TPM4siRNA transfecting on apoptosisSW480.Results:1After transfection TPM4siRNA TPM4mRNA expression of SW480decreased significantly.2After SW480cells transfected TPM4siRNA vectors, growth inhibition of cells was not obvious, even at the highest concentration of SW480inhibition rate probably only about20%. From time gradient map view, TPM4siRNA transfection vectors24h began significantly inhibited cell growth,48h effect is more pronounced.3After TPM4siRNA transfected cells SW480cell sub-structure changed.4After transfection TPM4siRNA SW480apoptotic index is increased. Summary:1Interfering SW480cell’s TPM4expression can effectively inhibit colorectal cancer cells TPM4mRNA expression.2Interfering SW480cell’s TPM4expression can effectively inhibit the proliferation of colorectal cancer cells.3Interfering SW480cell’s TPM4expression can change colorectal cancer cells sub-structure.4Interfering SW480cell’s TPM4expression is effective promotion of colon cancer cell’s apoptosis.Conclusion:1We successfully established different proteins spectrum of TNM III colorectal cancer tissues and adjacent tissues,among them,24proteins were differentially expressed,15up-regulated proteins and9down-regulated proteins.Western blot analysis showed TPM4protein in cancer tissues was significantly higher than that in adjacent tissues.2TPM4expression in carcinoma tissue was significantly higher than that in adjacent tissues by immunohistochemistry,and the expression level of TPM4was related to the differentiation of tumor.3The expression vector of TPM4gene was successfully constructed and was successfully transfected to SW480colorectal cancer cells.4TPM4siRNA transfected SW480cell biological behavior undergone significant changes:the expression of TPM4’s mRNA in SW480cells was decreased,the proliferation of SW480cells was inhibited and apoptosis was enhanced.