| Part Ⅰ Identification and Screening of molecular Biomarker in colorectal laterally spreading tumors Based on iTRAQBackground:Colorectal cancer (CRC) is currently one of the common malignant tumor in the world, its morbidity and mortality are also in the front rank of the malignant tumors. Due to the rapid adoption of Westernized lifestyles and diet structure, there is a rapid increase in colorectal cancer incidence and mortality in China. Colorectal laterally spreading tumors (LSTs) are defined as a relatively flat neoplastic lesion greater than 10 mm in diameter, which is an important precancerous lesion of colorectal cancer. As LSTs have no obvious clinical symptoms early, which might be found usually in conventional colonoscopy. LSTs accounted for about 6% of colorectal cancer in the early colorectal cancer. The data shown that the colonoscopy detection rate of LSTs in the endoscopy center of NangFang Hospital was 0.8%, the canceration rate of which was 11.5%. LSTs typically extends laterally rather than vertically along the colonic wall and which can also develop into a deeper submucosal invasive cancer. Thus, it is suited to be treated with endoscopic resection (such as endoscopic mucosal resection, piecemeal endoscopic mucosal resection, or endoscopic submucosal dissection). But the postoperative recurrence rate is high, which suffered relapses easily and could develope into cancer.Proteomics is applied in clinical diseases research such as cancer, which was used to identify the protein expression levels in the normal physiological condition and different disease states. Quantitative proteomics was quantitative analysis for gene expression on the base of the whole protein of cell, which was widely used in the studies of disease pathogenesis and pharmacological regulation mechanism. Isobaric tags for relative and absolute quantitation (iTRAQ) was independently developed by Applied Biosystems Incorporation of USA in 2004. The basic flow of the iTRAQ operation is as follows:after trypsin digestion, the peptides labeled with respective isobaric tags, then using high validity liquid chromatogram to separate labelled peptide, and LC-ESI-MSMS analysis was performed based on Mass spectrometer. In the first mass spectrometry, the same peptides which was marked show the same mass-to-charge ratio. In the secondary mass spectrometry, signal ion manifest as different peak of mass-to-charge ratio (114-121). According to the height and area of the peak, proteins were identified and the quantitative information of the same protein through different treatment was analysed. iTRAQ technique could label 8 components to the most in identification experiments simultaneously, and which had shown huge advantages in sensitivity, throughput and quantitative precision to conquer some disadvantages of traditional proteomic techniques and was served as a powerful alternative.Objective:To identify and analyze the specific expressed proteins of colorectal LSTs through proteomic technology. And to find the possible molecular mechanisms which lead to the lateral growth of LSTs. In additon, we further identified the potential biomarkers associated with LSTs progression. Methods:Tissue specimens for iTRAQ were obtained randomly from the endoscopy center of NangFang Hospital. The patients were divided into LSTs、 protruded-type colorectal adenomas、small flat adenomas (d< 1 cm)、colorectal cancer colon tissue and normal colon mucosa tissue groups, and each group has 20 cases. The iTRAQ process was as follow:protein extraction, enzymolysis, iTRAQ lable, mixed, SCX separation and LC-MS/MS. The protein identification and quantification for the results were accomplished with Mascot software (Matrix Science Inc, USA). The standard of bioinformatics analysis were also performed in NCBInr、SwissProt and UniProt databases.Result: With iTRAQ technique combining multi-dimensional solid/liquid chromatography with tandem mass spectrometry (LC/MS/MS) study strategy, a total of 4955 expression proteins、22587 peptides and 21095 Unique peptides were identified. Through comparative analysis, a total of 2001 proteins were identified as differentially expressed proteins, and 1026 proteins were up-regulated while 975 proteins were down-regulated. Base on the abundance of the protein,when the foldchange is above 1.2 and P<0.05,we define this protein is differentially expressed protein. Compared with other groups, the total number of the differential expressed proteins were 679、390、304 and 628 in LSTs respectively. Through further statistical analysis, there are 55 tissue specific proteins in colorectal LSTs compared to other groups, and 14 proteins were up-regulated while 41 proteins were down-regulated. According to the analysis of the GO functional annotation, most of differential proteins were located in more than one organelle or cellular component. In brief, 0.15%,19.02%,15.86%,5.75%,2.05%,1.01%, and 6.33% of the differential proteins were located in the nucleu, cytoplasm, organelle, cell membrane, cytoskeleton, cell junction and cell cavity, and 7.32% of which were part of macromolecular compound proteins, respectively. And most of the identified proteins were involved in the biology processes and molecular functions, such as binding(49.54%), enzyme catalytic activity (28.39%), enzyme regulator activity(4.51%). Some identified proteins participated in the immune defense responses and angiogenesis. The 55 differentially expressed proteins in colorectal LSTs were involved mainly in metabolism and transport.Conclusion:Through iTRAQ-based quantitative proteomic research, we have successfully identified the protein expression profiling of colorectal LSTs. And we have screened out a group of specific proteins expressed in colorectal LSTs. Most pecific proteins were involved mainly in binding, metabolism and transport, and which might play an important role in tumorigenesis and development of LSTs.Part II The validation and diagnostic assessment of molecular markers for colorectal laterally spreading tumorsBackground:The diagnosis of LSTs patients, including those in early stage, lacks marker with high specificity and sensitivity. The studies of genesis and development mechanism of colorectal LSTs, especially the studies in molecular level, confirm that the transformation from adenoma to carcinoma occurs with the change of varieties and numbers of proteins. And the specifically expressed proteins involved in regulation of differentiation of LSTs could be characteristic molecular markers and have high diagnostic value. The proteins could be quantified and located by Western blot, ELISA and immunohistochemistry, which might play an important role in the diagnosis and prognosis of the disease.Objective:The aim of this study is to identify and validate the proteins specifically expressed in colorectal LSTs.Methods:The serum samples of LSTs group and normal control group were randomly collected,30 cases in each group. The mRNA levels of the proteins that were differently expressed in the tissues of LSTs, protruded adenoma, small flat adenoma, colorectal cancer and normal colorectal mucosa were validated by RT-PCR. The LCN-2 and MMP-9 protein expression levels were validated by Western Blot in those groups. The protein expression levels of LCN-2 and MMP-9 in colorectal LSTs group and normal control group were validated by ELISA double sandwich method and immunohistochemistry.Results:We chose four proteins, LCN-2、SORD、RAB2 and CPA3, which were closely related to cancer progression and had rarely been reported. We first analyzed them by Real Time PCR. The results showed that the mRNA expression levels of LCN-2 and SORD were significantly increased and the tendency in the expression difference of LCN-2 mRNA was most consistent with that in proteomics, meanwhile the mRNA expression level of CPA3 was significantly decreased. We used Western blot and immunohistochemistry to validate the protein expression levels of LCN-2 and MMP-9, which is closely related to LCN-2’s function. The results shown that the protein expression levels of LCN-2 and MMP-9 were significantly up-regulated in LSTs tissues than that in other groups (P=0.000, P=0.000, respectively). Moreover, the expression intensity of LCN-2 and MMP-9 increased with the degree of pathological grading (rs=0.943,P=0.000; rs=0.684,P=0.000; respectively), and which was no relation to patient age, sex, lesion location, size and shape of LSTs. And the protein expression level of MMP-9 was positively correlated with that of LCN-2 (rs =0.815, P=0.00). The protein expression levels of serum LCN-2 and MMP2 in LSTs were significantly higher than that of normal control group and the expression intensity were positively associated with pathological grading degree (rs=0.927, P=0.000; rs= 0.924, P=0.000; respectively)Conclusion:Among the distinct proteins selected by mass spectrographic analysis, LCN-2 and MMP-9 were expressed significantly high in the tumor tissues and serum of colorectal LSTs patients and the expression intensity were positively correlated with pathological grading degree. The results shown that LCN-2 and MMP-9 might be closely related to the development of LSTs, and which provided new clues to the studies of colorectal LSTs development and tumor marker. LCN-2 might be of clinical worthy in terms of increasing the diagnostic rate of early colorectal LSTs and improving prognosis, and which could be an important marker in the diagnosis, prognosis assessment, surveillance, prevention and treatment of colorectal LSTs.Part Ⅲ The preliminary discussion of colorectal laterally spreading tumor growing mechanismBackground:Colorectal LSTs is a kind of flat adenomas with a special form, and it has characters below:the diameter of lesion is more than 10 mm; it grows along the lateral extension or ring lumen rather than straight down growth; on the basis of its surface morphology is divided into particles and non-particles. Colorectal LSTs are one of the most important precancerous lesions in advanced colorectal cancer and pathology is mainly tubular villous adenoma or tubular adenoma. Compared with protruded type of tumor, colorectal LSTs have higher apoptosis index and lower hyperplasia index. Such a special cell apoptosis and cell proliferation provide colorectal LSTs unique surface features, lateral development growth way, low malignant rate after submucous infiltration and relatively low malignant potential. But former molecular biology research mainly focused on the research of colorectal LSTs methylation, about its way of lateral growth mechanism is still unclear. In the early data analysis of proteomics research, we found that the protein expression levels of desmosomes glue glycoprotein family members (DSG2), Plakophilin3b (PKP3), and Junction Plakoglobin (JUP) were significantly increased in LSTs compared to normal colon mucosa, protruded adenoma and CRC. This finding indicated that desmosomes glue glycoprotein family members DSG2, PKP3 and JUP may play an important role in the growth process of colorectal LSTs.Desmosomes mainly consists of two types of protein:one type is a transmembrane protein, a member of calcium adhesive proteins family, consists of Desmoglein (DSG) and Desmocollin (DSC), respectively form the intercellular contact layer of desmosomes and electron dense layer; another type is desmosomes in the cytoplasm, it mainly consists of Desmoplakin (DP), Plakoglobin (PG) and Plakophilins (PP). One end of desmosomes attaches desmosomes transmembrane protein, the other end attaches Intermediate Filaments (IF). DSG2 mainly distributes in the epidermis in the basal cell layer, myocardial and intestinal epithelium, it’s a simple epithelium type. DSG2 has similar function with classic E-cadherin, and participates in the regulation of normal epithelial cell adhesion. A large number of clinical reports indicated that many diseases associate with abnormal expression of DSG. However, the mechanism of it’s abnormal expression is still unclear. Some studies reported the abnormality of DSG leads to dysfunction of desmosomes connection, which affects the structure and function of the corresponding organization integrity. In the cell, PKP3 and JUP combine with the intracellular function areas of DSG and DSC, participate in regulating intercellular adhesion and combining the functions of cytoskeleton, and desmosome is crucial for the IF attached to the bridge.Objective:Using proteomics technology to preliminarily discuss the growth mechanism of colorectal LSTs.Methods:By studying the ultrastructures of colorectal LSTs, protruded adenoma and normal colon mucosa with transmission electron microscope (TEM), we observed the intercellular connection of these tissues mentioned above, and to make sure whether we can find abnormal structures related to the colorectal LSTs lateral growth. And we use western blot and IHC to verify the expression of desmosomes DSG2 family proteins, PKP3, JUP in colorectal LSTs, protruded adenoma and normal colon tissue.Results:We used transmission electron microscopy (TEM) to study the ultrastructures colorectal LSTs, protruded adenoma and normal colon, and found that in the base layer, the expression of desmosomes density is normal, but the number and density of epithelial side desmosomes in colorectal LSTs increased significantly, compared with protruded adenoma and normal colon mucosa tissue (P=0.000,0.000, respectively), and the number and density of desmosomes in LSTs colorectal epithelial side are the highest. We confirmed that the protein expression levels of DSG2, PKP3 and JUP were significantly increased in colorectal LSTs and protruded adenoma than those in normal colon mucosa (P=0.000,0.000, respectively), and which have the highest expression levels in colorectal LSTs.Conclusion:As an important part of the desmosome, the overexpression of DSG2, PKP3 and JUP in colorectal LSTs confirmed that the difference of amount and density of the desmosome on the epithelial side, and which may lead to the growth of colorectal LSTs differ from that of protruded adenoma. |
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