Research On The Correlation Of Biological Periostin In Colorectal Cancer

Colorectal cancer (Colorectal cancer, CRC) is one of the fastest rising incidenceof cancer tumors in our country.Relapse and metastasis are the leading cause of poorprognosis and death of patients with colorectal cancer, but also a crucial factoraffecting survival, and therefore determine the risk factors for recurrence andmetastasis is very important.Nearly600,000new cases worldwide cases of humanhealth and pose a great threat to life each year.In the United States, the prevalence ofcolorectal cancer ranks third, behind skin cancer and lung cancer.The world ofcolorectal cancer incidence and mortality in sub-rise, with the continuousdevelopment of medical technology, five-year survival rate for colorectal cancer hasgreatly improved, but after nearly a decade of colorectal cancer5year survival ratehas been fluctuating between50%to70%[1].In recent years the incidence of colorectalcancer ranking fourth in China which after lung cancer, stomach cancer and livercancer[2-5]. Colorectal cancer patients without lymph node metastasis, survival rateafter five years, up to90%[6],the incidence of lymph node metastasis, survival rate ofonly65percent after five years[7,8].Therefore, the key to improving the efficacy andprognosis of colorectal cancer is early detection, early diagnosis and earlytreatment.Early detection, early diagnosis and early treatment to improve the qualityof life of patients, prolong the lives of patients with a very important significance.Medical researchers effort aims to explore effective methods for early diagnosisof malignant tumors.The current academic attention molecular marker for thediagnosis of malignant tumors since the treatment, and prognosis have a veryimportant role.Tumor marker (tumor marker, TM) is the tumor tissue and tumor cells due to the anti-cancer genes or oncogenes and other cancer-related genes and theirproducts and abnormal antigen expression of bioactive substances produced.Detecttheir presence or quantitative nature of the tumor can be prompted, you can also learnabout the tumor tissue, cell differentiation, cell function, to help diagnose tumors,classification, prognosis and treatment guidelines.Medical researchers are trying tofind a highly sensitive, highly specific tumor markers to help diagnose colon cancer,this study has very important significance.In this study, through the application oftwo-dimensional chromatography coupled with mass spectrometry techniques,analyzed and identified differences in colorectal cancer tissues and adjacent tissuesexpressed protein profiling, identification and found there are24differentproteins.The identification of the24differentially expressed proteins, the differentialprotein upregulation has15, there are differences in protein downregulation9. This isconsistent with the preliminary laboratory test results[18].These differences incolorectal cancer tumor protein is expected to be highly sensitive, highly specificmarkers, but need further clinical validation. The lab will be this one by24differentproteins for clinical validation of all, this study only Periostin (POSTN) protein forclinical validation.First, the general characteristics POSTNPOSTN having a molecular weight of about90kDa, primarily bone precursorcells and their secreted by the cells into the involved cell recruitment, adhesion,migration process is a bone adhesion molecules newly discovered in recentyears.POSTN can effectively promote osteoblast proliferation, differentiation,adhesion and spreading, adhesion and aggregation and promote the role of periostealprogenitor cells.POSTN originally cloned from a mouse osteoblastic cell lines.Encoding a protein βig-h3, MBP-70, Algal-CAM, stablin Ⅰ Ⅱ belong to the sameprotein family and is considered a marker of mesenchymal cells[19].POSTN may consist of transforming growth factor-β1(TGF-β1) induced toproduce this discovery in colon cancer cells has been confirmed[20].In addition to TGF-β1can induce POSTN, there are some potential factors can promote thesecretion of pancreatic stellate cells POSTN, such as: platelet-derived growth factor(PDGF-aa, PDGF-bb), bone morphogenetic protein (BMP-2) and fibroblast growthfactor (FGF-B, FGF-A)[21].Angiotensin II, and fibroblast growth factor (FGF-1) canpromote the expression of pulmonary artery smooth muscle cells POSTN[22].Inaddition, in the study of lung cancer cell line A549, we found that under hypoxicconditions, transforming growth factor A (TGF-A) and basic fibroblast growth factor(bFGF) signals by activating RTK/PI3K the increased expression POSTN[23].Alongresearchers found Twist, IL-3and IL-4can induce the expression or secretionPOSTN.Rodent POSTN located in chromosome3, about30kbp, containing811aminoacids, molecular weight of about90.2kDa.Rodent POSTN cDNA length is3187bp,comprising a733bp3’untranslated region, a18bp5’ untranslated region and a2436bp open reading frame which encodes a811amino acids and902protein [24].Due tothe presence of the carboxyl terminus of alternative splicing POSTN, five differentsubtypes are separated POSTN.People POSTN located in chromosome13, about36kbp, containing836amino acids, molecular weight of about93.3kDa[25].By humanosteosarcoma cDNA library and found that placental POSTN two subtypes, thehuman osteosarcoma POSTN an open reading frame encoding the subtypescontaining836amino acids and a molecular weight of93.3kD;. Another for humanplacental POSTN the open reading frame encoding isoforms containing779aminoacids and7kD.POSTN highly conserved, homologous human and mouse aminoPOSTN up89.2%.Compared with other structures POSTN carboxy terminal(C-terminal) conserved lower,85.5%.Structurally, POSTN include amino terminal (N-terminus), C-terminus, acysteine-rich region (EM I) and four homologous repeat region. There is a typicalN-terminal signal sequence is located, suggesting that it might be a secretory protein.POSTN is a highly conserved extracellular matrix proteins, the structure of the repeat region of homology with the four insect protein fasciclin I (FAS I) having homology,which may be associated with cell adhesion. EMI includes about75amino acids, maybe of the protein or multimeric protein-associated protein interactions. In recentyears, studies have found that each region contains a FAS I C-carboxylase and severalrecognition sites can be carboxylated glutamine residue, further comprising the FAS Iarea integrin binding sites, POSTN with integrin interactions mediated epithelial-mesenchymal transition (EMT), thereby regulating cell migration and adhesion[26]. Ahydrophilic C-terminus, the alternative splicing of the mRNA transcription level, thehuman tissue POSTN isoforms reported a total of8[27]is generally considered theexpression of isoforms POSTN and the occurrence of certain tumors[28].Many POSTN protein expression in normal tissues: stomach, colon, lung,adrenal gland, thyroid, vagina, ovaries, testes, prostate, etc.[20],and in the developingheart valves, the developing teeth, skeletal muscle injury after hypoxia afterpulmonary artery smooth muscle cells showed high expression[29].In recent years,studies suggest that the expression of POSTN has obvious differences between tumortissue and normal tissue, thus POSTN tumor metastasis is considered one of thecandidate genes associated with tumor invasion and metastasis.To explore the expression in colorectal cancer cases POSTN and POSTNfunction in colorectal cancer, and our main findings are as follows:Part I: Application of two-dimensional chromatography, mass spectrometrytechnique differences beside colorectal cancer tissue and proteomics researchMethod:1, drawn from clinical samples:65cases of patients with Dukes Cadenocarcinoma, adjacent tissues and cancer tissues.2, total protein samples were extracted protein concentration analysis.3, the sample preparation proteolytic peptide mixtures.4, the two-dimensional chromatography and mass spectrometry analysis, thedatabase retrieval. Results:1, carcinoma: MS data collection, collation, obtain identification information846protein.2, adjacent tissues: MS data collection, collation, identification data obtained535proteins.3, the cancer tissue and adjacent tissues were compared proteome analysis, thereare significant differences in protein24, which regulate the expression of protein incancer tissue15, down-regulated protein9.4Summary:1, two-dimensional chromatography and mass spectrometry with the successfulapplication of technology to complete colorectal cancer and adjacent tissuesproteomic data identification.2, through the identification data, comparative analysis of tumor tissues wereobtained and adjacent tissues of24different proteins, including15proteins wereup-regulated and9down-regulated protein expression.Part II:Plasma levels of colorectal cancer Periostin relationship withclinicopathological parameters between patientsMethod:Established clinical and pathological data of the database.Measured byELISAplasma POSTN.Results:1, POSTN plasma of patients with colorectal cancer were significantlyincreased.Dukes staging of colorectal cancer, plasma POSTN levels C and D of theperiod was significantly higher than theAand B stages (p <0.01).2, TNM staging of colorectal cancer, clinical expression level Ⅲ and Ⅳ wassignificantly higher than clinical and clinical Phase I Phase Ⅱ (p <0.01).3, POSTN levels in plasma of colorectal cancer with lymph node metastasis wassignificantly higher than those without lymph node metastasis of colorectal cancer (p =0.001).4, With distant metastases in colorectal cancer POSTN plasma levels weresignificantly higher than those without distant metastasis of colorectal cancer (p=0.003).5, Poorly differentiated type of colorectal cancer in plasma levels weresignificantly higher than in POSTN differentiation, poorly differentiated withcolorectal cancer (p=0.003).Summary:POSTN levels in plasma of colorectal cancer and tumor Dukes staging, TNM stage,lymph node metastasis and distant metastasis and differentiation, but not with patientgender and age (p>0.05).Part III: POSTN gene expression and function of the colon cancer cell linesw480Method:1, POSTN protein expression in colorectal cancer cell lines sw480.2,Build POSTN siRNAexpression vector and plasmid amplification.Results:1, POSTN sw480protein localized in the cytoplasm of cells, expressed as amoderate intensity.2, POSTN siRNA lentiviral construct the recombinant expression vectors canefficiently transfected sw480cells.Summary:1, successfully screened from colon cancer cell lines SW480POSTN highexpression cell lines, lay the foundation for further experiments.2, this part of the experiment successfully constructed efficiently inhibited gene expression vector POSTN, restriction enzyme digestion and sequencing confirmedplasmid entirely correct, that POSTN siRNA-1, postn siRNA-2.3, the successful use of liposome-mediated recombinant plasmid POSTNsiRNA-1, POSTN siRNA-2transfected into colon cancer cells.Part IV: POSTN siRNA transfection on the biological behavior of humancolon cancer cellsMethod:1, POSTN siRNAtransfection on sw480cells POSTN mRNAtranscription.2, POSTN siRNAtransfection protein expression of POSTN of sw480cells.3, POSTN RNAi on apoptosis sw480’s.4, POSTN siRNAtransfected cells sw480structural changes in subcellular.5, POSTN RNAi effects on sw480cell proliferation and cell cycle.Results:1, transfected siRNA-1plasmid, POSTN gene transcription decreased4.38timestransfected siRNA-2plasmid group, POSTN gene transcription decreased6.2times,compared with the control plasmid transfected group, a significant statisticaldifference.2, statistical analysis showed POSTN siRNA transfected cells, POSTN proteinexpression was significantly lower than the control plasmid transfected group.3, transfection of siRNA sw480apoptotic index was significantly higher than thecontrol plasmid transfected group, a significant difference between the two groups.4, POSTN siRNAtransfected cells sw480structural changes in subcellular.5, Sw480POSTN RNAi effects on cell proliferation and cell cycle.Summary:1, the interference can be reduced expression POSTN POSTN mRNA expressionin colon cancer cells and POSTN protein. 2, can inhibit the overexpression POSTN apoptosis in colon carcinoma cells.3, the interference POSTN colon cancer cells change expression substructure.4, the overexpression POSTN can promote the proliferation of colon cancer cells.5, After transfection of RNAi plasmid transfected cells survived significantlylower than the normal cells cultured sw480,also lower than the control plasmidtransfected group,the difference was statistically significant.6,The difference was statistical significant compared to G1and G2phasessiRNAtransfection group and control group.Part V: Conclusion:...