Study Of Expression Of EHD2, PDLIM7in Colorectal Cancer Through Proteomics-based Approaches

Background and ObjectiveColorectal cancer is common malignant tumors in the world. Colorectal cancer (CRC),including neoplasms of colon and rectum, is a much common malignant tumor which dogreat harm to people’s health. The mobidity of CRC has been rising year by year in Chinaalong with the improvement of people’s alteration of life style and living standard. Eachyear, more than1.2million people are diagnosed with CRC, who have a5year survival rateof55%.There are3causes responding for current situation:1. Un-identification of etiologicalfactors of CRC, which is essential obstruction of prevention of CRC;2. Absence ofefficient and easy diagonosing approach leads patients with CRC couldn’t receive anytreatment until advanced stage of this disease;3. Although many therapies have been usedto help patients, the result is not good enough, and moreover, pains is more than happinessfor patients received those therapies. Therefore, studying CRC about its etiological factors,screening, early detection, or therapies would bring hope to people with CRC or high-riskpopulation.Recently, there are many difficulties in early diagnosis, screening, and therapy ofCRC. Many techniques were used for diagnosis and screening of CRC, such as, fecaloccult blood tests(FOBT), colonoscopy, double contrast barium enema(DCBE), MRI,CT, PET, genetic screening, and so on. They respectively have their shortcomings and notsatisfactory on the whole. Therapies of CRC include surgery, chemotherapy, radiotherapy,biotherapeutics. Recently, individual clinicians and researchers pay more and moreattention to the treatment effects related to individual differences． Individualizedtreatment of CRC is becoming the focus of clinical treatment and basic researchincreasingly． The individualized treatment of surgery，chemotherapy with preoperativeradiotherapy and targeted therapy improve the clinical effects of colorectal carcinoma．Developments of proteomics, especially those of the disease proteomics open a newwindow for tumor research. Numerous specific proteins has been identified and some ofthem has been validated as potential biomarkers of diagonosis, prognosis and/or therapy.Upgrade in proteomics techniques greatly facilitates proteomics improving. TheLC/MS-based isobaric tags for relative and absolute quantification (iTRAQ) approach isemerging as one of methodologies which are more powerful in the research of disease biomarkers. In our study, LC/MS-based iTRAQ have been used to dig different proteinsbetween CRC normal tissue and tumor tissue, which potentially become biomarkers thatfacilitate clinical oncology improving.MethodsThe normal tissue samples and the paired cancer tissue samples of five cases of CRCwere collected. After total excision, these five normal tissue samples and the paired cancertissue samples were pooled and digested by trypsin, respectively. Then treated thesebiological samples through proteomics approach which was LC/MS-based labeled withisobaric mass tags for relative and absolute quantitation(iTRAQ) analysis, and then, thedifferent proteins profile was depicted. The molecular functions and locations ofdifferential proteins were analyzed through searching in Swiss-Prot andGeneOntcology(GO) database.Based on bioinformatics analysis, EHD2and PDLIM7were selected for furthervalidation by imunohistochemistry, western-blotting and q RT-PCR. The normal tissuesamples and the paired cancer tissue samples of58cases of CRC patients were collected.The imunohistochemistry were performed according to the following workflow: fixed byformalinâ†'embeded in paraffin waxâ†'slicedâ†'deparaffinage to waterâ†'antigenretrievalâ†'endogenous peroxydase blocked by hydrogen peroxideâ†'incubation in primaryantibodyâ†'incubation in secondary antibodyâ†'coloration with DABâ†'counterstain inhematoxylinâ†'dehydrationâ†'transparention. Two pathological doctors were invited todiagnose the results double blindly. The immunohistochemical localization was scored in asemiquantitative fashion incorporating both the distribution and the intensity of specificstaining. The grade of staining intensity was:3, strongly positive;2, moderately positive;1,weakly positive; and0, negative. The extent percentage of positive cells was graded asfollows:4,>80%positive cells;3,51%to80%positive cells;2,11%to50%positive cells;1,<10%positive cells;0, negative.After total protein excision, the normal tissue samples and the paired cancer tissuesamples of3cases of CRC were collected. Western-Blotting analysis and q RT-PCR wereperformed for further validation by above samples.30mg of total proteins were undergoneWestern-Blotting. After SDS-PAGEâ†'membrane-transferingâ†'incubating in primaryantibodyâ†'incubating in secondary antibodyâ†'electrochemiluminescence(ECL)â†' exposingâ†'developingâ†'fixingâ†'scanningâ†'semi-determinalion analysis by PDQUEST software.The q RT-PCR was performed to measure the expression levels of selected protein inCRC cancer tissue and intestinal normol mucosa.ResultsFirstly, via LC/MS-based iTRAQ proteomics approach mapping CRC proteomeprofile. There were802proteins been identification, of which68proteins were defined asdifferent proteins with the standard of P﹤0.05and the ratio of cancer tissuecontent/normal mucosa content≥1.5or≤0.5, and these68proteins include33up-regulatedproteins and35down-regulated proteins. According to annotations of the UniProtknowledgebase (Swiss-Prot/TrEMBL) and the Gene Ontology (GO) Database, theseproteins localizes in nuclear, plasma membrane, extracellular matrix, and so on. ThenEHD2and PDLIM7were selected to undergo validation.EHD2was verified to be downregulated in74.14%CRC cancer tissue byimmunohistochemistry. Via western-blotting EHD2were verified to be downregulated inCRC cancer tissue. The q RT-PCR demonstrated a result same as in Western-blotting.PDLIM7was verified to be downregulated in70.69â„…CRC cancer tissue byimmunohistochemistry. Via western-blotting PDLIM7were verified to be downregulatedin CRC cancer tissue. The q RT-PCR demonstrated a result same as in Western-blotting.Conclusions1. The different protein profile of CRC tissue has been obtained through LC/MS-basediTRAQ proteomic approach in this study, which would provide new information aboutthe characterize of CRC;2. EHD2down-regulated in CRC cancer tissue, which indicated EHD2potentially to be atumor suppressor gene which would act as a new therapy target.3. PDLIM7down-regulated in CRC cancer tissue, which indicated EHD2potentially tobe a tumor suppressor gene which would act as a new therapy target.