| Objectives:Cervical cancer has been occurred in Uighur women with high morbidity and mortality.Early detection will promise an effective treatment of a cancer,whereas the screening of tumor molecular markers is the prerequisite to establish methods for early diagnosis.The formation and survival of tumor cells is just the failure of the body in immune surveillance,represents the switch of the body into a pathological state,associated with the dynamic changes in whole body not only manifested in the subtle deregulation of internal gene expression network of tumor cells,but also integrated into plasma proteome as a fine tuning,in the form of plasma(serum)protein markers.Thus,the identification and the application of such molecular markers in plasma become a cutting edge in the establishment of non-invasive methods in clinical diagnosis.As a systems biology approach,the application of proteomics in the high-throughput,accurate and rapid separation of plasma proteome for the identification of tumor molecular markers followed by the establishment of methods for early diagnosis as well as studies of the molecular mechanisms of cervical carcinogenesis has a very high impact of social economy in the comprehensive prevention and treatment of the cancer in Xinjiang.In this study,we will focus on the plasma of Uighur women with cervical lesions in Xinjiang,apply screening techniques and strategies combining multiple proteomics approachs for the establishment of differential expression profile of plasma proteome followed by identification of candidate plasma protein markers as an important part of a systematic diagnostic profile for preventive screening and early detection of cervical cancer.Additionally,bioinformatic research tools,as a part of systems biology approach,will be applied to analyze the plasma protein marker profile for functional annotation and enrichment,construction of function networks,identify canonical pathways for further reveal the biological nature of cervical pathogenesis and carcinogenesis and provide scientific evidences for the prevention and early diagnosis of cervical cancer.Methods1.69 cases of plasma samples from Uighur women with cervical squamous cell carcinoma(CSCC),cervical intraepithelial neoplasia(CIN Ⅱ/Ⅲ)and cervicitis were collected for the preparation of the low abundant plasma proteome.The plasma proteome were analyzed by two-dimensional liquid phase chromatography(2-D HPLC),using the ProteomeLabTM PF-2D system for separation and quantitative analysis,to establish diferential expression profile of plasma proteins specific to early stage cervical carcinoma and precancerous lesions by setting up cervicitis as control,identify and collect differentially expressed protein components.The protein components were digested with tyripsin and analyzed by high-resolution mass spectrometry using LTQ-MS for the identification of target proteins,i.e.differentially expressed candidate plasma proteins.IPA@ online bioinformatics software was applied to the function analysis of candidate plasma proteins including the functional annotation,regulatory networks,biological function,canonical pathways and biomarker,for further understanding the association of cervical lesion pathogenesis with the normal or abnormal function of candidate plasma proteins.2.The two-dimensional differential in gel electrophoresis(2-D DIGE)and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF/TOF MS were applied for the separation and quantitative analysis of low abundant plasma proteome of CSCC,CIN Ⅱ/Ⅲ and cervicitis,to establish differential 2-D DIGE proteome profile,screen the differentially expressed protein spots and identify candidate plasma proteins.3.Based on the results obtained from the both analysis by proteomics and focused on so called core differential proteins,MetaCoreTM Analysis bioinformatics software was applied to analyze the core differential proteins in 5 aspects such as functional annotation and enrichment,cellular component,biological process,GeneGo network distribution and network construction and biomarker assessment for the regulatory network modeling and further screening of candidate tumor marker proteins.4.We focused on 6 candidate plasma proteins according to the selection criteria mentioned above,quantitatively analyzed the plasma samples from patients with cervical lesions by enzyme-linked immune sorbent assay(ELISA),for the assessment of quantitative and statistical differences of every single candidate protein in plasma,and evaluation of the impact of these plasma proteins as markers for early diagnosis in clinical practice.Results1.We established differentially expression profiles of plasma proteome specific to early stage cervical cancer and/or CIN Ⅱ/Ⅲ by analyzing low abundant plasma proteome samples with high quality,with cervicitis as control.By setting up the change in peak value more than 2 folds as a standard for quantitative differences of protein components in plasma proteome,we identified 8 ascending and 1 descending protein components in plasma of early stage cervical cancer compared to cervicitis with significant differences,while 3 ascending and 1 descending protein components 3 were specific to CIN Ⅱ/Ⅲ.By the specific enzyme cleavage of the protein components and subsequent analysis of their peptide profile with mass spectrometry,we identified 103 proteins specific to early stage cervical cancer and 31 proteins for CIN Ⅱ/Ⅲ.With an IPA@ online bioinformatics software analysis approach,we further screened the online database information of candidate plasma proteins,found that these proteins may be involved in 7 function modules such as inflammatory response etc.;biological functions such as cell assembly and organization,cancer etc.;typical cellular pathways involved in acute phase response pathway,JAK/Stat pathway,IL-4 pathway etc.we also found that biological functions of cervical cancer specific plasma specific serum differential protein are involved in 9 function modules such as cancer,inflammatory response and gene abnormality etc;biological functions involved in genetic defects,ophthalmic disease,cancer,reproductive system diseases etc.and activation of several receptor pathways,acute phase response pathway,B cell maturation and immune deficiency pathways etc.By further application of IPA@ software for biomarker screening,we found that 14 out of 103 candidate plasma proteins specific to early cervical cancer may have marker potential and involved in metabolism associated proteins(APOA1,DC,UBR4),cellular skeleton associated proteins(ADCY2,VIL1),transcription and translation associated proteins(ANK2,ANK3,RelB),signal transduction associated proteins(MADD,SHC1),proliferation and apoptosis associated proteins(BIRC6),immune function-associated proteins(HLA-DQB1).In case of CIN Ⅱ/Ⅲ,2 out of 31 candidate plasma proteins may also have biomarker potential and involved in lipid metabolism associated protein(APOA1)and signal transduction associated protein(mTOR).2.By a proteomics approach with 2D DIGE platform,and setting up cervicitis as controls,we established a differencial expression profile of plasma low abundant proteome specific to early stage cervical cancer and CIN Ⅱ/Ⅲ,identified 43 differencially expressed protein spots for early stage cancer compared to cervicitis,including 18 spots in ascending and 25 spots in descending concentration in plasma.respectively,whereas 16 spots for CIN Ⅱ/Ⅲ,including 7 and 9 spots with ascending and descending concentration,respectively.Further analysis by enzymatic cleavage of protein spots specific to early stage cervical cancer followed by detection with mass spectrometry,we identified 16 plasma proteins,in Which 7 and 9 proteins with ascending and descending content in plasma,respectively,compared to cervicitis.3.By applying the MetaCoreTM bioinformatics software and online database were applied for further analysis of differentially expressed plasma protein profile identified by both proteooics approach described above,we identified 43 core differential plasma proteins specific to early stage cervical cancer.These proteins are involved in various biological activities in the body:① Mainly associated with 15 kinds of diverse biological functions such as negative regulation of cellular composition,reverse transportation of cholesterol,negative regulation of stress response,etce;② Distributed in 12 intracellular compartments,mainly in high density lipoprotein particles;③Participated in lipid metabolism,inflammatory complement system,actin fiber of the cellular skeleton,ESR1 membrane receptor transduction pathways,and inflammatory response IL-6 pathway;④ Belong to 8 function network modules of whole network of protein-protein interaction;⑤ The screening with the key word "cervix disease"resulted in 4 candidate marker proteins closely associated with cervical cancer and lesions,including ATⅢ,CLU,VIL1,and IGK@.The analysis of protein interaction network showed that CLU,VIL and IGK@are involved in the activation of NFκB and COX-2,CLU,whereas CLU and IGK@may also participate in the inhibition of cellular apoptosis mediated by BCL-XL,BAX or BCL-6.4.The analysis of plasma level of 6 candidate proteins by ELISA showed that the development of cervical lesion was accompanied with the gradual decrease in plasma APOA1 and ATⅢ level and increase in APOE,CLU,mTOR and VIL1,with significant differences(P<0.05).Analysis based on clinical classification of the cancer showed that the plasma level of five proteins including APOA1,AT Ⅲ,CLU,mTOR and VIL1 was significantly changed with the development of cervical cancer from early to late stages(P<0.05).Conclusion1.The development of cervical cancer and precancerous lesions(CIN Ⅱ/Ⅲ)is closely associated with the dynamic change in plasma level of multiple low abundance plasma proteins.2.The candidate differentially expressed plasma proteins specific to cervical cancer or precancerous lesions may involved in multiple biological processes in the body,including inflammatory and stress response,the activation of inflammatory-related complement or siggnaling pathways,disruption of energy metabolism,deregulation of lipid metabolism,abnormalities in cellular signal transduction immune response,activation of several receptor pathways.Thus,this analysis may provide important evidences for revealing the molecular mechanisms of cervical carcinogenesis.3.The quantitative changes in plasma level of 6 proteins,including APOA1,APOE,CLU,ATⅢ,mTOR and VIL1,was associated with cervical cancer and its precursors,with high speciifcity and sensitivity,and may become clinical plasma protein markers for early diagnosis of the cancer.4.A combined application of high throughput technical approaches 2D HPLC,2D DIGE and high resolution mass roectrometry is an important strategy in cancer proteomics research,while bioinfonnatics softwares and databases such as IPA@and MetaCoreTM are important auxiliaiy analytic teols in providing with efective technical support on high tihroughput data processing and biomarker screening. |
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