| Objectives:Cervical cancer is gynecological malignancy.Early detection is crucial for efficient cancer treatment,while the screening of markers for early prognosis will provide evidence for early diagnosis of the cancer.The infection by human papillomaviruses?HPVs?is the main etiological factor in cervical carcinogenesis,and thus may associate with early predictive markers of cervical carcinoma.In this study,we focused on cervical cancer tissues that were positive for HPV16 and tissues of normal cervix or cervicitis negative for HPV,and screened candidate proteins that were differentially expressed in tumor tissues by novel iTRAQ-based proteomics,bioinformatics and quantitative verification of protein expression,to confirm the association of cervical carcinogenesis with the regulation of protein expression in tumor tissues and its dependence on HPV infection,and establish a profile for auxiliary diagnosis for cervical carcinoma in addition to HPV screening.The aim of this study is to provide evidence for diagnosis,clinical treatment and the application of HPV vacicines of cervical carcinoma.Methods:1)Seventy-six cases of fresh specimens of uterine cervix were collected from both ethnic Han and Uyghur women with cervical squamous cell carcinoma?CSCC?,cervical intraepithelial neoplasia?CIN?II or III and healthy individuals with normal cervix control?NCC?;2)After extraction of tissue DNAs,HPV infection and high-risk HPVs were detected by quantitative PCR;3)In accordance with the capacity of 8 flex-iTRAQ reagents of labeling and quantifying up to 8 protein samples,we constructed two groups of test samples,with 8 cases of tissue samples in each group,that contained 4 cases of HPV-positive tumor tissues and 4 cases of HPV-negative normal cervix collected either from ethnic Han or Uyghur women,respectively.After protein extraction,enzymatic digestion and labeling,the samples from tumor and controls were subjected to analysis by iTRAQ-based proteomics using the platform provided by Proteomocs Research Center of Beijing Hua-Da Company.By identification of the protein profile changed in,and specific to,cervical cancer tissues and HPV infection,the candidate proteins common to cercical carcinoma of ethnic Han and Uyghur women was evaluated;4)The dysfunction of candidate proteins associated with disease and tumorigenesis was analyzed by bioinformatics using MetaCoreTM software and its database;5)We designed primers specific to mRNAs coding for several candidate proteins that were upregulated in cervical carcinoma tissues and analyzed tissue totally RNAs by fluorescent quantitative RT-PCR to verify the data of proteomics and bioinformatics analysis;6)Several proteins were selected in line with of RT-PCR data and analyzed by immunohistochemistry using 116 cases of paraffin-embedded tissue specimens of cervical carcinoma,CIN and normal cervix?or cervicitis?that were collected by case review,for further verification of the specificity and accuracy of proteomics data.Results:1)By HPV testing and the typing of six high-risk HPVs,such HPV16,18,33,52,58 and 67 in 76 cases of tissue DNA,we detected HPV positivity in 36 cases of CSCC,11cases of CIN II-III,and 5 cases of normal controls,respectively.HPV16 positivity were detected in 28 cases of CSCC,11 cases of CIN II-III,and 5 cases of normal controls,respectively;2)Proteomic analysis identified 58 proteins that were differentially expressed in cervical carcinoma compared with controls,setting greater than 1.2fold-change for upregulation and less than 0.83 fold-change?1/1.2?for downregulation of protein expression as standard for quantitative comparison,and focusing on simultaneous changes in the protein profile of both Uyghur and Han women.Among them,33 and 25proteins were up-and downregulated,respectively;3)We evaluated the role of 58identified proteins by bioinformatics analysis using MetaCore?software and online databases,in several aspects:?1?biological process enrichment distribution:mainly the response to injury,injury repair,acute inflammation reaction and the blood coagulation;?2?subcellular localization enrichment distribution:a part of the protein is located in the extracellular matrix,others are in the collagen,in the extracellular space,and at the cell membrane;?3?The molecular function enrichment distribution:act as a part of molecular structure,extracellular matrix components,growth factor binding functions;?4?a protein enriched pathways:mainly in the glycolysis and gluconeogenesis,protein folding and maturation,dysfunction of mitochondria pathway;4)We selected 10 upregulated proteins?genes?,such as ASAH1,PCBP2,DDX5,MCM5,TAGLN2 and hnRNPA1,ENO1,TYMP,CYC and MCM4,and designed ans synthesized primers specific to mRNA coding for candidate proteins.Quantitative RT-PCR analysis of 76 cases of tissue RNAs showed that the expression of all genes significantly upregulated in tumor tissues compared with normal controls?P<0.05?,as an evidence for specificity and sensitivity of proteomics data,and the expression of PCBP2,MCM5,hnRNPA1,TYMP and CYC was significantly upregulated in CSCC compared with CIN?P<0.05?,whereas only the expression of DDX5 was significant between CIN and normal controls?P<0.05?;5)By immunochemical analysis of candidate upregulated proteins,ASAH1,PCBP2,DDX5 and TAGLN2 and hnRNPA1,in 116 cases of cervical lesions demonstr ated that the expression of ASAH1,PCBP2,DDX5 and hnRNPA1 was significant in CSCC and CIN compared with normal controls?P<0.05?,in consistent with the results of proteomics and RT-PCR analysis,but no difference was found for TAGLN2.Conclusion:1)Cervical carcinogenesis and HPV infection is causally associated with aberrent regulation of protein expression in tumor tissues among ethnic Han and Uyghur women;2)Fifty-eight proteins that were identified in this study are involved in diverse biological processes,while the upregulation of these proteins may promotes cancer development;3)The upregulation of ASAH1 PCBP2,DDX5 and hnRNPA1 may become auxiliary diagnostic profile of cervical carcinoma in addition to HPV screening. |
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