Studies Of The Expression Profile Of Downregulated Proteins Associated With Cervical Carcinoma And HPV Infection

Objective: Cervical cancer is a gynecological malignancy,which occurs in women of Uyghur ethnic group with very high morbidity and mortality,known as a cancer of high incidence in Xinjiang,China.Early detection is crucial for efficient cancer treatment,and thus the study of early prognostic markers is the prerequisite for the establishment of early diagnostic methods.Since the infection by human papillomavirus(HPV)is the main etiological factor in cervical carcinogenesis,HPV screening became an important test for early prognosis of cervical cancer.However,cervical cancer development after HPV infection may have a latent period of at least 5-10 years,and requere auxilliary diagnostic markers.Here,we analyzed tissue specimens of cervical cancer by proteomics,and studied the tissue specific downregulated protein profile associated with cervical carcinogenesis and HPV infection,and provide evidence for revealing the mechanism and early diagnosis of cervical cancer.Methods:(1)Collection of biological specimen: For proteomics analysis and detection of candidate proteins by quantitative RT-PCR,we collected 87 cases of fresh cervical tissue specimens from female patients of Uyghur and Han Chinese ethnic groups,including 53 cases of cervical squamous cell carcinoma(CSCC)and 34 cases of cervicitis or normal cervix(NC);In addition,116 cases of paraffin-embedded tissue samples,47 cases of CSCC,37 cases of CIN II-III and 32 cases of NC,were collected for immunohistochemistry tests;(2)DNA was extracted from tissues,HPV positivity and genotypes were detected.we choose HPV native cervicitis,positive CSCC,Then study the relationship between HPV infection and expression regulation of candidate markers of cervical cancer and precancerous lesions;(2)Proteomics analysis: For separate analysis of Uyghur and Han-Chinese women,two groups of fresh tissue samples,8 samples of each,were prepared by selecting 4 cases of HPV-positive samples of cervical carcinoma and 4 cases of HPV-negative samples of normal controls;These samples were subjected to proteomics analysis for identification of differentially expressed proteins,the platform of isobaric tags for relative and absolute quantitation(iTRAQ)provided by Huada Proteomics Research Center,Beijing China;(3)The specificity and accuracy of proteomics data were verified by analysis of some genes coding for candidate proteins in tissue RNAs from cervical carcinoma and normal controls at the mRNA expression level,using mRNA-specific primers and fluorescent qRT-PCR method;(4)The expression levels of candidate proteins were further verified by immunohistochemistry test.Results:(1)Proteomic analysis identified 66 proteins that were differentially expressed in cervical carcinoma compared with controls,setting greater than 1.2 fold-change of differential expression of proteins as standard for quantitative comparison,and focusing on simultaneous changes in the protein profile of both Uyghur and Han women.Among them,32 and 34 proteins were up-and downregulated,respectively.(2)In line with the fold-change of protein expression and combined with literiture search,we selected five proteins(genes),CAV1,FBN1,TGM2,KANK2 and AGT,as candidates for verification.qRT-PCR analysis confirmed a significant decrease in the transcription of these genes in cervical carcinoma compared with normal controls(P<0.05).ROC curve analysis showed the detection of these genes at transcriptionla level has relatively high sensitivity(70.6?88.9%),accuracy(65.6?77.5)and specificity(68.2%).In accordance with Pearson correlation analysis,the differential expression of KANK2 may have a medium correlation(r,0.40?0.55)with CAV1,FBN1 and AGT.Since 94% of cancer patients were infected with HPV,and most of which with HPV16,whereas this accounted for 11.7% in women with cervicitis or normal cervix,the dowregulation of genes described above may be associated with HPV infection,in particular with HPV16.(3)Immunohistochemistry analysis demonstrated that the proteins expression of KANK2,CAV1 and FBN1 were strong positive in cervicitis,and shifted to medium-or weak-positive expression in cancer and CIN tissues,with statistical significance among three groups(P<0.05).In addition,the expression of these proteins in cancer or CIN tissues were significant compared with cervicitis,but no difference was found between cancer and CIN tissues(P>0.05).Conclusion:(1)The development and progression of cervical cancer is closely associated with the differential expression of multiple proteins in a tissue-specific manner;(2)The downregulation of CAV1,FBN1,TGM2,KANK2 and AGT at transcription and protein level are prognostic markers for cervical cancer,which might be dependent on HPV16 infection;(3)This study provides evidence for revealing mechanisms and prognosis of cervical cancer development.