| Objective: To explore the changes of protein expressed levels in exfoliated cells of cervical epithelium in patients with HPV(Human papillomavirus)16 positive cervical cancer,and screen out the differentially expressed proteins in the HPV16 positive cervical cancer compared with the HPV negative normal cervix,to explore the relationship between the occurrence and development of HPV16 positive cervical cancer and protein expressed levels are dysregulated.The major functions,the major involved signaling pathways of these differentially expressed proteins,and the interaction between differentially expressed proteins were analyzed by bioinformatics,to preliminarily explore the specific target protein molecules related to the occurrence of HPV16 positive cervical cancer and the possible mechanism.Methods: 1)Clinical specimen collected: HPV genotyping was detected in the affiliated hospital of Guizhou Medical Uuniversity and the affiliated tumor hospital of Guizhou Medical University from December 2017 to July 2018,cervical exfoliated cells with only HPV16 subtype positive or HPV negative were collected and stored at-80°C.And the pathologic diagnosis was diagnosed as normal cervix or cervical cancer.2)Grouping: HPV negative normal cervix(group A)and HPV16 positive cervical cancer(group B),and three biological replications were performed in each group,A1,A2,A3.and B1,B2,B3 respectively.15 cases in each repeat group,and 90 cases in total.3)quantitative proteomics research based on TMT(Tandem Mass Tag)-LC-MS/MS(Liquid chromatography-mass spectrometry/mass spectrometry)technology was conducted on the HPV negative normal cervix and HPV16 positive cervical cancer group,and the differentially expressed proteins between the groups were screened out.Then analysis the main function,involving the main signaling pathways and the interaction of these differentially expressed proteins.3)To verify the accuracy and reliability of proteomics data,Western Blot was used in this study to detect the part of differential proteins.5)The correlation between candidate proteins and PI3K-Akt proliferation apoptosis-related proteins was preliminarily analyzed using the STRING technology platform.Results: 1)351 differentially expressed proteins were identified in the HPV16 positive cervical cancer,including 247 upregulated proteins and 104 down-regulated proteins.These differentially expressed proteins are mainly involved in "Single-multicellular organism process" 、"multicellular organismal process" and "negative regulation of biological process".extracellular membrane-bound organelle" and "extracellular exosome" of cell component;"Protein binding" and other functions of molecular functional.These differentially expressed proteins are mainly involved in "PI3K-Akt","coagulation and complement system"and "ECM-receptor interaction" signaling pathways.2)Western Blot results: Compared with HPV negative normal cervix,PRDX2,GRP94 and G6 PD protein levels were significantly up-regulated in HPV16 positive cervical cancer group,while CSTA and Involucrin were significantly down-regulated(P<0.05).The results were consistent with TMT-LC-MS/MS proteomics.3)PRDX2,GRP94 and G6 PD are correlated with Akt and P53 proteins of the PI3 K Akt signaling pathway,and the correlation between GRP94 and Akt is stronger than the other two proteins.Conclusion: 1)Compared with HPV16 negative normal cervix,HPV16 positive cervical cancer were found to have differentially expressed proteins.2)The up-or down-regulation of the expressed level of these differential proteins may be correlated with the occurrence and development of HPV16 positive cervical cancer.3)Bioinformatics analysis was used to understand the biological functions of these differential proteins,the involved signal pathways and the correlation between candidate proteins and PI3K-Akt pathway,and the key protein molecule GRP94 was screened,to provide a preliminary clue for further study on the mechanism of HPV16 positive cervical cancer. |
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