Function And Mechanism Studys Of LYN In Cervical Cancer

Cervical cancer is one of the most common malignancies in women around the world.Tumor occurrence and progression is very complex.Although HPV persistence infection is the main reason of tumorigenesis,but not all patients with HPV infection will develop into cervical cancer,suggesting that HPV infection is the necessary,but not sufficient conditions for the occurrence of cervical cancer.In the process of cervical cancer tumorigenesis,development and metastasis,there are many genes and proteins involved and their specific mechanism has not yet fully elucidated.So looking for the novel proteins of cervical cancer during tumor occurrence and progression can be served with new targets of tumor diagnosis and treatment.And also can provide new diagnosis and treatment ideas.iTRAQ is a kind of relative and absolute quantitative technique for in vitro isotope labeling,which has been widely used in many tumor biomarkers.In this study,iTRAQ technique was used to analyze the difference of differential protein expression between cervical cancer and normal cervical tissue.Search for the specific proteins in the differential proteins which were closely related to the occurrence and development of cervical cancer.The selected protein molecules in-depth analysis,clear its occurrence and development in the process of cervical cancer in the process.Therefore,the subject from the following three parts to study.In-depth analysis of selected protein molecules,and then study its mechanism in the development and progression of cervical cancer.Therefore,we will conduct this study in the following three part.PART ? DIFFERENTIAL PROTEOMICS RESEARCH OF CERVICAL CANCER TISSUES AND NORMAL CERVICAL TISSUESObjcetive: Proteomics were used to screen out the differential proteins of cancer and normal cervical cancer and explore the meaningful potential targets.Methods:(1)iTRAQ tandem mass spectrometry was used to identify and quantitatively analyze the differential proteins in cervical cancer tissues and normal cervical tissues.(2)Western blot was used to validate the results of iTRAQ.(3)GO-Analysis and Pathway-Analysis were used to analyze functional and related signal path of the differential proteins screened by iTRAQ.Results:(1)3300 aberrantly proteins with 95% confidence interval were identified by iTRAQ.Furthermore,in order to reduce the occurrence of false positives,at least 1.3 times as the standard for we select the differential proteins.Finally,330 differential proteins were selected,including 137 up-regulated proteins and 195 down-regulated proteins.(2)Western blot results showed that the expression of LYZ,ORM1,LYN and STMN1 in cervical cancer tissues were significantly higher than that in normal cervical tissues.The expression of LUM,BGN and KRT4 in cervical cancer tissues was significantly lower than that in normal cervical tissues.The results showed that the expression of differential protein was consistent with that of iTRAQ combined with mass spectrometry.Indicating the the results of Western blot was consistent with that of iTRAQ combined with mass spectrometry.(3)The results of GO-Analysis showed that the main function of up-regulated differentially expressed proteins was involved gene expression,mitotic cell cycle,apoptotic process and so on.The main function of down-regulated differentially expressed proteins was involved extracellular matrix organization,negative regulation of endopeptidase activity,muscle contraction and so on.The results of Pathway-Analysis showed that the up-regulated differentially expressed proteins are involved in many signaling pathways,among which the five signaling pathways are the most common,namely RNA transport,spliceosome,DNA replication,NF-kappa B signaling pathway and cell cycle.The five most common signaling pathways which were related to the down-regulated differentially expressed proteins are focal adhesion,complement and coagulation cascades,vascular smooth muscle contration African trypanosomiasis and amoebiasis.Conclusion:(1)iTRAQ is an important tool for the current proteomics research,and it is also a reliable tool and platform for studying the performance of cervical cancer-specific biomarkers.(2)The protein involved in the occurrence and progression of cervical cancer is extremely complex.PART ? THE EXPRESSION AND CLINICOPATHOLOGICAL ASSOCIATION OF LYN EXPRESSIONG IN CERVICAL CANCERObjective: This study aimed to detect the expression of LYN in cervical cancer tissues,para-cancerous tissues and normal cervical tissues,and explore the correlation between LYN expression and clinicopathological parameters of cervical cancer.Methods:(1)The expression of LYN in cervical cancer tissue microarray was detected by immunohistochemistry.(2)The correlation between the expression of LYN and the clinicopathological parameters of cervical cancer was statistically analyzed.Results:(1)Compared with para-cancerous tissues and normal cervical tissues,the expression of LYN was significantly up-regulated in cervical cancer tissues and the expression of LYN was gradually increased with the progression of tumor.(2)In cervical cancer tissues,the high expression of LYN was positively correlated with FIGO staging and tumor grade in cervical cancer.The difference was statistically significant.There was no significant correlation between age and tumor type.Conclusion:(1)LYN is up-regulated in cervical cancer and plays an oncogene role in cervical cancer;(2)The expression of LYN expression has positively correlated with FIGO staging and tumor grade in cervical cancer,indicating that LYN plays an important role in the development of cervical cancer.PART ? LYN INVOLVED IN REGULATION OF BIOLOGICAL BEHAVIOR OF CERVICAL CANCER CELLS AND THE POTENTIAL MECHANISMSObjective: To investigate the role of LYN in the biological behavior of cervical cancer,such as proliferation,invasion and migration,and its related mechanisms.Methods:(1)Western blot and RT-PCR were used to detect the expression of LYN in different cervical cancer cell lines.(2)Lentivirus were used to slience or overexpression LYN;(3)Plate clone formation assays and CCK-8 assays were used to detect the proliferation ability of cervical cancer cells after silencing or overexpression LYN.(4)Transwell migration experiment and scratch test were used to detect the migration ability of cervical cancer cells after silencing or overexpression LYN.(5)Transwell invasion assay was used to detect the invasion of cervical cancer cells after silencing or overexpression LYN.(6)The changes of cytoskeleton of cervical cancer cells after silencing or overexpression of LYN were detected by phalloidine(7)Co IP was used to detect the interaction between LYN and p-STAT3.(8)Western blot was used to detect the expression of IL-6R,STAT3 and p-STAT3 in IL-6 / STAT3 signaling pathway after silencing or overexpression LYN.(9)Western blot was used to detect the optimal concentration of IL-6 in cervical cancer cells.(10)Western blot was used to detect the expression of IL-6R,STAT3 and p-STAT3 after IL-6 treatment in cervical cancer cells;(11)Effect of subcutaneous tumorigenesis ability was detected by subcutaneous tumor formation in nude mice after silencing or overexpression of LYN.(12)Immunohistochemistry was used to detect the expression of LYN and p-STAT3.Results:(1)The results of RT-PCR and Western blot showed that the expression of LYN in Si Ha cells was the highest in Si Ha cells in cervical cancer He La,Si Ha and C33 a cell lines,and the expression level of LYN in C33 a cells was the lowest.We selected Si Ha cells as silencing experiments target,C33 a cells as the object of overexpression experiments;(2)GFP fluorescence,RT-PCR and Western blot showed that lentivirus could slience or overexpress LYN expression.(3)Plate clone formation assays and CCK-8 assays showed that silencing or overexpression of LYN could inhibit or promote the proliferation ability of cervical cancer cells.(4)Transwell migration assays and scratch assays showed that silencing or overexpression of LYN could inhibit or promote the migration ability of cervical cancer cells.(5)Transwell invasion assays showed that silencing or overexpression of LYN could inhibit or promote the invasion ability of cervical cancer cells.(6)Phalloidin labeled cytoskeleton of cervical cancer cells showed that: LYN could affect the cytoskeleton of cervical cancer.(7)Co IP experiments showed that p-STAT3 interacted with LYN in vivo.(8)Western blot results showed that silencing or overexpression LYN could regulate the biological behavior of cervical cancer cells through IL-6 / STAT3 signaling pathway.(9)Nude mice subcutaneous tumorigenesis experiments show that silencing or overexpression of LYN in vivo could promote or inhibit tumor growth.(10)Immunohistochemical results showed that: Compared with LV5-NC group,the expression of LYN and p-STAT3 in LV3-LYN group was significantly lower than that in LV5-NC group.Compared with LV5-NC group,the expression of LYN and p-STAT3 in LV5-LYN group was significantly increased.Conclusion:(1)LYN can regulate the proliferation,invasion and migration ability of cervical cancer cells;(2)LYN regulates the invasion and migration of cervical cancer cells through IL-6 / STAT3 signaling pathway,which affects the cytoskeleton of cervical cancer.