| Part ?:IFI16 can be used as a new indicator for the diagnosis of cervical precancerous lesionsThe latest data shows that the incidence and mortality of cervical cancer rank fourth among female tumors.Persistent high-risk HPV infection has been identified as the initial factor in the development of cervical cancer.Although HPV infection is an indispensable factor,it is not enough to cause cancer.Most HPV infections are transient,and it takes a long process from persistent high-risk HPV infection to the occurrence of cervical cancer:persistent viral infection,cervical intraepithelial neoplasia(LSIL-HSIL),and cervical cancer.The LSIL stage is still in the category of benign lesions,with a good prognosis,and more than 70%will resolve within one to two years.The HSIL stage is a monoclonal tumorous lesion with a poor prognosis.The main clinical treatment is surgical resection.Therefore,effectively blocking the progress of LSIL to HSIL will greatly reduce the incidence of cervical cancer,which is depended on early diagnosis and accurate classification of cervical intraepithelial neoplasia.Although there are standardized diagnostic criteria for the pathological diagnosis and classification of cervical intraepithelial neoplasia,they are too subjective to make same decisions in different medical units in China.In addition to morphological observations,p16 immunohistochemistry is a commonly used marker for pathological diagnosis.However,because of diverse modes of p16 staining,it is difficult to make a diagnosis for inexperience.Therefore,there is an urgent need to find and develop more reliable and stable molecular markers to assist in the accurate diagnosis and classification of pathology.Interferon gamma inducible protein 16(IFI16)is a human member protein in the HIN200 family.When foreign substances such as viruses and bacteria invade the body's environment,as an abnormal double-stranded DNA sensor in the nucleus and cytoplasm,IFI16 initiates a natural immune response.The purpose of this study is to reveal the expression characteristics of IFI16 in cervical intraepithelial neoplasia,and compare it with the current clinical application index p16,to formulate more clear and accurate diagnostic criteria.In this study,clinical paraffin specimens of patients with cervical intraepithelial neoplasia were taken as the research object,and the following results were as follows:1.IFI16 is significantly different in different stages of cervical intraepithelial neoplasiaThe research group selected 119 cases of cervicitis,LISL,and HSIL cases to construct tissue chips and then performed IFI16 immunohistochemistry.It was found that with the progress of cervical intraepithelial neoplasia,the expression of IFI16 gradually increased and the differences between the groups were obvious.The difference of IFI16 expression in different lesion types is not only manifested in different cases,while in the same slice field of view,the difference between different lesion areas is still significant.2.The clinical application value of IFI16 is better than p16The current gold standard for the diagnosis of cervical precancerous lesions is the change in cell morphology,which mainly relies on the molecular marker p16,so IFI16 and p16 are compared.Taking pathological diagnosis as the gold standard and dividing histochemical intensity into 3,we found that the sensitivity of p16 was higher than that of IFI16,but its specificity was lower than that of IFI16.ROC curve analysis shows that the area under the curve of p16 is 0.801,while IFI16 is 0.974,indicating that the clinical application value of IFI16 is higher than p16.3.IFI16 expression is normal in cases with abnormal p16 expressionWe selected cases with abnormal p16 expression,then performed immunohistochemical staining of IFI16.IFI16 was moderately positive in HSIL cases with low p16 expression,while in LSIL cases with high p16 expression,IFI16 was negative.In summary,IFI16 is significantly different in different stages of cervical intraepithelial neoplasia,and its clinical application value is better than p16,so IFI16 can be used as a new diagnostic indicator for cervical intraepithelial neoplasia.Part ?:Expression Characteristics and Biological Behavior of IFI16 in Cervical CancerAs we all know,the occurrence and development of cervical cancer are closely related to HPV infection.As a virus infection-related tumor,there must be an immune response in the body during its persistent viral infection.From the perspective of the body's antiviral immune system,looking for significant inhibitors of cervical cancer development may open up new directions for cervical cancer treatment.The abnormal expression of IFI16 is closely related to the occurrence of a variety of tumors,and its expression and biological effects have tissue differences in different tumors and have not been studied in cervical cancer.The occurrence and development of cervical cancer is accompanied by the persistent infection of HPV virus.In theory,the expression of IFI16 should also continue to increase in cervical cancer.Surprisingly,IFI16 showed a tendency to increase first and then decrease during the persistent HPV infection-LSIL-HSIL-CCa.And the expression of IFI16 was negatively correlated with the stage of cervical cancer patients,and negatively correlated with the maximum tumor area.Whether IFI16 is down-regulated,then the cervical tissue progresses from HSIL to CCa,or after progressing to CCa,IFI16 is down-regulated due to its role.Clarifying this issue will further demonstrate the molecular mechanism of CCa pathogenesis and seek new target for CCa.In this study,the biological effects of IFI16 were detected by tissue testing,cell experiments and animal experiments,and the results were as follows:1.IFI16 is down-regulated in cervical cancerImmunohistochemistry showed that compared with HSIL,IF116 expression decreased in cervical cancer.Not only in HSIL cases and CCa cases,but also in CCa cases with surrounding HSIL lesions,the expression of IFI16 in cancer tissues is still lower than that in HSIL tissues.2.IFI16 exerts suppressing effect in cervical cancerThe expression of IFI16 in CCa and its correlation with clinical indicators were analyzed by IFI16 immunohistochemistry.The results showed that the expression of IFI16 was negatively correlated with cervical cancer stages,which is not related with lymph node metastasis.We constructed IFI16 overexpression plasmids,siRNA and shRNA viruses,and respectively transfected siha and C33a.CCK8 and EDU were used to detect the proliferation ability of cells,transwell to detect the migration and invasion ability of cells,and clone formation to detect the cloning ability of cells.The results showed that the proliferation,migration,invasion,and cloning ability of cervical cancer cells were improved after interfering with IFI16,while overexpression of IFI16 reduced the proliferation of C33a cells.Subsequent tumor-bearing experiments in nude mice showed that compared with the control group,the tumor tissue of the knock-out IFI16 group was larger,heavier,and had stronger fluorescence intensity.The research group concluded that IFI16 exerts suppressing effect in cervical cancer.3.Preliminary exploration of the mechanism of down-regulation of IFI16 in cervical cancerWe performed immunohistochemistry on minimally invasive carcinoma,the initial stage of cervical cancer-cervical,which showed that compared with HSIL,there was no significant difference in the expression of IFI16 between HSIL lesions and micro-invasive lesions.This result suggests that due to the tumor suppressive effect of IFI16,it will be reduced in some way after HSIL progresses to a tumor.In summary,IFI16 exerts suppressing effect in cervical cancer.Compared with HSIL,the decreased expression level of IFI16 in cervical cancer is caused by a mechanism after cervical cancer.Part ?,TRIM21 promotes cervical cancer progression through ubiquitinated IFI16IFI16 plays a powerful role in suppressing cancer in the progress of cervical cancer.However,compared with HSIL,the expression of IFI16 in cervical cancer decreased,and the expression level of IFI16 was negatively correlated with the stage of cervical cancer.The mechanism of down-regulation of IFI16 may be the key link for tumor cells to escape from the virus immune mechanism.Clarifying and intervening the down-regulation mechanism of IFI 16 will play an important role in inhibiting the malignant progress of cervical diseases and is expected to become a new therapeutic target for related diseasesThrough database analysis and comparison of extracted mRNA from cervical lesion tissue,we found that there is no statistical difference between HSIL and cervical cancer at the mRNA level,which suggests that the decreased expression of IFI 16 in cervical cancer is related to protein stability.After determining that IFI 16 was degraded by the ubiquitination-proteasome pathway,we used protein immunoprecipitation-mass spectrometry to find E3 ubiquitin ligase.Through verification,we found that the E3 ubiquitin ligase TRIM21 binds to IFI 16.TRIM21 may be the core regulatory molecule for down-regulation of IFI 16 expression in cervical cancer.TRIM21 belongs to the TRIM family,which has more than 100 members currently.It is known as the E3 ubiquitin ligase family because it generally has a Ring-finger domain.TRIM21 is located on chromosome 11 and has a molecular weight of 52.It is also called Ro52 and includes four domains:RING,B-box,coiled-coil,and PRYSPRY.The abnormal expression of TRIM21 is closely related to the occurrence of a variety of tumors,but its role in tumors has tissue differences.It exerts a cancer-promoting effect in tumors such as nasopharyngeal carcinoma,bladder cancer,and glioma,and a cancer-suppressing effect in tumors such as breast cancer and liver cancer.The role of TRIM21 in cervical cancer has not been studied.There are three problems to solve:the biological effect of TRIM21 in cervical cancer,the regulatory effect and specific mechanism of TRIM21 on IFI16 and the role of TRIM21-IFI16 axis in the progress of cervical cancer.It is helpful to understand the influence of antiviral immune response in the occurrence and progression of malignant diseases caused by infection HPV.In this study,the biological effects of IFI16 were detected by tissue testing,cell experiments and animal experiments,and results were as follows:1.Down-regulation of IFI16 expression in cervical cancer is associated with ubiquitinationThrough database analysis and comparison of extracted mRNA from cervical lesion tissue,we found that there is no statistical difference between HSIL and cervical cancer at the mRNA level,which suggests that the decreased expression of IFI16 in cervical cancer is related to protein stability.The degradation pathway of IFI16 was confirmed by adding the ubiquitinated proteasome inhibitor MG 132 and the autophagolysosome pathway inhibitor chloroquine.Therefore,the down-regulation of IFI16 expression in cervical cancer is related to ubiquitination.2.TRIM21 binds to IFI16 and regulates IFI16 at the protein levelWe verified that IFI16 and TRIM21 were combined in siha and 293t tool cells through the Co-IP experiment,and then GST-pull down experiment proved that they can combined directly.After interfering with TRIM21 in siha,we found that compared with the control group,IFI16 protein levels were up-regulated in interfering with TRIM21 group,and vice versa.In immunofluorescence,the relationship betweenTRIM21 and IFI16 is same.It shows that TRIM21 and IFI16 have a certain co-localization.Immunohistochemistry showed a negative correlation between the two at the tissue level.The above results indicate that TRIM21 can negatively regulate x IFI16.3.The biological effect of TRIM21 in cervical cancerThe expression characteristics of TRIM21 in CCa and its correlation with clinical indicators were analyzed by TRIM21 immunohistochemistry.The results show that TRIM21 is highly expressed in cervical cancer and is positively correlated with cervical cancer stage.CCK8 and EDU showed that the proliferation capacity of cells was weakened after TRIM21 interference.Transwell showed that the migration and invasion ability of cells was weakened after TRIM21 interference,and clone formation showed that the ability of clone formation was weakened after TRIM21 interference.The nude mice tumor-bearing experiment showed that the tumor tissue volume,weight and fluorescence intensity were enhanced after TRIM21 was knocked out.So far,the research group has concluded that TRIM21 exerts a cancer-promoting effect in cervical cancer.4.TRIM21 promotes cervical cancer progression by reducing IFI16In order to prove that TRIM21 promotes cervical cancer progression by regulating IFI16,we first conducted the regulation of TRIM21 on IFI16 to affect its downstream.WB showed that after interfering with IFI16,pro-IL-1? increased,and TRIM21 overexpression showed the same effect after.Subsequent rescue experiments showed that the up-regulation of IFI16 caused by the interference of TRIM21 was backfilled by the interference of IFI16.The knock-down of TRIM21 cell reduced the proliferation,invasion,migration,clone formation ability in siha,which is compensated by knock-down of IFI16.Nude mice tumor-bearing experiments showed that knock-down of TRIM21 cell reduced the volume,weight and fluorescence intensity,which is compensated by knock-down of IFI16.Based on this,it is concluded that TRIM21 promotes cervical cancer progression through IFI16.5.TRIM21 regulates IFI16 through ubiquitinationAfter overexpressing or interfering with TRIM21,IFI16 protein level showed a negative correlation and mRNA level did not change.In addition,TRIM21 is a well-known E3 ubiquitin ligase,so that we considered the ubiquitination pathway.The cycloheximide protein tracing experiment showed that the protein stability of IFI16 was up-regulated after interfering with TRIM21,and the ubiquitination experiment confirmed that overexpression of TRIM21 caused the increase of IFI16's ubiquitination level.It is concluded that TRIM21 regulates IFI16 through ubiquitination.To sum up,it is concluded that TRIM21 can down-regulate IFI16 through ubiquitination and promote cervical cancer progression. |
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