| Peptidyl-prolyl cis-trans isomerase(Pinidyl-prolyl cis-trans isomerase)is abnormally activated in a variety of tumors,and acts on its substrate or related signaling pathways to promote tumorigenesis and development.Cervical squamous intraepithelial lesions(SIL)are precancerous lesions.Low-grade squamous intraepithelial lesions(LSIL)have a good prognosis,but the progression of LSIL to high-grade squamous intraepithelial lesions(HSIL)greatly increases the risk of cervical cancer.There is currently no indicator for effective risk assessment.Due to the unique role of Pin1 in the initial stage of tumors,detecting Pin1 expression in the precancerous lesion stage is of great significance in developing the risk stratification of the precancerous lesion stage.Cervical cancer metastasis and decreased chemical sensitivity are the main causes of treatment failure.Pin1 can bind to downstream substrates,catalyze cis-trans isomerization changes,regulate downstream targets,and play an important role in promoting tumorigenesis.Pin1 has a wide range of substrates,including c-Jun,AKT,cyclinD1,?-catenin,and STAT3.Pin1 activates a variety of oncogenes and growth promoters to promote carcinogenesis,while Pin1 inhibitors can significantly inhibit its carcinogenesisPin1 targeted therapy has shown good results in human acute promyelocytic leukemia,liver cancer and breast cancer.KPT-6566 is a novel Pin1 specific inhibitor,which covalently binds Pin1 to lead to the degradation of Pin1 structure.KPT-6566 exert high degree of targeting and small effect on non-tumor cells,and has good prospects for transformation.Cisplatin is the first-line chemotherapeutic drug for the treatment of cervical cancer in China.It has the problems of high-dose side effects and the decreased chemical sensitivity of cervical cancer cells to cisplatin.Explore the feasibility of the combined application of KPT-6566 and cisplatin can provide new ideas for clinical cervical cancer medication.This essay intends to examine the expression of Pin1 in cervical SIL and cervical cancer tissues,analyze the risk stratification of LSIL and its clinical significance;discover the effect of Pin1 on the proliferation and metastasis of cervical cancer cells,evaluate the possibility of Pin1 and its therapeutic target;explore KPT-6566 The feasibility of improving the efficacy of cisplatin and providing a scientific basis for improving the diagnosis and treatment of cervical cancer.Method1.Expression and clinical significance of Pin1 and c-Jun in cervical SIL and cervical cancer tissues(1)Expression and clinical significance of Pin1 and c-Jun in SIL tissue: Immunohistochemical staining was used to detect the expression of Pin1 and c-Jun in 72 SIL tissues,and the relationship with the progression of LSIL was analyzed,as well as the relationship between Pin1 and c-Jun expression.(2)Expression and clinical significance of Pin1 and c-Jun in cervical cancer tissues: Immunohistochemical staining was used to detect the expression of Pin1 and c-Jun in 28 cervical cancer tissues,the relationship with clinical parameters of patients as well as the correlation between Pin1 and c-Jun were analyzed.Western Blot method was used to further verify the expression of Pin1 and c-Jun in 12 patients with different stages of cervical cancer.2.Establishment and identification of Pin1 inhibition system(1)Knockdown Pin1 in Hela and Siha cells: Liposome transfection to silence Pin1 in cervical cancer Hela and Siha cells,establish Hela-shPin1 and Siha-shPin1 cell lines,and identificated by RT-PCR and Western Blot.(2)Synthesis and identification of new Pin1 inhibitor KPT-6566: Chemical synthesis of new Pin1 inhibitor KPT-6566,and identification of molecular weight and chemical structure of the compound by mass spectrometry and nuclear magnetic resonance proton spectroscopy.(3)KPT-6566 inhibits the expression of Pin1 and related proteins: Western blot was used to detect the expression of Pin1 and related signaling pathways and some substrate proteins after KPT-6566 treated Hela and Siha cells.3.Pin1 as a novel target for cervical cancer treatment(1)Inhibit Pin1 on the proliferation of cervical cancer cells: CCK8 cell proliferation curve,two-dimensional clonal colony formation test to detect the effect of silencing Pin1 on cervical cancer cells proliferation.(2)Inhibit Pin1 on the migration and invasion ability of cervical cancer cells: Cell scratch test and Transwell invasion test were used to detect the effects of inhibited Pin1 on the migration and invasion ability of Hela and Siha cells.Nude mouse tumor cell liver metastasis model was used to observe the effect of inhibition of Pin1 expression on cervical cancer cell metastasis.(3)Effect of Pin1 on EMT of cervical cancer cells via c-Jun / Slug: Western blot was used to detect the protein expression levels of c-Jun,Slug,E-cadherin,N-cadherin and Vimentin.Overexpressed c-Jun inHela-shPin1 and Siha-shPin1 cell lines.Western Blot was used to detect the expression of EMT-related proteins.4.Effect of KPT-6566 on the cisplatin killing effect of cervical cancer cells(1)KPT-6566 affects the killing effect of cisplatin on cervical cancer cells: Transmission electron microscopy to observe the ultra-morphological structure of cells treated with KPT-6566 and cisplatin;flow cytometry to detect cell apoptosis;Western Blot to detect apoptosis-related Protein cleaved-caspase3 and cleaved-PARP expression.(2)Effect of KPT-6566 on the anti-tumor effect of cisplatin in nude mice xenograft tumor model: The effect of KPT-6566 combined with cisplatin on the tumorigenic ability of cervical cancer cells in vivo was tested in nude mice;immunohistochemistry Staining was used to detect Pin1 expression in transplanted tumor tissue;H&E staining was used to observe xenograft tumor tissues and mouse main oxygen tissues;TUNEL test was used to detect apoptosis in transplanted tumor tissue.Result1.Expression of Pin1 and c-Jun in cervical SIL tissue and cervical cancer tissue and its clinical significance(1)Expression and clinical significance of Pin1 and c-Jun in cervical SIL tissue: 35 cases of Pin1 positive in 72 LSIL,of which 14 cases progressed to high grade(40%);37 cases were Pin1 negative,3 cases progressed HSIL(8%);Pin1 expression was associated with progression from LSIL to HSIL(P= 0.001).The above results suggest that Pin1 can be used as an indicator to judge the progress of LSIL.In 72 LSIL tissues,25 were c-Jun negative,5 of which progressed to HSIL;of the 47 c-Jun positive LSIL tissues,12 progressed to HSIL(25%).The above results indicate that c-Jun expression has nothing to do with the progression from LSIL to HSIL(P = 0.599).Moreover,in LSIL tissues,Pin1 was positively correlated with c-Jun expression levels.(2)Expression and clinical significance of Pin1 and c-Jun in cervical cancer tissues: Pin1 was positive in 18 cases of 28 cervical cancer tissues.Pin1 expression was related to cervical cancer lymph node metastasis(P= 0.039)and proliferation index Ki67(P= 0.014);it was not related to the patient's stage and tumor size of patients.Of the 28 cases of cervical cancer,26 were c-Jun positive and 2 were negative.The expression of c-Jun was related to the cervical cancer proliferation index Ki67,and was not related to the age of cervical cancer patients,lymph node metastasis,and tumor volume(P= 0.002).The correlation of immunohistochemical staining results showed that Pin1 and c-Jun expression levels were positively correlated in cervical cancer tissues(P= 0.049).Western Blot results showed that the expression levels of Pin1 and c-Jun proteins in cervical cancer tissues were significantly higher than those in cervical inflammation tissues.The above results suggest that Pin1 can be used as an index for judging the progression of LSIL;abnormal Pin1 expression in cervical cancer tissues may be related to malignant phenotypes such as proliferation and metastasis of cervical cancer cells.2.Establishment and identification of Pin1 inhibition system(1)Knockdown Pin1 in Hela and Sihacells: Hela-shPin1/Hela-shNC and Siha-shPin1/Siha-shNC cell lines were successfully obtained and identified.(2)Synthesis and identification of the new Pin1 inhibitor KPT-6566: KPT-6566 was synthesized and obtained,the molecular weight was confirmed by mass spectrometry,and the chemical structure was confirmed by nuclear magnetic resonance hydrogen spectrum.(3)KPT-6566 suppressed Pin1 related protein expression: Western blot results showed that after KPT-6566 treatment,the expression of Pin1,c-Jun,?-catenin,cyclinD1,AKT-p473,ERK1/2,p65/NF-?B,and GSTP1 were decreased,and ?-H2 AX was increased.These results show that we successfully obtained silent Pin1 cell lines and synthesis of KPT-6566 for subsequent experiments.3.Pin1 as a novel target for cervical cancer treatment(1)Inhibit Pin1 on the proliferation of cervical cancer cells: Hela-shPin1 and Siha-shPin1 cells viability and colony-forming ability were significantly lower than control group;KPT-6566 treated Hela and Siha cells vitality and clone-forming ability were significantly lower than that in the control group.(2)Inhibit Pin1 on the migration and invasion ability of cervical cancer cells: The scratch test showed that the healing distance of Hela-shPin1 and Siha-shPin1 cells were lower than that of the control group;Transwell experiment showed that the cell number of passing through the compartment was significantly less than the control group.The wound healing distance of Hela and Siha cells after KPT-6566 treatment was significantly lower than that of the control group of the control group;the cell number of passing through the chamber was significantly less than that of the control group.(3)Effect of Pin1 on EMT of cervical cancer cells via c-Jun / Slug: Compared with the control group,the expressions of c-Jun,Slug,N-cadherin and Vimentin in Hela-shPin1 and Siha-shPin1 cells were decreased,and E-cadherin was increased compared with the control group,the expression of c-Jun,Slug,N-cadherin and Vimentin decreased and the expression levels of E-cadherin increased after KPT-6566 treatment.After Hela-shPin1 and Siha-shPin1 cells overexpressed c-Jun,the expression of Slug,N-cadherin and Vimentin increased,and E-cadherin was decreased.The above results show that inhibition of Pin1 significantly reduces the proliferation and exercise capacity of cervical cancer cells,and Pin1 can be used as a potential target for cervical cancer treatment.4.KPT-6566 enhances the killing effect of cisplatin on cervical cancer cells(1)KPT-6566 significantly enhanced the killing effect of cisplatin on cervical cancer cells: Transmission electron microscopy showed that low-dose cisplatin combined with KPT-6566 caused cervical cancer cell membranes to rupture,apoptotic bodies appeared,and the cells disintegrated.Flow cytometry showed that KPT-6566 significantly increased the cisplatin-mediated apoptosis rate;Western Blot showed that the expression of cleaved-caspase3 and cleaved-PARP in the combined treatment group was significantly increased.The above results indicate that KPT-6566 enhances the response of cervical cancer cells to cisplatin treatment.(2)KPT-6566 enhances the anti-tumor effect of cisplatin in nude mice xenograft tumor models: The results of nude mice xenograft tumor experiments show that the volume and quality of xenograft tumors in the combined treatment group are significantly smaller than those in the control group,and the necrosis area of the transplanted tumor is larger than the control group,and the number of apoptotic cells is more than that of the control group.H&E staining results showed that the combined treatment had no side effect to the main oxygens of the mice.The above results show that KPT-6566 effectively enhances the responsiveness of cisplatin to cervical cancer cells in vivo.It has potential as a new treatment for cervical cancer.Conclusion1.Pin1 can be used as a biomarker to evaluate the risk of cervical intraepithelial neoplasia.2.KPT-6566 significantly enhances the responsiveness of cervical cancer cells to cisplatin therapy and has potential application value. |
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