| Backgrounds and ObjectivesOvarian cancer is one of the most common malignant tumors in female genital organs.The incidence rate is inferior to cervical cancer and endometrial cancer.However,the mortality rate is the first place in the female reproductive system malignant tumors.Among the ovarian malignant tumors,epithelial ovarian cancer is the most common.Because of the occult onset,70% of the patients with ovarian cancer are in the advanced stage when they see a doctor.At present,comprehensive treatment of surgery and chemotherapy is the main method for the treatment of ovarian cancer.Chemotherapy resistance is the main reason that affects the therapeutic effect of ovarian cancer.Therefore,it is urgent to explore the molecular mechanism of chemotherapy resistance in ovarian cancer and explore effective diagnosis and treatment methods.Crystallin is the main structural protein in mammalian lens cytoplasm,which is mainly divided into three categories: α,β,γ crystallin family.β B2 crystallin(Crybb2)is the highest protein component in the β-crystallin family.Crybb2 was initially considered as a lensspecific protein,and its stability and solubility are the keys to maintain the transparency and longevity of the lens.Studies have shown that Crybb2 not only acts in the lens but also is a multifunctional protein with physiological and pathological characteristics in non-lens.We found that β B2 crystallin can influence the structure and function of the ovary by inducing proliferation,apoptosis and hormone secretion of granulocyte.In recent years,the unique role of Crybb2 in tumors has been gradually recognized.Crybb2 also exists in malignant tumor tissues such as breast cancer,colorectal cancer,prostate cancer,neuroblastoma and others.Studies are indicating that Crybb2 is related to the growth,differentiation,invasion and metastasis of tumor cells.The overexpression of Crybb2 is closely related to the poor prognosis of malignant tumors.There are no reports on the role of crybb2 in ovarian cancer.Based on the research basis of Crybb2 in the ovary,Crybb2 has important research value in ovarian cancer.Based on the gene analysis,we found that the expression level of Crybb2 in ovarian cancer tissues was higher than that in normal tissues.Pretesting study found that the sensitivity of ovarian cancer cells to chemotherapeutic drugs decreased after the upregulation of the expression level of Crybb2.Under the treatment of chemotherapeutic drugs,the apoptosis rate of ovarian cancer cells overexpressing Crybb2 was lower than that of the control group.Therefore,we will first explore the effect of Crybb2 on the chemotherapy resistance of ovarian cancer cells.The stemness of ovarian cancer cells is closely related to chemotherapy resistance,tumor metastasis and recurrence.Wnt signaling pathway is an important way of cell survival and is closely related to the drug resistance mechanism of ovarian cancer stem cells.Further studies showed that overexpression of Crybb2 could promote the EMT(epithelialmesenchymal transition)ability of ovarian cancer cells,and the stemness level of ovarian cancer cells was significantly increased.These results suggest that the resistance of ovarian cancer cells overexpressing Crybb2 to chemotherapeutic drugs may be closely related to the regulation of stem cells.In conclusion,we will explore the role of overexpression of Crybb2 on ovarian cell invasion,metastasis and chemotherapy resistance,and then further explore the specific mechanism of the effect of Crybb2 on them.Part 1 Effect of Crybb2 on invasion,metastasis and chemoresistance of ovarian cancer cellsAfter the lentivirus transfection of HO-8910 and SKOV-3 ovarian cancer cells,the ovarian cancer cell model with overexpression of Crybb2 was successfully constructed.The control group and experimental group were constructed.HO-8910,HO-LV-Ctrl,HO-LVCrybb2;SKOV-3,SK-LV-Ctrl,SK-LV-Crybb2 were virus-free group,control virus group and experimental virus group respectively.Firstly,ovarian cancer cells in HO-8910,HOLV-Ctrl,HO-LV-Crybb2;SKOV-3,SK-LV-Ctrl,SK-LV-Crybb2 groups were treated with scratch test and cell migration changes were recorded at 0h,12 h,24h.The change of metastasis ability of ovarian cancer cells with over expression of crybb2 was observed by the transwell test.Secondly,HO-8910 and SKOV-3 ovarian cancer cells with overexpression of crybb2 and normal expression of crybb2 were treated with cisplatin and paclitaxel.CCK-8 experiment was used to observe the changes of cell proliferation of HO-8910 and SKOV-3 ovarian cancer cells after 48 hours of treatment with different concentrations of cisplatin and paclitaxel.Flow cytometry was used to verify the results of the CCK8 experiment.Finally,we used nude mice to construct a subcutaneous tumor model of ovarian cancer in nude mice,measured the changes of subcutaneous tumor size after chemotherapy with cisplatin and paclitaxel.The results showed that the invasion and metastasis of ovarian cancer cells in HO-LVCrybb2 and SK-LV-Crybb2 overexpression groups were higher than control groups.HO-8910 and SKOV-3 Crybb2 overexpression groups show a lower apoptosis rate after chemotherapy with cisplatin and paclitaxel.The results of subcutaneous tumorigenesis in nude mice showed that ovarian cancer cells overexpressing Crybb2 had larger tumorigenic volume and lower sensitivity to chemotherapeutic drugs.In conclusion,the results suggest that HO-8910 and SKOV-3 ovarian cancer cells overexpressing Crybb2 have stronger invasion and metastasis ability and lower chemosensitivity.Part 2 The Mechanism of Crybb2 in Ovarian Cancer Cell Invasion,Metastasis and Chemotherapy ResistanceThe invasion,metastasis and chemoresistance of ovarian cancer cells are closely related to the stem characteristics of ovarian cancer cells.The EMT ability of ovarian cancer cells is also an important factor affecting the invasion and metastasis of ovarian cancer cells.In this part of the experiment,we will preliminarily explore the specific mechanism of the effect of Crybb2 on the invasion,metastasis and chemoresistance of ovarian cancer cells.First,we detected the difference of E-cadherin and vimentin expression in ovarian cancer cells by cell immunofluorescence assay.The difference in ovarian cancer cell cloning ability was observed by the cell cloning experiment.The expression of CD133,Nanog and Sox2 in HO-8910 and SKOV-3 ovarian cancer cells were detected by q RT-PCR and Western blot.Then the subcutaneous tumorigenesis test in nude mice was processed.After subcutaneous tumorigenesis of nude mice,the expression of ovarian cancer stem markers in ovarian cancer tissues of nude mice was detected by immunohistochemical staining.The results showed that the EMT ability and the stemness of HO-8910 and SKOV-3 ovarian cancer cells overexpressing crybb2 were significantly enhanced.Secondly,we detected the expression levels of Cyclin-D1,c-Myc and Wnt3 a in ovarian cancer cells by Western blot.Finally,we used wnt-c59,an inhibitor of the Wnt pathway,to reversely interfere with HO-8910 and SKOV-3 ovarian cancer cells overexpressing crybb2.We used CCK8 to observe the changes of cell proliferation and apoptosis of ovarian cancer cells overexpressing crybb2 after inhibition of the Wnt pathway and treated with cisplatin and paclitaxel.We also observed the stemness change of ovarian cancer cells after inhibition of the Wnt pathway.The results showed that the expressions of Cyclin-D1,c-Myc and Wnt3 a were increased in ovarian cancer cells with overexpression of crybb2.Moreover,after treatment with Wnt pathway inhibitor wnt-c59,the cell survival rate was decreased and the apoptosis rate was increased after treatment with cisplatin and paclitaxel,suggesting the important role of the Wnt pathway in this process.In summary,our research draws the following conclusions.First,Crybb2 can improve the EMT ability of ovarian cancer cells,thus promoting the invasion and metastasis of ovarian cancer cells.Second,Crybb2 can promote the occurrence of chemotherapy resistance of ovarian cancer cells by enhancing the level of ovarian cancer cell stemness.Third,the wnt signaling transduction pathway may mediate the effect of Crybb2 on chemotherapy resistance of ovarian cancer cells. |
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