| Epithelial ovarian cancer(EOC)is one of the most common reproductive system malignancies in women.At present,the EOC mortality rate in gynecologic tumors has been the first in developed countries.For malignant tumors,it is not only shows an active proliferative state but also shows the invasion and migration characteristics which is the most serious threat.At present,some genes which have influence on the biological function of tumor cells have been tested and explored at home and abroad.Interventions provide more for targeted therapy reference,at the same time also promote the development of tumor therapy.For EOC,it is important to study whether the abnormal expression of a certain gene can cause the biological behavior changes and then to explore its regulatory mechanism in epithelial ovarian cancer cells.All of this is important to study etiology and long-term targeted therapy of EOC.The transcription factor FOXC1(forkhead box C1)is a member of the Fragment Box Transcription Factor Gene Family(FOX family),which binds the target gene fragment through its own DNA region of the fork region to initiate the transcription of the relevant gene.FOXC1 is involved in biological processes such as cell or organ differentiation and metabolism.It has been confirmed that FOXC1 can also regulate a variety of tumor cell signaling pathways such as NF-?B,Wnt / ?-Catenin.These cell signaling pathways are closely related to cell proliferation,invasion,migration and apoptosis.Therefore,FOXC1 has become a hotspot in the pathogenesis of tumor.Microchromosomal maintenance protein 2(MCM2)is a new indicator of cell proliferation activity,which is used to reflect the specificity and sensitivity of cell proliferation.Matrix metalloproteinase(MMP9)as the main proteolytic enzyme for extracellular matrix degradation and it is closely related to the degradation of extracellular matrix which is associated with tumor cells invasion and metastasis.Therefore,when the tumor cells changes its proliferation and invasion and other biological behavior in the EOC at the molecular level,it is often able to detect MCM and MMP9 changes.In recent years there have been reports that in ovarian tumor tissue the expression rate of FOXC1 in benign epithelial ovarian tumors is 84% and that in epithelial ovarian cancer is 37.5%.The preliminary study of this group also found that the expression of mRNA in epithelial ovarian cancer tissue was lower than that in borderline ovarian epithelial tumor tissue and benign ovarian epithelial tumor tissue and the mRNA expression of FOXC1 in tissue was correlated with lymph node metastasis.It Suggest that the lack of FOXC1 expression in the organization may be important for the development of epithelial ovarian cancer.However,whether the abnormal expression of FOXC1 can cause changes in the biological function of epithelial ovarian cancer cells and how to participate in the development of EOC is not clear.In addition,a large number of studies have shown that in the EOC NF-?B signaling pathway can participate in MCM2,MMP9 regulation.Therefore,This topic will be involved in the FOXC1 which has influences on the malignant biological behavior in epithelial ovarian cancer and whether the NF-?B signaling pathway can be used to explore its regulation.ObjectiveBy lentivirus infection we investigate the effect of FOXC1 on the proliferation and invasion of SKOV3 cells in the epithelial ovarian cancer and then determine whether FOXC1 can activate NF-?B signaling pathway.Analysis the role of FOXC1 in EOC.Methods 1 Study object and groupsThis study selected human epithelial ovarian cancer cell line SKOV3 cells(Fourth Military Medical University Department of pathogen biology Professor Zhao Ya gift),which application by RPMI-1640 complete medium.The cells placed at 37 ?,5% CO2 in the incubator.The experiment is divided into three groups: The experimental group(FOXC1 overexpression group,FOXC1 shRNA group),vehicle control group(Overexpress Control group,shRNA control group)and blank control group(Control group).2 methodIn the epithelial ovarian cancer SKOV3 cells were infected with FOXC1 lentiviral vector interference plasmid,FOXC1 lentiviral vector overexpression plasmid.Stable cell lines were constructed and verified at mRNA and protein levels,respectively.The proliferation,invasion and migration of SKOV3 cells were observed by CCK-8 experiment,Transwell experiment and scratch test.The mRNA levels of FOXC1,MCM2 and MMP9 were detected by qRT-PCR.The protein levels of FOXC1,MCM2,MMP9,p65 and p-p65 were detected by Western blot.3 statistical analysisSPSS21.0 software was used for statistical analysis.The experimental data were expressed as x ąs.Comparison of data between groups,the use of one-way variance analysis of the normal distribution and independent samples of the t test,the Kruskal-Wallis test and rank sum test were used to compare the data from each group.The test level is ? = 0.05.Results 1 stable strain construction and identification of the situationThree FOXC1 interference sequences are identified by qRT-PCR and the FOXC1 shRNA1 is optimized.The results showes that FOXC1 protein expression in FOXC1 shRNA group is lower than that in control group and blank control group Group and the difference is statistically significant(P<0.05).The FOXC1 interference stabilizer is successfully constructed in SKOV3 cells.The SKOV3 cells are successfully infected with FOXC1 lentiviral vector overexpression plasmid.They are screened by puromycin and verified by qRT-PCR and Western blot.The expression level and protein level of FOXC1 in FOXC1 overexpression group are significantly higher than those in the control group and the blank control group,the difference is statistically significant(P<0.05).We successfully obtaine SKOV3 cell line with stable expression of FOXC1.2 FOXC1 on the proliferation and invasion of SKOV3 cells 2.1 FOXC1 on the proliferation of SKOV3 cells in each groupThe proliferation of SKOV3 cells is detected by CCK-8.The results showes that there is no difference in the absorbance of each group at 0h.The proliferation activity of SKOV3 cells is significantly higher than that of control group and blank control group(P<0.05),especially after 48 h.As the same,the proliferation activity of SKOV3 cells is significantly lower than that of the control group and the blank control group(P<0.05).2.2 FOXC1 on the invasion of SKOV3 cells in each groupAfter 24 hours of SKOV3 cell count,the number of transmembrane cells are counted under light microscope.The number of transmembrane in FOXC1 group is significantly higher than that in control group and blank control group(P<0.05),FOXC1 overexpression group compared with the corresponding control group and blank control group,the number of cell penetration is significantly reduced,and the difference is statistically significant(P<0.001).3 The expression of the expression of MMP9 and MCM2 mRNA and protein in each group 3.1 Changes of MMP9 and MCM2 mRNA and protein levels in SKOV3 cells after interference with FOXC1The expression of MMP9 and MCM2 mRNA in SKOV3 cells treated with FOXC1 is significantly higher than that in the control group(P<0.05).The expression of MMP9 and MCM2 is significantly increased by qRT-PCR.Western blot analysis showes that the expression level of MCM2 protein in SKOV3 cells is significantly higher than that in control group and blank control group(P<0.05),and the difference is statistically significant(P<0.05).3.2 Changes of MMP9 and MCM2 mRNA and protein levels in SKOV3 cells after overexpression of FOXC1The mRNA expression of MMP9 and MCM2 in SKOV3 cells which has overexpressing FOXC1 is decreased by qRT-PCR,and the difference is statistically significant(P<0.05).Western blot analysis showes that the expression of MMP9 and MCM2 in SKOV3 cells which has overexpressing FOXC1 is significantly lower than that in the control group and the blank control group(P<0.05).4 The expression of p65 and p-p65 protein in each group 4.1 Changes of p65 and p-p65 protein levels in SKOV3 cells after FOXC1 interferenceWestern blot analysis showes that the expression of p65 protein in SKOV3 cells which has transfected with FOXC1 is increased compared with the control group and the blank control group,but the difference is not statistically significant(P>0.05).The expression of p-p65 protein in FOXC1 interference group is significantly higher than that in the control group(P<0.05).4.2 Changes of p65 and p-p65 protein levels in SKOV3 cells after overexpression of FOXC1Western blot analysis showes that the expression of p65 protein in FOXC1 overexpression group is lower than that in control group and blank control group,but the difference is not statistically significant(P>0.05).The expression of p-p65 protein in FOXC1 overexpression group is significantly lower than that in control group(P<0.05).ConclusionsRespectively,successfully constructed FOXC1 interference and overexpression of SKOV3 cell stabilizer.In the epithelial ovarian cancer SKOV3 cells,downregulation of FOXC1 can promote the proliferation and invasion of tumor cells,overexpression of FOXC1 can inhibit the proliferation and invasion of tumor cells.FOXC1 may regulate the biological function of epithelial ovarian tumor cells by activating NF-?B signaling pathway and FOXC1 may participate in the development of ovarian cancer to a certain extent. |
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