The Function And Mechanism Of FAM83D In Ovarian Cancer

Ovarian cancer is one of the most common malignancies in the world and the deadliest gynecologic malignancy.About 70% of cases are diagnosed at an advanced stage.Lack of early effective screening methods and typical clinical symptoms are the most important reasons for delaying the diagnosis of ovarian cancer.The high mortality rate of ovarian cancer is attributed to its distant metastasis.Therefore,there is an urgent need to find more reliable tumor markers for early diagnosis and targeted therapy.FAM83D(The family with sequence similarity 83,member D)is located on chromosome 20 q and contains a highly conserved domain(DUF1669)that localizes to the spindle during mitosis to regulate the spindle and maintain cell division.FAM83 D is highly expressed in a variety of cancers,but its significance in ovarian cancer is not clear.This topic will explore the role of FAM83 D in ovarian cancer and its possible molecular mechanisms.PART ?EXPRESSION OF FAM83 D IN OVARIAN CANCEROBJECTIVE: To investigate the expression of FAM83 D in ovarian cancer tissues and to analyze the correlation between its expression level and clinicopathological features.METHODS: Tissue microarray was stained by immunohistochemical staining.The expression of FAM83 D in tissues was observed and recorded.The correlation between FAM83 D expression and clinicopathological features was statistically analyzed.RESULTS: FAM83 D was primarily localized to the cytoplasm.Compared with normal ovarian tissue,the expression of FAM83 D was significantly up-regulated in ovarian cancer tissues.Compared with early ovarian cancer(stage I,II),FAM83 D was more highly expressed in advanced stage(III,IV)(P < 0.05).No significant correlation was found between the expression level of FAM83 D and age or tumor grade.CONCLUSION: FAM83 D is highly expressed in ovarian cancer tissues.The expression of FAM83 D is associated with ovarian cancer staging,and the higher the stage,the higher its expression.PART ?EFFECT OF FAM83 D ON THE MALIGNANT BIOLOGICAL BEHAVIOR OF OVARIAN CANCEROBJECTIVE: To investigate the changes in autophagy,proliferation and invasion of ovarian cancer cells SKOV3 and A2780 after silencing and overexpressing FAM83 D.METHODS:(1)RT-PCR was used to detect the basal expression of FAM83 D m RNA in different ovarian cancer cell lines;lentivirus was transfected into ovarian cancer cells to construct silencing and over-stable cell lines,and the transfection efficiency was detected by WB.(2)WB and RFP-GFP tandem fluorescent labeling LC3 were used to detect the effect of FAM83 D on autophagy of ovarian cancer cells.(3)Edu test to detect the effect of FAM83 D on the proliferation of ovarian cancer cells.(4)Transwell assay to detect the effect of FAM83 D on the invasive ability of ovarian cancer cells.(5)The effect of FAM83 D on the tumorigenic ability of ovarian cancer cells in nude mice by subcutaneous tumor formation model in nude mice.RESULTS:(1)The expression level of FAM83 D was relatively high in SKOV3 cells,while the expression level of FAM83 D was relatively low in A2780 cells.In SKOV3 cells,it was set to silencing group LVRU6P-FAM83D-01,LVRU6P-FAM83D-02 and control group.LVRU6P-FAM83D-NC in the A2780 cells,set the overexpression group LV121-FAM83 D and the control group LV121-NC.The expression of FAM83 D protein in silencing group LVRU6P-FAM83D-01 and LVRU6P-FAM83D-02 was lower than that in control group LVRU6P-FAM83D-NC,while the expression of FAM83 D protein in overexpression group LV121-FAM83 D was higher than that in control group LV121-NC.(2)In the silencing group LVRU6P-FAM83D-01 and LVRU6P-FAM83D-02,the LC3 II protein was significantly up-regulated,and the expression of LC3 II protein was decreased in the overexpressing group LV121-FAM83 D.In the silencing group of FAM83 D,the number of red fluorescence was significantly higher than that of the control group.In the LV121-FAM83 D group overexpressing FAM83 D,the amount of red fluorescence was significantly reduced.(3)In the silent group LVRU6P-FAM83D-01 and LVRU6P-FAM83D-02,the positive rate of cells was significantly lower than that of the control group;while in the overexpressed group LV121-FAM83 D,the positive rate of cells was significantly higher than that of the control group.(4)In the silencing group LVRU6P-FAM83D-01 and LVRU6P-FAM83D-02,the number of cells passing through was significantly less than that in the control group;in the overexpression group LV121-FAM83 D,the number of cells passing through was significantly greater than that in the control group.(5)Compared with the control group LVRU6P-FAM83D-NC,the weight and volume of the subcutaneous tumor in the silent group were smaller than those in the control group;the overexpression group LV121-FAM83 D was compared with the control group LV121-NC,the weight and volume of the subcutaneous tumor are greater than the control group.CONCLUSION: In vitro experiments showed that after silencing FAM83 D,the autophagy level of SKOV3 cells increased,and the proliferation and invasion ability decreased.After overexpression of FAM83 D,the autophagy level of A2780 cells decreased,and the proliferation and invasion ability increased.In vivo,after silencing FAM83 D It inhibited the tumor-forming ability of SKOV3 cells in nude mice,and the over-expression of FAM83 D increased the tumorigenic ability of A2780 cells in nude mice.PART ?THE MECHANISM OF FAM83 D PROMOTING MALIGNANT BIOLOGICAL BEHAVIOR OF OVARIAN CANCEROBJECTIVE: To explore the potential mechanism of FAM83 D in promoting the malignant biological behavior of ovarian cancer cells.METHODS:(1)WB detects the expression level of PI3K/AKT/m TOR pathway protein;(2)Immunohistochemistry to detect the expression of FAM83 D,p-AKT and p-m TOR in nude mice;(3)WB,Edu and Transwell detect whether the m TOR inhibitor Torin1 can reverse the effect of FAM83 D on autophagy,proliferation and invasion of ovarian cancer A2780 cells.RESULTS:(1)The protein expression levels of PI3 K,p-AKT and p-m TOR in LVRU6P-FAM83D-01 and LVRU6P-FAM83D-02 groups were lower than those in LVRU6P-FAM83D-NC group,and PI3 K,p-AKT,p in LV121-FAM83 D group.-m TOR protein expression was higher than LV121-NC group.(2)In nude mice,the expression of FAM83 D,p-AKT and p-m TOR in LVRU6P-FAM83D-01 group was lower than that in LVRU6P-FAM83D-NC group;FAM83D,p-AKT and p-m TOR in LV121-FAM83 D group.The expression of m TOR was enhanced compared with the LV121-NC group.(3)After adding the m TOR inhibitor Torin1,the expression level of LC3 II in LV121-FAM83D+Torin1 group was higher than that in LV121-FAM83 D group,while the positive rate of cell proliferation and the number of cell passage in LV121-FAM83D+Torin1 group were lower than those in LV121-FAM83 D group.CONCLUSION: FAM83 D affects the malignant biological behavior of ovarian cancer cells through the PI3K/AKT/m TOR signaling pathway.After blocking m TOR,it can reverse the effect of FAM83 D on autophagy,proliferation and invasion of ovarian cancer cells.