Functional And Mechanistic Studies Of MARCH7 In The Tegulation Of Malignant Progress In Epithelial Ovarian Cancer

Ovarian cancer mortality rate is arranged first in gynecologic malignant tumors; the high mortality rate is mainly related to ovarian cancer metastasis. Ovaries located in the pelvic cavity, anatomical location is deep, hidden disease at the same time, no specific symptoms, early and lack of effective laboratory detection means. So it is difficult to early detection and diagnosis. About 70% of the patients with ovarian cancer have occurred abdominal cavity or distant metastasis for the first time diagnosis. Dualistic model for the origin of ovarian cancer divided into two categories, high grade and low grade serous carcinoma. High grade of ovarian epithelial carcinoma accounts for 75% of ovarian cancer, accounting for 90% of the ovarian cancer mortality. It is rapid, highly aggressive, often with a wide range of pelvic metastasis and planting. The prognosis is poor. Low grade of ovarian epithelial carcinoma is in a relatively slow stepwise process. It experienced benign, border and low-grade malignant development process, more for early diagnosis. So the prognosis is good. High grade ovarian epithelial carcinoma usually accompanies invasion and metastasis. It is a malignant tumor of the threat to the health of women. However, the occurrence mechanism of invasion and metastasis is unknown.E3 ubiquitin ligase MARCH7 is related to zinc finger protein membrane(RING- CH) a member of the family. The present study found MARCH7 is involved in sperm development and microtubule protein ubiquitin. Microtubules regulates cell movement, plays an important role in tumor invasion and autophagy. So we speculated that MARCH7 may play an important role in the cancer. Currently, there is no study about MARCH7 in cancer. Therefore, this study will explore the function and the underlying mechanisms of MARCH7 in epithelial ovarian cancer, for uncovering the role of MARCH7 in ovarian cancer and provide a theoretical basis and accurate treatment.Part one The expression and significance of MARCH7 in epithelial ovarian cancerObjective To detect the expression of MARCH7 in epithelial ovarian cancer tissues, explore the relationship between the expression of MARCH7 in epithelial ovarian cancer and the clinical pathological parameters.Methods We detected the expression of MARCH7 in 20 cases of normal ovarian tissue and 138 cases of epithelial ovarian cancer tissue samples using immunohistochemical staining; then analysis the correlation of MARCH7 expression and the clinical pathological parameters. The expression of MARCH7 m RNA in ovarian cancer cell line(A2780, C30, COC1, COC1 / DDP, ES- 2, OVCA3 WT, SKOV3) was detected by q PCR;Results(1)The expression of MARCH7 in ovarian epithelial significantly higher than that in normal ovarian tissue(P<0.05). MARCH7 was predominantly localized on the plasma membrane, and cytoplasm. MARCH7 expression was significantly higher in epithelial ovarian cancer samples than that in normal ovary tissues(P<0.05). To determine the correlation of MARCH7 expression with cancer type and cancer stage, all cancer samples were grouped into histologic types(serous papillary adenocarcinoma, mucinous adenocarcinoma, and endometrioid adenocarcinoma). The differently expression of MARCH7 between serous adenocarcinoma and other histologic type of the tumor was not significant(P>0.05). MARCH7 immunostaining was significantly higher in tumor samples in advanced stages(stage III/IV) as compared to those in the early stages(stage I/II) disease(P <0.001). Further, the staining intensity significantly correlated with the tumor grade(grades 2–3 versus 1, P<0.05). However, the associations between MARCH7 expression and age were not significant(P > 0.05).(2) The expression of MARCH7 m RNA in ovarian cancer SKOV3, ES-2 and OVCA3 WT was significantly higher in that of the A2780 cells;Conclusion MARCH7 was higher expression in epithelial ovarian cancer tissues and the expression of MARCH7 was related to the stage and grade of ovarian cancer.Part two MARCH7 regulated malignant behavior of the ovarian cancer A2780 and SKOV3 cell linesObjective Explore the malignant biological behavior including cell proliferation, metastasis, and invasion when silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells.Methods(1) The CCK-8, Ed U cell proliferation and Colony formation assays was used to detected cell proliferation ability after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;(2) The scratch test and transwell assay was used to detected cell migration and invasive ability after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;(3) The F-actin staining was to detect cellular cytoskeleton after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;(4) The tumor growth in vivo was evaluated by subcutaneous transplantation of the tumor after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;Results(1) The CCK-8, Ed U cell proliferation and Colony formation assays results showed that the cell proliferation ability was inhibited in SKOV3 cells infected LV3-sh MARCH7-1 or sh MARCH7-1 compared to that of SKOV3 cells infected LV3-NC(P < 0.05). However, the cell proliferation ability was enhanced when ectopic expression MARCH7 in A2780 cells(P < 0.05).(2) Wound-healing and trans-well invasion assays both demonstrated that the migration and invasion capabilities of SKOV3 cell were significantly suppressed when MARCH7 was silenced by LV3-sh MARCH7-1 or LV3-sh MARCH7-2(p<0.05) At the same time, we found that the migration and invasion capabilities of A2780 cells were significantly promoted when MARCH7 was overexpressed with a lentiviral vector expressing MARCH7(LV5-MARCH7)(P < 0.05).(3) In LV3-NC infected SKOV3 cells, F-actin staining was predominantly localized in the cellular outgrowth and projections. In contrast, in LV3-sh MARCH7-1 or LV3-sh MARCH7-2 infected SKOV3 cells, F-actin staining was homogenous throughout the cytoplasm, and the formation of membrane ruffles and lamellipodia was prevented. LV5-GFP-infected A2780 cells displayed some small lamellipodia and ruffles. In contrast, LV5-MARCH7 infected A2780 cells showed F-actin reorganization in membrane ruffles and lamellipodia.(4) The average weight of tumors was significantly lower in LV3-sh MARCH7-1 infected group than that in LV3-NC infected group(P<0.001).The average volume of tumors in LV3-sh MARCH7-1 infected group was significantly lower than that of the LV3-NC infected group(P < 0.001). A2780 cells infected LV5-MARCH7 increased the average weight of tumors and the average volume of tumors than that of the LV5-GFP infected group(P < 0.001).Conclusion The cell proliferation, metastasis and invasion ability was inhibited when silencing MARCH7 in ovarian cancer SKOV3; the cell proliferation, metastasis and invasion ability was enhanced when over-expression in A2780 cells.Part three The mechanism of MARCH7 regulate malignant behavior in epithelial ovarian cancerObjective To explore the potential mechanism of MARCH7 regulate malignant behavior in epithelial ovarian cancerMethods(1) The expression of MARCH7 m RNA was detected by q PCR after adding TGF-beta, IL-1 beta, or TNF alpha in SKOV3 cells; the expression of MARCH7 protein was detected by Western blot after adding TGF-beta, IL- 1 beta or TNF alpha in SKOV3 cells;(2) The Nfkb, Topflash, P53, Stata3 and Notch signaling pathway Reporter activity was detected using Dual luciferase Reporter gene detection system after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;(3) The expression of Nfkb P50, Nfkb P65, beta-catenin and E-cadherin was detected by Western blot after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;(4) The MARCH7 m RNA and protein level was detected when adding Nfkb pathway inhibitor PDTC or silence the Nfkb P50 and Nfkb P65;(5) The location of the Nfkb P50, Nfkb P65 and beta-catenin was detected by immunofluorescence staining after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;(6) The expression of LEF1, MYC and SP5 m RNA was detected by q PCR after silencing MARCH7 in SKOV3 cells or ectopic expression MARCH7 in A2780 cells;(7) The expression of Nfkb P50, Nfkb P65 and beta-catenin in subcutaneous transplantation of the tumor was detected by immunohistochemical staining.Results(1) MARCH7 m RNA and protein expression were up-regulated when adding 10ng/m L TGF-beta1, IL-1 beta or TNF alpha into SKOV3 cells. However, the expression of MARCH7 m RNA and protein was down-regulated when adding 20ng/m L or 30ng/m L.(2) The Nfkb, Topflash, Stata3 and Notch signaling pathway reporter activity was increased and P53 signaling pathway Reporter activity was inhibited after ectopic expression MARCH7 in A2780 cells(P<0.001). The Nfkb and Topflash signaling pathway reporter activity decreased significantly in SKOV3 cells infected LV3-sh MARCH7-1and LV3-sh MARCH7-2 groups compared to that of LV3-NC group(P < 0.05).(3) The expression of Nfkb P50, Nfkb P65 and beta-catenin was decreased in SKOV3 cells infected LV3-sh MARCH7-1 and LV3-sh MARCH7-2 compared to that of LV3-NC group; however, the expression of E-cadherin expression was increased in SKOV3 cells infected LV3-sh MARCH7-1 and LV3-sh MARCH7-2 compared to that of LV3-NC group(P < 0.05). The expression of Nfkb P50, Nfkb P65 and beta-catenin was increased in A2780 cells infected LV5-MARCH7 compared to that of LV5-GFP group; however, the expression of E-cadherin expression was decreased in A2780 cells infected LV5-MARCH7 compared to that of LV5-GFP group(P < 0.05).(4) The expression of MARCH7 m RNA and protein level were decreased when adding Nfkb pathway inhibitor PDTC(10 um, um 30 and 50 um) into SKOV3 cells. With the concentration increased, the expression of MARCH7 m RNA and protein decreased more(P < 0.05). When silencing Nfkb P50 or Nfkb P65, the expression of MARCH7 m RNA and protein was decreased(P < 0.05).(5) A2780 cells exposed to LV5-MARCH7 resulted in significantly higher levels of β-catenin proteins in both the cytoplasm and the nuclei, relative to LV5-GFP-infected A2780 cells(P < 0.05). Conversely, LV3-sh MARCH7-1 or LV3-sh MARCH7-2 infected SKOV3 cells showed a decrease in both, the cytoplasmic and nuclear expression levels of β-catenin relative to LV3-NCinfected SKOV3 cells(P < 0.05). The decrease in nuclear translocation of NF k B p65 and P50 after transfection to SKOV3 cells were observed with LV3-sh MARCH7-1 or LV3-sh MARCH7-2 as compared to that of LV3-NC(P<0.05). Increase in nuclear translocation of NF k B p65 and P50 were observed after infection of A2780 cells with LV5-MARCH7 as compared with that of LV5-GFP(P < 0.05).(6) The m RNA expression of LEF1, MYC and SP5 was lower in SKOV3 cells infected LV3-sh MARCH7-1 and LV3-sh MARCH7-2 compared to that of LV3-NC group; however, the m RNA expression of LEF1, MYC and SP5 was increased in A2780 cells infected LV5-MARCH7 compared to that of LV5-GFP group(P < 0.05).(7)Immunohistochemical staining results revealed that the expression of MARCH7, P50, P65 and beta-catenin in tumors from LV3-sh MARCH7-1 infected group was lower than that in the LV3-NC infected group(P < 0.05). IHC revealed that the expression of MARCH7, P50, P65 and betacatenin in tumors from LV5-MARCH7 infected group was increased than that in the LV5-GFP infected group(P < 0.05)Conclusion MARCH7 promoted proliferation, invasion and metastasis of ovarian epithelial cancer maybe through activating the Nfkb and Wnt/beta-catenin signaling pathway. MARCH7 may be the potential regulatory factor of Nfkb and Wnt/beta-catenin signaling pathway.Part four Mi R-101 regulated the malignant behavior of SKOV3 cells in epithelial ovarian cancer by targeting MARCH7Objective To study mi R-101 regulated the malignant behavior of SKOV3 cells in epithelial ovarian cancer and mi R-101 regulated the expression of MARCH7.Methods(1) The combination site between MARCH7 and mi R-101 was predicted through the bioinformatics;(2) The expression of MARCH7 m RNA and protein when transfection mi R-101 mimics;(3) For MARCH7 3’ UTR luciferase reporter assay, wild type or mutant reporter constructs(termed WT or Mut). Transfection mi R-101 mimics, then detected the luciferase activity using dual luciferase reporter gene detection system.(4) Transfection mi R-101 mimics, then detection of ovarian cancer SKOV3 cells proliferation, metastasis and invasion ability.Results(1) We predicted MARCH7 is a predictive target of mi R-101(http://www.targetscan.org/) through the bioinformatics;(2) The expression of MARCH7 m RNA and protein was significantly decreased after transfection mi R-101 mimics in ovarian cancer SKOV3 cells(P < 0.001);(3) mi R-101 markedly inhibited luciferase activity when MARCH7 3’UTR was inserted downstream of luciferase c DNA in our reporter vector(p MIR-MARCH73UTR)(P<0.05). In contrast, no significant suppressive effect on luciferase activity was observed in cells transfected with a control vector with mutant MARCH7 3’UTR(MIR- MARCH73UTRm), when mi R-101 expression was elevated(P>0.05).(4) Expression of mi R-101 inhibited proliferation, migration and invasion of SKOV3 cells. The phenotypes can be partially restored expression of a mi R-101 resistant MARCH7(P<0.05).Conclusion Mi R-101 regulated of ovarian cancer proliferation, metastasis and invasion by targeting MARCH7.