【摘要】：目的：通過(guò)對(duì)上皮性卵巢癌組織和細(xì)胞的體內(nèi)外實(shí)驗(yàn)研究，探討細(xì)胞骨架蛋白MICAL-L2及其相互作用蛋白α-輔肌蛋白4（ACTN4）在卵巢癌發(fā)生發(fā)展和侵襲轉(zhuǎn)移中的作用及可能機(jī)制。 方法：（1）用免疫組化法檢測(cè)人上皮性卵巢癌組織芯片中MICAL-L2蛋白的表達(dá)，探討MICAL-L2與人卵巢癌臨床病理特征之間的關(guān)系。（2）利用慢病毒轉(zhuǎn)染技術(shù)構(gòu)建穩(wěn)定干擾MICAL-L2的人卵巢癌細(xì)胞株，采用CCK8方法、劃痕實(shí)驗(yàn)、空穿實(shí)驗(yàn)、膠穿實(shí)驗(yàn)等方法檢測(cè)干擾MICAL-L2后對(duì)細(xì)胞增殖，遷移及侵襲功能的影響。（3）利用細(xì)胞免疫熒光技術(shù)檢測(cè)干擾MICAL-L2后細(xì)胞形態(tài)變化及EMT相關(guān)分子標(biāo)志物的變化。（4）利用western blotting方法檢測(cè)EMT相關(guān)分子標(biāo)志物及下游通路中相關(guān)分子在干擾MICAL-L2組和對(duì)照組中的變化情況。（5）利用RT-PCR方法檢測(cè)EMT過(guò)程中相關(guān)轉(zhuǎn)錄因子的變化。（6）利用熒光素酶報(bào)告基因檢測(cè)Wnt通路中TOP/FOP的活性變化情況。（7）利用RT-PCR方法及siRNA干擾方法初步驗(yàn)證MICAL-L2與ACTN4的相互作用關(guān)系。（8）利用TCGA數(shù)據(jù)庫(kù)分析ACTN4與卵巢癌預(yù)后之間的關(guān)系。（9）利用MTT和流式細(xì)胞術(shù)的方法分析ACTN4與卵巢癌耐藥的關(guān)系及可能機(jī)制。 結(jié)果：（1）MICAL-L2在上皮性卵巢癌中的高表達(dá)率（68.60%）明顯高于正常對(duì)照組（16.67%），差異有顯著性（P0.0001）。MICAL-L2在卵巢癌III-IV期中的高表達(dá)率（81.40%）顯著高于I-II期（55.81%）（P=0.011）；在組織學(xué)分級(jí)為G2、G3中的高表達(dá)率顯著高于G1期（P=0.009）。（2）穩(wěn)定干擾MICAL-L2基因后，與對(duì)照組比較，細(xì)胞增殖、遷移及侵襲能力均明顯減弱，差異有顯著性（p值均＜0.01）。(3)細(xì)胞免疫熒光檢測(cè)F-actin、上皮分子標(biāo)志物E-cadherin、間質(zhì)分子標(biāo)志物vimentin發(fā)現(xiàn)，干擾穩(wěn)定MICAL-L2后細(xì)胞形態(tài)發(fā)生重要變化，由梭形變?yōu)閳A形，細(xì)胞與細(xì)胞間連接更加緊密，，上皮分子標(biāo)志物E-cadherin明顯增多，間質(zhì)分子標(biāo)志物vimentin表達(dá)明顯下降。（4）western blotting結(jié)果顯示：上皮分子標(biāo)志物E-cadherin表達(dá)升高，間質(zhì)分子標(biāo)志物vimentin表達(dá)明顯下降，胞核中β-catenin表達(dá)明顯降低，胞漿中無(wú)變化，p-β-catenin無(wú)變化，Wnt/β-catenin通路下游Cyclin-D1表達(dá)降低。(5) RT-PCR方法檢測(cè)EMT轉(zhuǎn)錄因子SNAIL、SLUG、TWIST、ZEB1和ZEB2在干擾MICAL-L2組和對(duì)照組變化，結(jié)果顯示干擾MICAL-L2后SNAIL和SLUG無(wú)變化，TWIST、ZEB1和ZEB2在mRNA水平表達(dá)明顯降低且有統(tǒng)計(jì)學(xué)意義（P0.05）。（6）TOP/FOP熒光素酶報(bào)告基因檢測(cè)Wnt通路中轉(zhuǎn)錄活性的變化，結(jié)果顯示干擾MICAL-L2后TOP活性較對(duì)照組明顯降低，F(xiàn)OP活性無(wú)明顯變化。（7）RT-PCR方法及siRNA干擾方法分析MICAL-L2與ACTN4的關(guān)系，結(jié)果發(fā)現(xiàn)在高表達(dá)MICAL-L2的細(xì)胞株中ACTN4表達(dá)也相應(yīng)較高，瞬時(shí)干擾MICAL-L2后ACTN4表達(dá)也明顯降低（8）TCGA數(shù)據(jù)庫(kù)分析ACTN4與卵巢癌患者預(yù)后明顯相關(guān)，ACTN4基因改變的患者預(yù)后較差，且有統(tǒng)計(jì)學(xué)意義（P0.01）。(9)轉(zhuǎn)染ACTN4-siRNA的卵巢癌細(xì)胞對(duì)化療藥紫杉醇敏感性增加（P＜0.05)，凋亡率增加（P＜0.05)。 結(jié)論： 1、卵巢癌組織中MICAL-L2高表達(dá)與卵巢癌臨床分期和組織學(xué)分級(jí)有關(guān)。 2、MCIAL-L2可促進(jìn)卵巢癌細(xì)胞的增殖，遷移和侵襲。 3、MICAL-L2能促進(jìn)卵巢癌細(xì)胞EMT的發(fā)生。 4、激活經(jīng)典的Wnt/β-catenin通路以及促進(jìn)Rac1的活化可能是MICAL-L2引起卵巢癌細(xì)胞EMT的發(fā)生的機(jī)制之一 5、ACTN4與卵巢癌不良預(yù)后關(guān)系密切，其可能通過(guò)抑制卵巢癌細(xì)胞的凋亡參與對(duì)紫杉醇的化療抵抗。 6、抑制MCIAL-L2和ACTN4將為卵巢癌的臨床治療提供一個(gè)新的靶點(diǎn)。
[Abstract]:Objective: To investigate the role of cytoskeleton protein MICAL-L2 and its interacting protein alpha-comyosin 4 (ACTN4) in the development, invasion and metastasis of epithelial ovarian cancer (EOC) in vitro and in vivo.
Methods: (1) The expression of MICAL-L2 protein in human epithelial ovarian cancer tissue microarray was detected by immunohistochemistry, and the relationship between MICAL-L2 and clinicopathological characteristics of human ovarian cancer was studied. (2) The human ovarian cancer cell line stably interfering with MICAL-L2 was constructed by lentivirus transfection technique. CCK8 method, scratch test, empty puncture test and adhesive puncture test were used. Methods The effects of interfering with MICAL-L2 on cell proliferation, migration and invasion were examined. (3) Cell morphology and EMT-related molecular markers were detected by immunofluorescence assay. (4) EMT-related molecular markers and related molecules in downstream pathway were detected by Western blotting. (6) The activity of TOP/FOP in Wnt pathway was detected by luciferase reporter gene. (7) The interaction between MICAL-L2 and ACTN4 was preliminarily verified by RT-PCR and siRNA interference. (8) The interaction between MICAL-L2 and ACTN4 was analyzed by TCGA database. The relationship between TN4 and the prognosis of ovarian cancer. (9) The relationship between ACTN4 and drug resistance in ovarian cancer was analyzed by MTT and flow cytometry.
Results: (1) The high expression rate of MICAL-L2 in epithelial ovarian cancer (68.60%) was significantly higher than that in normal control group (16.67%). The high expression rate of MICAL-L2 in ovarian cancer stage III-IV (81.40%) was significantly higher than that in stage I-II (55.81%) (P = 0.011); the high expression rate in histological grade G2 and G3 was significantly higher than that in stage G1 (P = 0.009). After stably interfering with MICAL-L2 gene, the proliferation, migration and invasiveness of the cells were significantly decreased compared with the control group (p < 0.01). (3) F-actin, E-cadherin and vimentin were detected by immunofluorescence, and the morphology of the cells was significantly changed after interfering with the stabilization of MICAL-L2. The results of Western blotting showed that the expression of E-cadherin increased, the expression of vimentin decreased, the expression of vimentin decreased and the expression of E-cadherin decreased. The expression of Cyclin-D1 in the downstream of Wnt/beta-catenin pathway was decreased. (5) The expression of SNAIL, SLUG, TWIST, ZEB1 and ZEB2 in the interfering MICAL-L2 group and control group was detected by RT-PCR. The results showed that SNAIL and SLUG did not change after interfering with MICAL-L2, and the expression of TWIST, ZEB1 and ZEB2 decreased significantly at mRNA level. Low and statistically significant (P 0.05). (6) TOP / FOP luciferase reporter gene detection of Wnt pathway transcriptional activity, the results showed that after interfering with MICAL-L2, TOP activity was significantly lower than the control group, FOP activity was not significantly changed. (7) RT-PCR and siRNA interference analysis of the relationship between MICAL-L2 and ACTN4, the results showed that the high expression of MICAL-L2 fine. The expression of ACTN4 was also higher in the cell lines, and the expression of ACTN4 was significantly lower after transient interference with MICAL-L2. (8) TCGA database analysis showed that ACTN4 was significantly correlated with the prognosis of ovarian cancer patients, and the prognosis of patients with ACTN4 gene change was poor, and there was statistical significance (P 0.01). (9) The sensitivity of ovarian cancer cells transfected with ACTN4-siRNA to paclitaxel increased (P < 0.0). 5) apoptosis rate increased (P < 0.05).
Conclusion:
1, the high expression of MICAL-L2 in ovarian cancer is related to the clinical stage and histological grading of ovarian cancer.
2, MCIAL-L2 can promote the proliferation, migration and invasion of ovarian cancer cells.
3, MICAL-L2 can promote the occurrence of EMT in ovarian cancer cells.
4. Activating the classical Wnt/beta-catenin pathway and promoting the activation of Rac1 may be one of the mechanisms of EMT induced by MICAL-L2 in ovarian cancer cells.
5. ACTN4 is closely related to the poor prognosis of ovarian cancer. It may participate in the chemotherapeutic resistance to paclitaxel by inhibiting the apoptosis of ovarian cancer cells.
6, inhibition of MCIAL-L2 and ACTN4 will provide a new target for clinical treatment of ovarian cancer.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

【共引文獻(xiàn)】

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