發(fā)布時(shí)間：2018-06-26 05:04

  本文選題：結(jié)腸癌轉(zhuǎn)移相關(guān)基因1 + 卵巢癌��； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：背景 作為一種嚴(yán)重威脅女性健康和生命的惡性腫瘤，卵巢癌死亡率居?jì)D科惡性腫瘤第一位。卵巢癌具有起病隱匿、發(fā)現(xiàn)晚、轉(zhuǎn)移早、進(jìn)展快及預(yù)后差的特點(diǎn)。目前卵巢癌的發(fā)病機(jī)理仍不十分清楚。由于卵巢居于盆腔底部，所以卵巢癌缺乏早期特異性癥狀，患者因腹脹等原因就診時(shí)約70%已是晚期。晚期卵巢上皮性癌行腫瘤細(xì)胞減滅術(shù)和以鉑類為基礎(chǔ)的化學(xué)治療。腫瘤細(xì)胞減滅術(shù)旨在最大限度和范圍的切除腫瘤組織，但術(shù)后可能殘留的病灶仍然無法避免。因此，化療對(duì)晚期卵巢癌患者來說必不可少。但部分患者對(duì)化療藥物尤其一線藥物順鉑耐藥卻造成治療失敗�；熌退幱挚煞譃閮�(nèi)在性耐藥和獲得性耐藥兩大類：約15％-25％的卵巢癌患者對(duì)以鉑類為基礎(chǔ)的聯(lián)合化療方案內(nèi)在性耐藥，即為原發(fā)耐藥；而至少80％的接受化療的患者在治療過程中逐漸對(duì)順鉑產(chǎn)生耐藥性，稱為獲得性耐藥，即為繼發(fā)性耐藥�；熌退巼�(yán)重制約著卵巢癌患者的生活質(zhì)量和生存期。因此，近年來，科學(xué)研究者在不斷尋找改善患者化療敏感性的方法。 2009年，Stein等在結(jié)腸癌中發(fā)現(xiàn)了一個(gè)新的基因，因其與結(jié)腸癌的轉(zhuǎn)移密切相關(guān)，因而這個(gè)基因被稱為結(jié)腸癌轉(zhuǎn)移相關(guān)基因-1(Metastasis-associated in colon cancer-1，MACCl)。MACC1是肝細(xì)胞生長(zhǎng)因子（hepatocyte growth factor，HGF）／C-MET信號(hào)轉(zhuǎn)導(dǎo)途徑的一個(gè)重要調(diào)節(jié)因子，通過提高c-met的表達(dá)，在結(jié)腸癌的侵襲、轉(zhuǎn)移過程中起著重要作用。后來，國(guó)內(nèi)外科學(xué)研究者在多種人類惡性腫瘤中發(fā)現(xiàn)了MACC1的高表達(dá)，比如肝細(xì)胞癌、膽囊癌、胃癌、食管癌、乳腺癌等。本課題組前期研究發(fā)現(xiàn)，卵巢上皮性癌中檢測(cè)到異于卵巢良性腫瘤及正常卵巢組織中的MACC1mRNA及蛋白的高表達(dá)，并合成了MACC1基因特異性shRNA，成功轉(zhuǎn)染人卵巢癌細(xì)胞OVCAR-3，通過一系列功能實(shí)驗(yàn)發(fā)現(xiàn)轉(zhuǎn)染后OVCAR-3細(xì)胞的增殖、遷移、侵襲能力減弱，而凋亡增加；通過RT-PCR和Western Blot發(fā)現(xiàn)轉(zhuǎn)染細(xì)胞的C-met、p-ERK1/2、cyclinD1、survivin、MMP9、MMP2及VEGFA等表達(dá)顯著減少，提示MACC1可能通過HGF/C-met信號(hào)通路及下游的MAPK通路來調(diào)節(jié)腫瘤相關(guān)的一系列基因表達(dá)來參與卵巢癌的惡性進(jìn)展過程。但MACC1是否參與卵巢癌對(duì)順鉑敏感性的調(diào)節(jié)，目前國(guó)內(nèi)外尚無文獻(xiàn)顯示。 本研究通過RNA干擾（RNA interferring，RNAi）技術(shù)分別抑制人上皮性卵巢癌細(xì)胞親本株SKOV3和相應(yīng)的順鉑耐藥株SKOV3/DDP細(xì)胞中MACC1表達(dá)，應(yīng)用RT-PCR檢測(cè)細(xì)胞中MACC1基因的mRNA的表達(dá)情況，同時(shí)采用western blot方法檢測(cè)MACC1、ERK1/2、p-ERK1/2、caspase-3及cleaved caspase-3蛋白的表達(dá)，MTT法檢測(cè)細(xì)胞增殖及順鉑敏感性變化，Transwell法研究細(xì)胞體外侵襲能力的變化，探討MACC1基因抑制對(duì)上皮性卵巢癌細(xì)胞順鉑敏感性的影響，以及MACC1基因抑制后ERK通路蛋白的變化，試圖進(jìn)一步了解MACC1是否通過ERK通路對(duì)卵巢癌順鉑敏感性產(chǎn)生影響，并為尋求臨床上有效逆轉(zhuǎn)卵巢癌耐藥提供新的思路。 目的 通過RNA干擾技術(shù)分別抑制SKOV3和SKOV3/DDP細(xì)胞中MACC1表達(dá)，，分析MACC1基因抑制后卵巢癌細(xì)胞增殖、凋亡及順鉑敏感性的變化，分析MACC1影響卵巢癌上述生物學(xué)行為的分子機(jī)制，為臨床上逆轉(zhuǎn)卵巢癌耐藥提供一個(gè)新的思路。 方法 1.前期設(shè)計(jì)的針對(duì)MACC1mRNA的小發(fā)卡狀RNA轉(zhuǎn)染卵巢癌細(xì)胞SKOV3，RT-PCR及Western blot分別檢測(cè)MACC1mRNA和蛋白表達(dá)，并檢測(cè)ERK1/2及p-ERK1/2蛋白含量的變化以及ERK通路抑制劑PD98059對(duì)上述蛋白水平的影響。MTT法評(píng)價(jià)細(xì)胞對(duì)順鉑化療敏感性的變化。 2.前期設(shè)計(jì)的針對(duì)MACC1mRNA的小發(fā)卡狀RNA轉(zhuǎn)染卵巢癌耐藥細(xì)胞SKOV3/DDP，RT-PCR及Western blot檢測(cè)MACC1mRNA和蛋白表達(dá)，并檢測(cè)ERK1/2及p-ERK1/2、caspase-3及cleaved caspase-3蛋白含量的變化以及ERK通路抑制劑PD98059對(duì)上述蛋白水平的影響。MTT法、FCM法和Transwell實(shí)驗(yàn)分別評(píng)價(jià)細(xì)胞增殖能力和順鉑化療敏感性、凋亡的變化及細(xì)胞體外遷移能力的變化。 3.統(tǒng)計(jì)學(xué)處理：利用SPSS17.0軟件進(jìn)行統(tǒng)計(jì)分析。采用單因素方差分析比較各組細(xì)胞MACC1mRNA及蛋白、下游蛋白、細(xì)胞IC50、細(xì)胞凋亡率差異，采用LSD-t法行兩兩比較。檢驗(yàn)水準(zhǔn)=0.05。 結(jié)果 1.在SKOV3中，轉(zhuǎn)染MACC1shRNA的細(xì)胞可檢測(cè)到MACC1mRNA和蛋白的低表達(dá)；p-ERK1/2表達(dá)明顯降低，細(xì)胞的順鉑半數(shù)抑制濃度（IC50）明顯低于空白對(duì)照組和轉(zhuǎn)染空質(zhì)粒組。在此基礎(chǔ)上加入PD98059后p-ERK1/2表達(dá)及IC50改變更為明顯。 2. MACC1沉默的SKOV3/DDP細(xì)胞中，MACC1mRNA和蛋白表達(dá)降低，p-ERK1/2及caspase-3表達(dá)降低，cleaved caspase-3升高。同時(shí)，MACC1沉默的SKOV3/DDP細(xì)胞增殖和遷移能力減弱、凋亡增加、對(duì)順鉑敏感性提高。PD98059能夠進(jìn)一步促進(jìn)以上變化。 結(jié)論 1、MACC1可能參與卵巢癌對(duì)順鉑的敏感性調(diào)節(jié) 2、MACC1可能通過下游的ERK通路調(diào)節(jié)卵巢癌的增殖、凋亡、遷移及順鉑敏感性
[Abstract]:background
As a malignant tumor that seriously threatens the health and life of women, the mortality of ovarian cancer is the first in gynecologic malignancies. Ovarian cancer has the characteristics of insidious onset, late discovery, early metastasis, rapid progress and poor prognosis. The pathogenesis of ovarian cancer is still not very clear. "Heterosexual symptoms, about 70% of patients with abdominal distention are advanced. Advanced ovarian epithelial cancer is treated with tumor cell subtraction and platinum based chemotherapy. Tumor cell subtraction is designed to maximize and range the tumor tissue, but the possible residual focus remains unavoidable. Therefore, chemotherapy for advanced ovary." Cancer patients are essential. But some patients are resistant to chemotherapy, especially cisplatin, which can be divided into two major categories: intrinsic and acquired resistance: approximately 15%-25% of ovarian cancer patients are resistant to platinum based combined chemotherapy, that is, primary drug resistance; at least 80% of patients receiving chemotherapy are gradually resistant to cisplatin during the treatment, known as acquired resistance, namely, secondary resistance. Chemotherapeutic resistance severely restricts the quality of life and life of ovarian cancer patients. Therefore, in recent years, scientific researchers are constantly looking for ways to improve chemotherapy sensitivity.
In 2009, Stein found a new gene in colon cancer, which was closely related to the metastasis of colon cancer, so the gene called the colon cancer metastasis related gene -1 (Metastasis-associated in colon cancer-1, MACCl).MACC1 is a hepatocyte growth factor (hepatocyte growth factor, HGF) / signal transduction pathway. Important regulatory factors play an important role in the invasion and metastasis of colon cancer by improving the expression of c-met. Later, researchers at home and abroad found high expression of MACC1 in many human malignant tumors, such as hepatocellular carcinoma, gallbladder cancer, gastric cancer, esophagus cancer, breast cancer, etc. The high expression of MACC1mRNA and protein in the ovarian benign tumor and normal ovarian tissue was detected, and the MACC1 gene specific shRNA was synthesized, and the human ovarian cancer cell OVCAR-3 was transfected successfully. Through a series of functional experiments, the proliferation, migration, emplacement ability and apoptosis of OVCAR-3 cells after transfection were found to be increased by RT-PCR and Weste. RN Blot found that the expression of C-met, p-ERK1/2, cyclinD1, survivin, MMP9, MMP2 and VEGFA in transfected cells decreased significantly, suggesting that MACC1 may regulate a series of tumor related gene expressions through the HGF/C-met signaling pathway and downstream MAPK pathway to participate in the malignant progression of ovarian cancer. There is no literature at home and abroad at present.
In this study, the expression of MACC1 in human epithelial ovarian cancer cell line SKOV3 and corresponding cisplatin resistant strain SKOV3/DDP cells was suppressed by RNA interference (RNA interferring, RNAi). RT-PCR was used to detect the expression of mRNA in the MACC1 gene in the cells, and the Western blot method was used to detect the expression of mRNA. The expression of ED caspase-3 protein, the change of cell proliferation and cisplatin sensitivity by MTT method, the change of cell invasiveness in vitro by Transwell method, the effect of MACC1 gene inhibition on cisplatin sensitivity of epithelial ovarian cancer cells, and the changes of ERK pathway protein after the inhibition of MACC1 gene, and try to further understand whether MACC1 is through ERK pass. It has an effect on cisplatin sensitivity of ovarian cancer and provides a new way of thinking to effectively reverse the drug resistance of ovarian cancer.
objective
The expression of MACC1 in SKOV3 and SKOV3/DDP cells was inhibited by RNA interference technique, and the changes of proliferation, apoptosis and cisplatin sensitivity of ovarian cancer cells after the inhibition of MACC1 gene were analyzed. The molecular mechanism of MACC1 affecting the biological behavior of ovarian cancer was analyzed, which provided a new idea for clinical reversal of ovarian cancer tolerance.
Method
1. a small hairpin RNA transfected into ovarian cancer cell SKOV3, RT-PCR and Western blot to detect the expression of MACC1mRNA and protein respectively, and to detect the changes of ERK1/2 and p-ERK1/2 protein content and the effect of ERK pathway inhibitor PD98059 on the above protein level, and the changes in sensitivity of cell to cisplatin chemotherapy were evaluated in 1..
2. the small hairpin RNA transfected to MACC1mRNA, SKOV3/DDP, RT-PCR and Western blot were designed to detect the expression of MACC1mRNA and protein, and the changes of ERK1/2 and p-ERK1/2, caspase-3 and cleaved caspase-3 protein content and the effect of the inhibitor on the protein level were detected. Well assay was used to evaluate cell proliferation and cisplatin chemosensitivity, apoptosis and cell migration in vitro.
3. statistical analysis: statistical analysis was carried out by SPSS17.0 software. Single factor analysis of variance was used to compare MACC1mRNA and protein, downstream protein, cell IC50, cell apoptosis rate, and LSD-t method was used to compare 22. Test level =0.05.
Result
1. in SKOV3, the cells transfected with MACC1shRNA could detect the low expression of MACC1mRNA and protein, the expression of p-ERK1/2 was obviously decreased, and the half inhibitory concentration of cisplatin (IC50) was significantly lower than that of the blank control group and the transfected empty plasmid group. On this basis, the expression of p-ERK1/2 and the change of IC50 were more obvious after adding PD98059.
In 2. MACC1 silent SKOV3/DDP cells, the expression of MACC1mRNA and protein decreased, the expression of p-ERK1/2 and caspase-3 decreased, and cleaved caspase-3 increased. At the same time, the proliferation and migration ability of MACC1 silenced SKOV3/DDP cells decreased, the apoptosis increased, and the sensitivity of cisplatin was enhanced by.PD98059.
conclusion
1, MACC1 may be involved in the sensitivity of ovarian cancer to cisplatin.
2, MACC1 may regulate the proliferation, apoptosis, migration and cisplatin sensitivity of ovarian cancer through the downstream ERK pathway.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 張瑞濤;史惠蓉;黃好亮;陳志敏;劉惠娜;苑中甫;;MACC1、HGF和C-met蛋白在卵巢上皮性癌中的表達(dá)及其意義[J];南方醫(yī)科大學(xué)學(xué)報(bào);2011年09期

2 ;ROLE OF ERK1/2 KINASE IN CISPLATIN-INDUCED APOPTOSIS IN HUMAN OVARIAN CARCINOMA CELLS[J];Chinese Medical Sciences Journal;2004年02期

3 黃好亮;史惠蓉;張瑞濤;陳志敏;劉惠娜;苑中甫;;卵巢上皮性癌組織中結(jié)腸癌轉(zhuǎn)移相關(guān)基因1的表達(dá)及其臨床意義[J];腫瘤;2011年06期

4 劉芳;;乳腺癌惡性程度與HGF/c-Met、端粒酶、微血管密度相關(guān)性研究[J];中國(guó)婦幼保健;2012年36期

本文編號(hào)：2069230