TGF-β1誘導(dǎo)卵巢癌EMT過程中miR-99家族的變化及其對(duì)增殖、遷移侵襲的影響
發(fā)布時(shí)間：2018-07-28 15:42
【摘要】：目的檢測(cè)TGF-β1誘導(dǎo)卵巢癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化過程中miR-99家族表達(dá)水平的變化及其對(duì)卵巢癌細(xì)胞增殖、遷移侵襲的影響,探討miR-99家族作為判斷卵巢癌惡性程度評(píng)價(jià)指標(biāo)及其作為治療靶點(diǎn)的可能性。方法TGF-β1以濃度梯度及時(shí)間梯度分別刺激卵巢癌細(xì)胞株A2780及SKOV3,Real-time q RT-PCR檢測(cè)miR-99a、miR-99b及miR-100表達(dá)水平變化,Western-blot檢測(cè)上皮性標(biāo)志物E-cadherin及間質(zhì)性標(biāo)志物Vimentin表達(dá)水平變化,CCK-8法檢測(cè)細(xì)胞增殖能力變化,劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞遷移及侵襲能力變化,分析miR-99家族表達(dá)水平與EMT進(jìn)程及卵巢癌增殖、遷移侵襲的相關(guān)性;miR-99家族各miRNA mimic/inhibitor分別或聯(lián)合轉(zhuǎn)染卵巢癌細(xì)胞株,探索miR-99家族mimic或inhibitor能否抑制TGF-β1對(duì)卵巢癌增殖、遷移侵襲的影響。結(jié)果TGF-β1刺激卵巢癌細(xì)胞株A2780及SKOV3后:⑴細(xì)胞增殖能力呈時(shí)間/劑量依賴性增強(qiáng),最小作用濃度2 ng/m L和最小作用時(shí)間12 h即與空白對(duì)照組差異有統(tǒng)計(jì)學(xué)意義(P0.01);⑵與空白對(duì)照組相比,miR-99家族各miRNA表達(dá)量均明顯上升,差異具有統(tǒng)計(jì)學(xué)意義(P0.01);⑶上皮性標(biāo)志物E-cadherin較空白對(duì)照組表達(dá)下降,間質(zhì)性標(biāo)志物Vimentin較空白對(duì)照組表達(dá)上升;⑷細(xì)胞遷移侵襲能力均較空白組增強(qiáng),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。MiR-99家族各miRNA mimic/inhibitor分別或聯(lián)合轉(zhuǎn)染卵巢癌細(xì)胞株后:⑴與空白對(duì)照或陰性對(duì)照組相比,miR-99家族各miRNA inhibitor轉(zhuǎn)染組卵巢癌細(xì)胞增殖能力增強(qiáng),且聯(lián)合轉(zhuǎn)染組的促增殖能力高于各miRNA inhibitor單獨(dú)轉(zhuǎn)染組,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05);⑵與空白對(duì)照或陰性對(duì)照組相比,miR-99家族各miRNA mimic轉(zhuǎn)染組卵巢癌細(xì)胞增殖能力減弱,且聯(lián)合轉(zhuǎn)染組的抑制增殖能力高于各miRNA mimic單獨(dú)轉(zhuǎn)染組,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05);⑶與TGF-β1處理組相比,miR-99家族各miRNA mimic分別與TGF-β1共處理卵巢癌細(xì)胞時(shí),各miRNA mimic不能有效抵抗TGF-β1的促增殖作用(P0.05),而miR-99家族mimic聯(lián)合轉(zhuǎn)染與TGF-β1共處理卵巢癌細(xì)胞時(shí),可在一定程度上抵抗TGF-β1的促增值作用,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);⑷與空白對(duì)照或陰性對(duì)照組相比,miR-99家族mimic轉(zhuǎn)染組可增強(qiáng)卵巢癌細(xì)胞的遷移能力、inhibitor轉(zhuǎn)染組可抑制卵巢癌細(xì)胞的遷移能力,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);⑸與TGF-β1處理組相比,miR-99家族inhibitor聯(lián)合轉(zhuǎn)染組可抵抗TGF-β1的促遷移作用,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);⑹與空白對(duì)照組或陰性對(duì)照組相比,miR-99家族mimic轉(zhuǎn)染組可增強(qiáng)卵巢癌細(xì)胞的侵襲能力、inhibitor轉(zhuǎn)染組可抑制卵巢癌細(xì)胞的侵襲能力,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);⑺與TGF-β1處理組相比,miR-99家族inhibitor聯(lián)合轉(zhuǎn)染組可部分抵抗TGF-β1的促侵襲作用,但差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論TGF-β1誘導(dǎo)卵巢癌細(xì)胞發(fā)生上皮-間質(zhì)轉(zhuǎn)化從而進(jìn)一步增強(qiáng)其增殖、遷移侵襲過程中miR-99家族表達(dá)水平升高,提示miR-99家族表達(dá)水平可作為判斷卵巢癌惡性程度的潛在指標(biāo);使用miR-99家族mimic或inhibitor可抵抗TGF-β1的促增殖及促遷移作用,提示miR-99家族具有作為卵巢癌治療靶點(diǎn)的可能性。
[Abstract]:Objective to detect the changes of the expression level of miR-99 family in the epithelial mesenchymal transition of ovarian cancer cells induced by TGF- beta 1 and the effect on the proliferation and migration of ovarian cancer cells, and to explore the miR-99 family as a marker for evaluating the malignancy of ovarian cancer and the possibility of being a target for treatment of ovarian cancer. Methods TGF- beta 1 is based on the gradient of concentration and time gradient. A2780 and SKOV3, Real-time Q RT-PCR were used to detect the changes in the expression of miR-99a, miR-99b and miR-100, Western-blot detected the Vimentin expression of the epithelial markers E-cadherin and interstitial markers. The CCK-8 method was used to detect the cell proliferation, and the scratch test and the invasion test were used to detect the migration and invasion of the cells. Analysis of miR-99 family expression level with EMT process and ovarian cancer proliferation and migration and invasion; miR-99 family miRNA mimic/inhibitor or co transfection of ovarian cancer cell lines to explore the effect of miR-99 family mimic or inhibitor on the effect of TGF- beta 1 on ovarian cancer growth and migration and invasion. Results TGF- beta 1 stimulates the ovary. Cancer cell lines A2780 and SKOV3: (1) cell proliferation was time / dose dependent, the minimum concentration of 2 ng/m L and the minimum action time 12 h were statistically significant (P0.01) with the blank control group (P0.01); (2) the miRNA expression in the miR-99 family increased significantly, and the difference was statistically significant (P0.01); 3 The expression of E-cadherin in the epithelial marker was lower than that in the blank control group, and the expression of interstitial marker Vimentin was higher than that in the blank control group; (4) the migration and invasion ability of the cells increased more than that in the blank group. The difference was statistically significant (P0.05).MiR-99 family miRNA mimic/inhibitor or co transfection of ovarian cancer cell lines: (1) and blank control Compared with the negative control group, the proliferation ability of ovarian cancer cells in the miRNA inhibitor transfected group of the miR-99 family was enhanced, and the proliferation promoting ability of the combined transfection group was higher than that of the miRNA inhibitor alone transfection group. The difference was statistically significant (P0.05). (2) compared with the blank control or negative group, the ovarian cancer of each miRNA mimic transfection group of the miR-99 family was in the miR-99 family. The proliferation ability of cell proliferation was weakened, and the inhibitory proliferation ability of the combined transfection group was higher than that of the miRNA mimic alone transfection group. The difference was statistically significant (P0.05). 3. Compared with the TGF- beta 1 treatment group, miRNA mimic in the miR-99 family and TGF- beta 1 co treated ovarian cancer cells respectively, and the miRNA mimic did not effectively resist the proliferation promoting effect of TGF- beta 1 (P0.0). 5), when miR-99 family mimic co transfected with TGF- beta 1 co treated ovarian cancer cells, it could resist the increment of TGF- beta 1 to a certain extent, and the difference was statistically significant (P0.05). 4. Compared with the blank control group or negative control group, the mimic transfection group of the miR-99 family could enhance the migration ability of ovarian cancer cells, and the inhibitor transfection group could inhibit the migration of ovarian cancer cells. The difference of migration ability of ovarian cancer cells was statistically significant (P0.05). Compared with TGF- beta 1 treatment group, the miR-99 family inhibitor combined transfection group could resist TGF- beta 1, the difference was statistically significant (P0.05). Compared with the blank control group or negative control group, the miR-99 family mimic transfection group could enhance ovarian cancer cells. The invasion ability of inhibitor transfected group inhibited the invasive ability of ovarian cancer cells, and the difference was statistically significant (P0.05). Compared with the TGF- beta 1 treatment group, the miR-99 family inhibitor combined transfection group could partially resist the invasion promoting effect of TGF- beta 1, but the difference was not statistically significant (P0.05). Conclusion TGF- beta 1 induced ovarian cancer cells to occur. The expression level of miR-99 family increased in the process of migration and invasion, suggesting that the expression level of miR-99 family can be a potential index for judging the malignant degree of ovarian cancer. The use of miR-99 family mimic or inhibitor can resist the proliferation and migration of TGF- beta 1, suggesting that the miR-99 family has the ovarian function as the ovary. The possibility of cancer treatment for targets.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.31
本文編號(hào)：2150755
[Abstract]:Objective to detect the changes of the expression level of miR-99 family in the epithelial mesenchymal transition of ovarian cancer cells induced by TGF- beta 1 and the effect on the proliferation and migration of ovarian cancer cells, and to explore the miR-99 family as a marker for evaluating the malignancy of ovarian cancer and the possibility of being a target for treatment of ovarian cancer. Methods TGF- beta 1 is based on the gradient of concentration and time gradient. A2780 and SKOV3, Real-time Q RT-PCR were used to detect the changes in the expression of miR-99a, miR-99b and miR-100, Western-blot detected the Vimentin expression of the epithelial markers E-cadherin and interstitial markers. The CCK-8 method was used to detect the cell proliferation, and the scratch test and the invasion test were used to detect the migration and invasion of the cells. Analysis of miR-99 family expression level with EMT process and ovarian cancer proliferation and migration and invasion; miR-99 family miRNA mimic/inhibitor or co transfection of ovarian cancer cell lines to explore the effect of miR-99 family mimic or inhibitor on the effect of TGF- beta 1 on ovarian cancer growth and migration and invasion. Results TGF- beta 1 stimulates the ovary. Cancer cell lines A2780 and SKOV3: (1) cell proliferation was time / dose dependent, the minimum concentration of 2 ng/m L and the minimum action time 12 h were statistically significant (P0.01) with the blank control group (P0.01); (2) the miRNA expression in the miR-99 family increased significantly, and the difference was statistically significant (P0.01); 3 The expression of E-cadherin in the epithelial marker was lower than that in the blank control group, and the expression of interstitial marker Vimentin was higher than that in the blank control group; (4) the migration and invasion ability of the cells increased more than that in the blank group. The difference was statistically significant (P0.05).MiR-99 family miRNA mimic/inhibitor or co transfection of ovarian cancer cell lines: (1) and blank control Compared with the negative control group, the proliferation ability of ovarian cancer cells in the miRNA inhibitor transfected group of the miR-99 family was enhanced, and the proliferation promoting ability of the combined transfection group was higher than that of the miRNA inhibitor alone transfection group. The difference was statistically significant (P0.05). (2) compared with the blank control or negative group, the ovarian cancer of each miRNA mimic transfection group of the miR-99 family was in the miR-99 family. The proliferation ability of cell proliferation was weakened, and the inhibitory proliferation ability of the combined transfection group was higher than that of the miRNA mimic alone transfection group. The difference was statistically significant (P0.05). 3. Compared with the TGF- beta 1 treatment group, miRNA mimic in the miR-99 family and TGF- beta 1 co treated ovarian cancer cells respectively, and the miRNA mimic did not effectively resist the proliferation promoting effect of TGF- beta 1 (P0.0). 5), when miR-99 family mimic co transfected with TGF- beta 1 co treated ovarian cancer cells, it could resist the increment of TGF- beta 1 to a certain extent, and the difference was statistically significant (P0.05). 4. Compared with the blank control group or negative control group, the mimic transfection group of the miR-99 family could enhance the migration ability of ovarian cancer cells, and the inhibitor transfection group could inhibit the migration of ovarian cancer cells. The difference of migration ability of ovarian cancer cells was statistically significant (P0.05). Compared with TGF- beta 1 treatment group, the miR-99 family inhibitor combined transfection group could resist TGF- beta 1, the difference was statistically significant (P0.05). Compared with the blank control group or negative control group, the miR-99 family mimic transfection group could enhance ovarian cancer cells. The invasion ability of inhibitor transfected group inhibited the invasive ability of ovarian cancer cells, and the difference was statistically significant (P0.05). Compared with the TGF- beta 1 treatment group, the miR-99 family inhibitor combined transfection group could partially resist the invasion promoting effect of TGF- beta 1, but the difference was not statistically significant (P0.05). Conclusion TGF- beta 1 induced ovarian cancer cells to occur. The expression level of miR-99 family increased in the process of migration and invasion, suggesting that the expression level of miR-99 family can be a potential index for judging the malignant degree of ovarian cancer. The use of miR-99 family mimic or inhibitor can resist the proliferation and migration of TGF- beta 1, suggesting that the miR-99 family has the ovarian function as the ovary. The possibility of cancer treatment for targets.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.31
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