TGF-β1诱导卵巢癌EMT过程中miR-99家族的变化及其对增殖、迁移侵袭的影响
发布时间：2018-07-28 15:42
【摘要】：目的检测TGF-β1诱导卵巢癌细胞上皮-间质转化过程中miR-99家族表达水平的变化及其对卵巢癌细胞增殖、迁移侵袭的影响,探讨miR-99家族作为判断卵巢癌恶性程度评价指标及其作为治疗靶点的可能性。方法TGF-β1以浓度梯度及时间梯度分别刺激卵巢癌细胞株A2780及SKOV3,Real-time q RT-PCR检测miR-99a、miR-99b及miR-100表达水平变化,Western-blot检测上皮性标志物E-cadherin及间质性标志物Vimentin表达水平变化,CCK-8法检测细胞增殖能力变化,划痕实验和Transwell实验检测细胞迁移及侵袭能力变化,分析miR-99家族表达水平与EMT进程及卵巢癌增殖、迁移侵袭的相关性;miR-99家族各miRNA mimic/inhibitor分别或联合转染卵巢癌细胞株,探索miR-99家族mimic或inhibitor能否抑制TGF-β1对卵巢癌增殖、迁移侵袭的影响。结果TGF-β1刺激卵巢癌细胞株A2780及SKOV3后:⑴细胞增殖能力呈时间/剂量依赖性增强,最小作用浓度2 ng/m L和最小作用时间12 h即与空白对照组差异有统计学意义(P0.01);⑵与空白对照组相比,miR-99家族各miRNA表达量均明显上升,差异具有统计学意义(P0.01);⑶上皮性标志物E-cadherin较空白对照组表达下降,间质性标志物Vimentin较空白对照组表达上升;⑷细胞迁移侵袭能力均较空白组增强,差异具有统计学意义(P0.05)。MiR-99家族各miRNA mimic/inhibitor分别或联合转染卵巢癌细胞株后:⑴与空白对照或阴性对照组相比,miR-99家族各miRNA inhibitor转染组卵巢癌细胞增殖能力增强,且联合转染组的促增殖能力高于各miRNA inhibitor单独转染组,差异均具有统计学意义(P0.05);⑵与空白对照或阴性对照组相比,miR-99家族各miRNA mimic转染组卵巢癌细胞增殖能力减弱,且联合转染组的抑制增殖能力高于各miRNA mimic单独转染组,差异均具有统计学意义(P0.05);⑶与TGF-β1处理组相比,miR-99家族各miRNA mimic分别与TGF-β1共处理卵巢癌细胞时,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联合转染与TGF-β1共处理卵巢癌细胞时,可在一定程度上抵抗TGF-β1的促增值作用,差异具有统计学意义(P0.05);⑷与空白对照或阴性对照组相比,miR-99家族mimic转染组可增强卵巢癌细胞的迁移能力、inhibitor转染组可抑制卵巢癌细胞的迁移能力,差异具有统计学意义(P0.05);⑸与TGF-β1处理组相比,miR-99家族inhibitor联合转染组可抵抗TGF-β1的促迁移作用,差异具有统计学意义(P0.05);⑹与空白对照组或阴性对照组相比,miR-99家族mimic转染组可增强卵巢癌细胞的侵袭能力、inhibitor转染组可抑制卵巢癌细胞的侵袭能力,差异具有统计学意义(P0.05);⑺与TGF-β1处理组相比,miR-99家族inhibitor联合转染组可部分抵抗TGF-β1的促侵袭作用,但差异不具有统计学意义(P0.05)。结论TGF-β1诱导卵巢癌细胞发生上皮-间质转化从而进一步增强其增殖、迁移侵袭过程中miR-99家族表达水平升高,提示miR-99家族表达水平可作为判断卵巢癌恶性程度的潜在指标;使用miR-99家族mimic或inhibitor可抵抗TGF-β1的促增殖及促迁移作用,提示miR-99家族具有作为卵巢癌治疗靶点的可能性。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
本文编号：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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
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