【作者】 韦敏；

【导师】 卢艳；

【作者基本信息】 广西医科大学 ， 妇产科学， 2022， 硕士

【摘要】 目的和意义:宫颈癌是女性第四大常见癌症,发病逐渐趋于年轻化和复杂化,其死亡原因主要在于肿瘤的治疗失败、复发和转移等,严重威胁着女性的生命健康,并因此造成巨大的经济损失。因此,寻找宫颈癌新型诊断、治疗及预后监测手段是其为重要的。近年来,越来越多的研究证明肿瘤相关EMT在肿瘤侵袭迁移过程中发挥了重要作用,PI3K/AKT信号通路在调节肿瘤EMT中扮演着重要的角色;而外泌体因其能参与细胞分子间的信息交流而参与癌症相关EMT的调控,进一步影响癌症的侵袭迁移能力。我们前期的研究发现,miR-1246在宫颈癌可能充当“促癌因子”的作用。因此,本研究目的是在探讨miR-1246对人宫颈癌EMT及其增殖、侵袭、迁移能力的影响的基础上进一步探讨外泌体miR-1246通过PI3K/AKT通路对人宫颈癌EMT及其增殖、迁移、侵袭能力的影响。方法:本研究通过体外实验完成,为了达到上述实验目的,将实验分为2个部分。1.miR-1246对宫颈癌Si Ha细胞EMT及增殖、侵袭和迁移的影响(1)构建过表达、沉默miR-1246及其对应空载慢病毒,转染宫颈癌Si Ha细胞获取稳转株细胞。(2)通过RT-PCR实验:1)验证各组细胞转染效果;2)评价各组细胞miR-1246相对表达水平。(3)通过细胞流式术:1)检测各组细胞转染效率;2)评价miR-1246水平对宫颈癌Si Ha细胞体外凋亡能力的影响。(4)通过Western blot实验:评价miR-1246水平对EMT相关蛋白表达的影响情况。(5)通过CCK-8实验:评价miR-1246水平对宫颈癌Si Ha细胞体外增殖能力的影响。(6)通过Transwell侵袭实验:评价miR-1246水平对宫颈癌Si Ha细胞体外侵袭能力的影响。(7)通过细胞划痕实验和Transwell迁移实验:评价miR-1246对宫颈癌Si Ha细胞体外迁移能力的影响。2.外泌体miR-1246通过PI3K/AKT通路对宫颈癌Si Ha细胞EMT及增殖、侵袭和迁移的影响(1)差速超速离心法提取过表达miR-1246及其空载稳转细胞上清外泌体,通过Western blot、NTA和透射电镜法验证外泌体表征、大小及形状,CCK-8法建立共培养体系。(2)通过RT-PCR实验:1)验证各组细胞上清外泌体中miR-1246相对表达水平;2)评价外泌体共培养后各组细胞miR-1246相对表达水平。(3)通过CCK-8实验:评价外泌体miR-1246对宫颈癌Si Ha细胞体外增殖能力的影响。(4)通过Transwell侵袭实验:评价外泌体miR-1246对宫颈癌Si Ha细胞体外侵袭能力的影响。(5)通过细胞划痕实验和Transwell迁移实验:评价外泌体miR-1246对宫颈癌Si Ha细胞体外迁移能力的影响。(6)通过Western blot实验:1)评价外泌体miR-1246对PI3K/AKT信号通路的影响;2)评价外泌体miR-1246通过PI3K/AKT信号通路对EMT相关蛋白的影响。结果:1.miR-1246对宫颈癌Si Ha细胞EMT及增殖、侵袭和迁移的影响(1)流式细胞术及RT-PCR实验结果提示,各组细胞慢病毒转染效率达90%以上,各组细胞miR-1246表达水平有显著差异,差异有统计学意义(P=0.042<0.05)。(2)Western blot实验结果提示,过表达miR-1246可以下调Ecadherin(P=0.004<0.01)表达,上调N-cadherin(P=0.000034<0.01)、Vimentin(P=0.000024<0.01)和Twist-2(P=0.000158<0.01)的表达,而敲低miR-1246则出现相反结果。(3)细胞流式凋亡实验结果提示,过表达miR-1246可抑制细胞凋亡(P=0.000146<0.001),而敲低miR-1246则可诱导细胞凋亡(P=0.000001<0.001)。(4)CCK-8实验结果提示:过表达miR-1246可增强宫颈癌Si Ha细胞增殖能力(P=0.0000<0.001),而敲低miR-1246则出现相反结果(P=0.0000<0.001)。(5)Transwell侵袭实验结果提示:对比miR-1246-mimics、miR-1246-inhibitor和各对照组细胞穿基底膜数量,差异有统计学意义(P=0.0000<0.001)。(6)Transwell迁移实验结果提示:对比miR-1246-mimics、miR-1246-inhibitor和各对照组细胞穿小室膜数量,差异有统计学意义(P=0.0000<0.001)。(7)细胞划痕实验结果提示:对比miR-1246-mimics、miR-1246-inhibitor和各对照组细胞划痕愈合能力,差异有统计学意义(P=0.000086<0.001)。2.外泌体miR-1246通过PI3K/AKT通路对宫颈癌Si Ha细胞EMT及增殖、侵袭和迁移的影响(1)Western blot、NTA和透射电镜实验验证外泌体提取成功,CCK-8实验确定了外泌体共培养浓度为10ug/ml。(2)RT-PCR实验结果提示,各组细胞上清外泌体miR-1246表达水平有显著差异,差异有统计学意义(P=0.016<0.05);外泌体共培养后各组细胞miR-1246表达水平有显著差异,差异有统计学意义(P=0.012<0.05)。(3)CCK-8实验结果提示:外泌体miR-1246可增强宫颈癌Si Ha细胞的增殖能力,且在共培养后48h差异显著,差异有统计学意义(P=0.027<0.05)。(4)Transwell侵袭实验结果提示:EX-miR-1246-mimics组细胞穿基底膜数量与空白对照组和EX-mimic-NC组不同,差异有统计学意义(P=0.0000<0.001)。(5)Transwell迁移实验结果提示:EX-miR-1246-mimics组细胞穿小室膜数量与空白对照组和EX-mimic-NC组不同,差异有统计学意义(P=0.0000<0.001)。(6)细胞划痕实验结果提示:EX-miR-1246-mimics组细胞划痕愈合能力较空白对照组和EX-mimic-NC组强,差异有统计学意义(P=0.001<0.01)。(7)Western blot实验结果提示,外泌体miR-1246可激活PI3K/AKT信号通路,p-PIK和p-AKT蛋白表达水平表现差异,差异有统计学意义(P值分别为0.000278、0.029均小于0.05)。(8)Western blot实验结果提示,外泌体miR-1246通过激活PI3K/AKT信号通路促进宫颈癌Si Ha细胞EMT,表现为E-cadherin表达降低(P=0.0000<0.001);而N-cadherin(P=0.000039<0.001)、Vimentin(P=0.000069<0.001)、Twist-2(P=0.000000<0.001)和Snail(P=0.000000<0.001)表达增高。结论:1.miR-1246可促进宫颈癌Si Ha细胞EMT(E-cadherin表达降低,而N-cadherin、Vimentin和Twist-2表达增强)其增殖、侵袭、迁移能力增强,可抑制宫颈癌Si Ha细胞凋亡能力。2.外泌体miR-1246可激活PI3K/AKT信号通路,PI3K和AKT蛋白磷酸化,p-PI3K和p-AKT表达增强。3.外泌体miR-1246可通过激活PI3K/AKT信号通路,促进宫颈癌Si Ha细胞EMT(表现为Twist-2、Snail、Vimentin和N-cadherin蛋白表达增强,而E-cadherin蛋白表达降低),其增殖、侵袭、迁移能力增强。更多还原

【Abstract】 Objectives Cervical cancer is the fourth most common cancer in women,and its incidence tends to be younger and more complicated.The main cause of death is the failure of tumor treatment,recurrence and metastasis,which seriously threatens the women’s life and health,and thus causes huge economic losses.Therefore,it is important to find new diagnostic,treatment and prognostic monitoring methods for cervical cancer.In recent years,more and more studies have proved that tumor-related EMT plays an important role in the process of tumor invasion and migration,and exosomes participate in the regulation of cancer-related EMT because of their ability to participate in the exchange of information between cells and molecules,which further affects cancer.Ability to invade and migrate.Our previous research found that miR1246 may act as a "cancer-promoting factor" in cervical cancer.Therefore,the purpose of this study is to explore the effects of miR-1246 on human cervical cancer EMT and its proliferation,invasion and migration capabilities on the basis of further exploring exosomal miR-1246 on human cervical cancer EMT and its proliferation through PI3K/AKT pathway,Migration and invasion capabilities.Methods This study was done by in vitro experiments.In order too achieve the above experimental purposes,the experiments were divided into two parts.1.The effect of miR-1246 on EMT and proliferation,invasion and migration of cervical cancer SiHa cells(1)The overexpressed and silenced miR-1246 and its corresponding empty lentivirus were constructed and transfected into cervical cancer SiHa cells to obtain stable transfected cells.(2)Through RT-PCR experiments:1)Verified the transfection effect of each group of cells;2)Evaluated the relative expression level of miR-1246 in each group of cells.(3)By flow cytometry:1)Detected the transfection efficiency of each group of cells;2)Evaluated the effect of miR-1246 level on the apoptosis ability of cervical cancer SiHa cells in vitro.(4)By Western blot experiment:Evaluated the effect of miR-1246 levels on the expression of EMT-related proteins.(5)Through CCK-8 experiment:Evaluated the effect of miR-1246 level on the proliferation ability of cervical cancer SiHa cells in vitro(6)Through Transwell invasion experiment:Evaluated the effect of miR1246 levels on the invasion ability of cervical cancer SiHa cells in vitro.(7)Through cell scratch test and Transwell migration test:Evaluated the effect of miR-1246 on the migration ability of cervical cancer SiHa cells in vitro.2.The effects of exosomal miR-1246 on EMT and proliferation,invasion and migration of cervical cancer SiHa cells through PI3K/AKT pathway(1)Differential ultracentrifugation was used to extract exosomes from the supernatant of cells overexpressing miR-1246 and its empty stably transfected cells,the characterization,size and shape of the exosomes were verified by Western blot,NTA and transmission electron microscopy,and a co-culture system was established by CCK-8 method.(2)Through RT-PCR experiments:1)Evaluate the relative expression level of exosomal miR-1246 in the supernatant of each group of cells;2)Evaluate the relative expression level of miR-1246 in each group of cells after exosome coculture.(3)Through CCK-8 experiment:Evaluated the effect of exosomal miR1246 on the proliferation ability of cervical cancer SiHa cells in vitro.(4)Through Transwell invasion experiment:Evaluated the effect of exosomal miR-1246 on the invasion ability of cervical cancer SiHa cells in vitro.(5)Through cell scratch test and Transwell migration test:Evaluated the influence of exosomal miR-1246 on the migration ability of cervical cancer SiHa cells in vitro.(6)Through Western blot experiments:1)Evaluated the effect of exosomal miR-1246 on PI3K/AKT signaling pathway;2)Evaluated the effect of exosomal miR-1246 on EMT-related proteins through PI3K/AKT signaling pathway.Results 1.The effect of miR-1246 on EMT and proliferation,invasion and migration of cervical cancer SiHa cells(1)The results of flow cytometry and RT-PCR experiments indicated that the lentivirus transfection efficiency of cells in each group of was over 90%,and the expression levels of miR-1246 in cells of each group were significantly different,and the difference was statistically significant(P=0.042<0.05).(2)Western blot experiment results indicated that overexpression of miR1246 could down-regulate the expression of E-cadherin(P=0.004<0.01)and upregulate the expression of N-cadherin(P=0.000034<0.01),Vimentin(P=0.000024<0.01)and Twist-2(P=0.000158<0.01),While knockdown of miR1246 showed the opposite result.(3)The results of cell flow cytometric experiments indicated that overexpression of miR-1246 could inhibit apoptosis(P=0.000146<0.001),while knockdown of miR-1246 could induce apoptosis(P=0.000001<0.001).(4)The results of CCK-8 experiment results indicated that overexpression of miR-1246 could enhance the proliferation of cervical cancer SiHa cells(P=0.0000<0.001),while knockdown of miR-1246 showed the opposite result(P=0.0000<0.001).(5)The results of the Transwell invasion assay showed that there was a statistically significant difference in the number of cells penetrating the basement membrane between miR-1246-mimics,miR-1246-inhibitor and each control group(P=0.0000<0.001).(6)The results of the Transwell migration assay that there was a statistically significant difference in the number of cells penetrating the cell membrane between miR-1246-mimics,miR-1246-inhibitor and each control group(P=0.0000<0.001).(7)The results of the cell scratch experiment showed thar the difference in the healing ability of miR-1246-mimics,miR-1246-inhibitor and the control group was statistically significant(P=0.000086<0.001).2.The effects of exosomal miR-1246 on EMT and proliferation,invasion and migration of cervical cancer SiHa cells through PI3K/AKT pathway(1)Western blot,NTA and transmission electron microscopy experiments verified that the exosomes were successfully extracted,and the CCK-8 experiment determined that the co-culture concentration of exosomes was 10ug/ml.(2)The results of RT-PCR experiments indicated that the expression levels of exosomal miR-1246 in the supernatant of cells in each group were significantly different,and the difference was statistically significant(P=0.016<0.05);and the expression levels of miR-1246 in each group cells after co-culture of cell supernatant exosomes were significantly different,and the difference was statistically significant(P=0.012<0.05).(3)The results of CCK-8 experiment results indicated that exosomal miR1246 could enhance the proliferation ability of cervical cancer SiHa cells,and the difference was significant at 48h after co-culture(P=0.027<0.05).(4)The results of the Transwell invasion assay showed that the number of cells penetrating the basement membrane in EX-miR-1246-mimics group was different from that in blank control group and EX-mimic-NC group,and the difference was statistically significant(P=0.0000<0.001).(5)The results of Transwell migration assay showed that the number of cells penetrating the membrane in the EX-miR-1246-mimics group was different from that in the blank control group and the EX-mimic-NC group,and the difference was statistically significant(P=0.0000<0.001).(6)The results of cell scratch experiment showed that the cell scratch healing ability of EX-miR-1246-mimics group was stronger than that of blank control group and EX-mimic-NC group,and the difference was statistically significant(P=0.001<0.01).(7)The results of Western blot experiments indicated that exosomal miR1246 could activate the PI3K/AKT signaling pathway,and the expression levels of p-PIK and p-AKT proteins were different,and the difference were statistically significant(The P values were 0.000278 and 0.029,both less than 0.05).(8)The results of Western blot experiments indicated that exosomal miR1246 promoted EMT in cervical cancer SiHa cells by activating the PI3K/AKT signaling pathway,which showed that the expression of E-cadherin decreased(P=0.0000<0.001),while N-cadherin(P=0.000039<0.001),Vimentin(P=0.000069<0.001),Twist-2(P=0.000000<0.001)and Snail(P=0.000000<0.001)were increased.Conclusions 1.miR-1246 can promote the EMT of cervical cancer SiHa cells(the expression of E-cadherin is decreased,while the expression of N-cadherin,Vimentin and Twist-2 is increased),and its proliferation,invasion and migration ability are enhanced,and it can inhibit the apoptosis of cervical cancer SiHa cells.2.Exosomal miR-1246 can activate the PI3K/AKT signaling pathway,phosphorylation of PI3K and AKT proteins,and enhanced expression of p-PI3K and p-AKT.3.Exosomes miR-1246 can activate the PI3K/AKT signaling pathway to promote EMT of cervical cancer SiHa cells(indicated by increased expression of Twist-2,Snail,Vimentin,and N-cadherin protein,but decreased expression of E-cadherin protein).The ability to multiply,invade and migrate is enhanced.更多还原