FOXC1对上皮性卵巢癌SKOV3细胞生物学功能的影响及相关机制的初步研究

发布时间：2018-05-20 04:40

本文选题：上皮性卵巢癌 + 叉头框转录因子FOXC1　；参考：《郑州大学》2017年硕士论文

【摘要】：上皮性卵巢癌(Epithelial ovarian cancer,EOC)是女性最常见的生殖系统恶性肿瘤之一。目前,EOC在发达国家的病死率已居妇科肿瘤之首。对恶性肿瘤而言,肿瘤细胞除了呈现活跃的增殖状态外,受到威胁最严重的是肿瘤细胞所表现出来的侵袭迁移特性。目前,国内外对影响肿瘤细胞的生物学功能的部分基因已进行了相关靶点实验及探索,对其靶点进行干预的同时也促进了肿瘤治疗的发展,为靶向治疗提供了更多的参考。对于EOC而言,通过明确某些基因的异常表达是否能够引起上皮性卵巢癌细胞生物学行为的改变,进而探讨其调控机制,对EOC病因机制的深入研究及长远靶向治疗具有重要意义。转录因子FOXC1(forkhead box C1)是叉头框转录因子基因家族(FOX家族)的一员,是通过自身的叉头区DNA结合域结合目的基因片段从而启动相关基因转录。FOXC1除了参与细胞或器官分化及代谢等生物学过程外,目前证实FOXC1还可以调控多种肿瘤细胞信号传导途径,如NF-κB、Wnt/β-Catenin等。这些细胞信号通路与细胞增殖、侵袭、迁移、凋亡等密切相关,因此,FOXC1已成为肿瘤发病机制研究的热点。微小染色体维持蛋白2(minichromosomemai n-tenance proteins 2,MCM2)是细胞增殖活性判断的新指标,用来反映细胞增殖状态时能表现出更强的特异性及敏感性。基质金属蛋白酶9(matrix metallopro teinase,MMP9)作为细胞外基质降解的主要蛋白水解酶,与肿瘤细胞侵袭、转移相关的细胞外基质降解密切相关。因此当EOC中的肿瘤细胞发生增殖及侵袭等生物学行为时在分子水平常常能够检测到MCM2、MMP9的改变。近年来有报道称,在卵巢肿瘤组织中,FOXC1在良性上皮性卵巢肿瘤的表达率为84%,在上皮性卵巢癌中的表达率为37.5%。本课题组前期研究也发现,上皮性卵巢癌组织中的FOXC1 mRNA表达量较交界性卵巢上皮性肿瘤组织及良性卵巢上皮性肿瘤组织中减少,且FOXC1在组织中的mRNA表达量与淋巴结转移相关,提示FOXC1在组织中表达的缺失可能对上皮性卵巢癌的发生发展有重要意义。然而,FOXC1的异常表达能否引起上皮性卵巢癌细胞生物学功能的改变,如何参与EOC的发生发展尚不清楚。另外,大量研究显示,在EOC中NF-κB信号传导通路可以参与MCM2、MMP9的调控。因此,本课题将对FOXC1是否参与了上皮性卵巢癌细胞的恶性生物学行为,是否能通过NF-κB信号通路对其调控进行初步探讨。目的通过慢病毒感染方式在上皮性卵巢癌细胞SKOV3中构建FOXC1过表达及干扰稳定细胞株后,探讨FOXC1对SKOV3细胞增殖及侵袭能力的影响,并初步判断FOXC1是否可以通过激活NF-κB信号通路对其进行调控,分析EOC中FOXC1异常表达所扮演的角色。材料和方法1研究对象及分组本研究选用人上皮性卵巢癌细胞系SKOV3细胞(第四军医大学病原生物学教研室赵亚教授馈赠),应用RPMI-1640完全培养基,将细胞置于37℃、5%CO2的培养箱中进行培养。实验分组:实验组(FOXC1 Overexpress组、FOXC1shRNA组),载体对照组(Overexpress Control组、shRNA Control组)及空白对照组(Control组)。2实验方法在上皮性卵巢癌SKOV3细胞中分别感染慢病毒FOXC1 shRNA、FOXC1慢病毒载体过表达质粒,构建稳定细胞株后分别在mRNA和蛋白水平进行验证。应用CCK-8实验、Transwell实验,分别观察SKOV3细胞的增殖、侵袭能力的变化;通过qRT-PCR法检测比较各组MCM2、MMP9的mRNA水平的变化;采用Western blot法检测各组MCM2、MMP9、NF-κB p65、NF-κB p-p65的蛋白表达水平情况。3统计学处理采用SPSS21.0软件进行统计学分析。实验数据均以x±s表示,各组间数据的比较,正态分布的采用单因素方差分析和独立样本的t检验。偏态分布的计量资料各组间比较采用Kruskal-Wallis检验和秩和检验。检验水准α=0.05。结果1稳定株构建及鉴定情况经qRT-PCR筛选鉴定3条FOXC1干扰序列,FOXC1 shRNA1抑制效果最佳,选取该序列,通过感染及嘌呤霉素筛选后,应用Western blot验证,结果显示实验组FOXC1蛋白表达量较载体对照组及空白对照组明显减少,差异具有统计学意义(P0.05)。在SKOV3细胞中成功构建了FOXC1干扰稳定株。应用FOXC1慢病毒载体过表达质粒成功感染SKOV3细胞,经嘌呤霉素筛选,通过qRT-PCR和Western blot验证,实验组中FOXC1在mRNA的表达水平及蛋白水平均明显高于2组对照组,且差异有统计学意义(P0.05)。成功获得稳定过表达FOXC1的SKOV3细胞株。2 FOXC1对SKOV3细胞增殖、侵袭能力的影响情况2.1 FOXC1对各组SKOV3细胞增殖的影响情况通过CCK-8检测SKOV3细胞增殖能力结果显示:0h各组细胞吸光度值无差异。下调FOXC1组SKOV3细胞的增殖活性高于2组对照组,尤其48h后增殖活性明显增高,经统计学分析,差异均有统计学意义(P0.05)。当上调FOXC1后,SKOV3细胞的增殖活性较2组对照组降低,且差异具有统计学意义(P0.05)。2.2 FOXC1对各组SKOV3细胞侵袭能力的影响情况各组SKOV3细胞种板24h后,在光镜下计数各组细胞穿膜数,下调FOXC1组穿膜数较2组对照组相比明显增多,差异具有统计学意义(P0.05)。上调FOXC1组与2组对照组相比细胞穿膜数明显减少,且差异具有统计学意义(P0.001)。3 FOXC1各组细胞MMP9、MCM2的mRNA和蛋白的表达情况3.1干扰FOXC1后SKOV3细胞中MMP9、MCM2的mRNA及蛋白水平变化情况经qRT-PCR检测,结果显示:下调FOXC1组的SKOV3细胞与2组对照组相比,MMP9、MCM2的mRNA表达量明显增加,差异有统计学意义(P0.05)。应用Western blot检测显示:下调FOXC1组的SKOV3细胞与2组对照组相比,MCM2的蛋白水平表达量增加,差异具有统计学意义(P0.05)。3.2过表达FOXC1后SKOV3细胞中MMP9、MCM2的mRNA及蛋白水平变化情况经qRT-PCR检测,上调FOXC1组的SKOV3细胞较2组对照组相比MMP9、MCM2的mRNA表达量减少,差异均有统计学意义(P0.05)。经Western blot检测,结果显示:上调FOXC1组的SKOV3细胞中MMP9、MCM2的蛋白表达量较2组对照组相比明显降低,差异有统计学意义(P0.05)。4 FOXC1各组细胞p65、p-p65的蛋白表达情况4.1干扰FOXC1后SKOV3细胞中p65、p-p65蛋白水平变化情况经Western blot检测,结果显示,下调FOXC1组的SKOV3细胞中p65的蛋白表达量较2组对照组相比有所增多,但差异无统计学意义(P0.05)。下调FOXC1组中p-p65的蛋白表达量较2组对照组明显增加,差异具有统计学意义(P0.05)。4.2过表达FOXC1后SKOV3细胞中p65、p-p65蛋白水平变化情况经Western blot检测,结果显示:SKOV3细胞中上调FOXC1组p65的蛋白表达量较2组对照组降低,但差异无统计学意义(P0.05)。上调FOXC1组p-p65的蛋白表达量较2组对照组明显降低,差异具有统计学意义(P0.05)。结论分别成功构建了FOXC1干扰及过表达SKOV3细胞稳定株。在上皮性卵巢癌SKOV3细胞中,下调FOXC1能够促进肿瘤细胞的增殖侵袭能力,上调FOX C1能够抑制肿瘤细胞的增殖侵袭能力。FOXC1可能是通过激活NF-κB信号传导通路实现对上皮性卵巢肿瘤细胞生物学功能的调控,在一定程度上参与卵巢癌的发生发展。
[Abstract]:Epithelial ovarian cancer (EOC) is one of the most common malignant tumor of reproductive system in women. At present, the mortality rate of EOC in developed countries is the first in gynecologic tumors. For malignant tumors, tumor cells are most threatened by the invasion of tumor cells in addition to active proliferation. At present, some target experiments and exploration of some genes affecting the biological function of tumor cells have been carried out at home and abroad. Intervention on its targets also promotes the development of tumor therapy and provides more reference for targeted therapy. For EOC, it is clear whether the abnormal expression of some genes can be expressed. The changes in biological behavior of epithelial ovarian cancer cells and further study of its regulatory mechanism are of great significance for the in-depth study of the etiological mechanism of EOC and long-term targeting therapy. The transcription factor FOXC1 (forkhead box C1) is a member of the forkhead transcription factor gene family (FOX family), which combines the binding domain of the DNA binding domain of the forked region of the DNA. In addition to participating in biological processes such as cell or organ differentiation and metabolism, the gene fragment, in addition to the biological processes such as cell or organ differentiation and metabolism, has shown that FOXC1 can also regulate the signal transduction pathways of various tumor cells, such as NF- kappa B, Wnt/ beta -Catenin, and so on. These cell signaling pathways are closely related to cell proliferation, invasion, migration, and apoptosis, thus, FOXC1, FOXC1 The micro chromosome maintenance protein 2 (minichromosomemai n-tenance proteins 2, MCM2) is a new indicator of cell proliferation activity, which is more specific and sensitive when it is used to reflect cell proliferation. Matrix metal egg white enzyme 9 (matrix metallopro teinase, MMP9) is used as a cell. The main protein hydrolase degrading of the external matrix is closely related to the invasion of tumor cells and the degradation of extracellular matrix related to metastasis. Therefore, MCM2, MMP9 changes are often detected at the molecular level when the proliferation and invasion of tumor cells in EOC are at the molecular level. In recent years, it has been reported that FOXC1 is benign in ovarian tumor tissues. The expression rate of epithelial ovarian tumor was 84%. The expression rate in epithelial ovarian cancer was 37.5%. in the previous study group. The expression of FOXC1 mRNA in epithelial ovarian cancer tissue was less than that of borderline ovarian epithelial tumor tissue and benign ovarian epithelial tumor tissue, and the mRNA expression of FOXC1 in the tissues and lymph nodes The deletion of FOXC1 expression in the tissue may be of great significance to the development of epithelial ovarian cancer. However, it is not clear whether the abnormal expression of FOXC1 can cause the biological function of epithelial ovarian cancer cells and how to participate in the development of EOC. In addition, a large number of studies have shown that the NF- kappa B signal transduction pathway in EOC has been shown. The road can be involved in the regulation of MCM2 and MMP9. Therefore, this topic will discuss whether FOXC1 is involved in the malignant biological behavior of epithelial ovarian cancer cells and whether it can be regulated by the NF- kappa B signaling pathway. The purpose of this study is to construct FOXC1 overexpression and interfere with stable cells in the SKOV3 of epithelial ovarian cancer cells by the way of lentivirus infection. After the study, the effects of FOXC1 on the proliferation and invasion of SKOV3 cells were investigated, and whether FOXC1 could be regulated by activating NF- kappa B signaling pathway and analyzing the role of abnormal FOXC1 expression in EOC. Materials and methods 1 research subjects and groups selected human epithelial ovarian cancer cell line SKOV3 cells (Fourth Military Medical doctors) Professor Zhao Ya, Professor Kui Zeng of the Institute of pathogenic biology of the University, used the RPMI-1640 complete culture medium to place cells in the incubator of 37 C and 5%CO2. Experimental groups: experimental group (group FOXC1 Overexpress, FOXC1shRNA group), carrier control group (Overexpress Control group, shRNA Control group) and.2 control group (Control group).2 experiment method in The SKOV3 cells of epithelial ovarian cancer were infected with lentivirus FOXC1 shRNA, FOXC1 lentivirus vector overexpressed plasmids, and the stable cell lines were constructed to verify the level of mRNA and protein respectively. CCK-8 experiment and Transwell experiment were used to observe the proliferation of SKOV3 cells and the changes of invasion ability. MCM2, MMP9 of each group were detected by qRT-PCR method. Western blot method was used to detect the protein expression level of MCM2, MMP9, NF- kappa B p65 and NF- kappa B p-p65 in each group. The statistical analysis of.3 statistical processing was carried out by mRNA software. The experimental data were compared with each other, and the normal distribution was analyzed by single factor analysis of variance and independent sample test. Kruskal-Wallis test and rank sum test were used in the measurement data of partial distribution. Test level alpha =0.05. results 1 stable strain construction and identification of 3 FOXC1 interference sequences by qRT-PCR screening, FOXC1 shRNA1 inhibition effect is the best, select the sequence, through infection and purinomycin screening, Western blot validation, knot The results showed that the expression of FOXC1 protein in the experimental group was significantly lower than that in the vector control group and the blank control group. The difference was statistically significant (P0.05). The FOXC1 interference stable strain was successfully constructed in the SKOV3 cells. The SKOV3 cells were successfully infected with the FOXC1 lentivirus vector overexpression plasmid, and it was screened by purinicamycin and verified by qRT-PCR and Western blot. In the experimental group, the expression level and protein level of FOXC1 in mRNA were significantly higher than those in the 2 groups, and the difference was statistically significant (P0.05). The effect of.2 FOXC1 on the proliferation of SKOV3 cells and the invasiveness of SKOV3 cells were successfully obtained. 2.1 FOXC1 on the proliferation of SKOV3 cells in each group by CCK-8 detection of SKOV3 thin. The results of cell proliferation showed that the cell absorbency of 0h groups was not different. The proliferation activity of SKOV3 cells in the FOXC1 group was higher than that of the 2 groups, especially after 48h, the proliferation activity was significantly higher, and the difference was statistically significant (P0.05). The proliferation activity of SKOV3 cells was lower than that of the control group of the 2 groups after the up regulation of FOXC1, and the difference was found. The effect of statistical significance (P0.05).2.2 FOXC1 on the invasive ability of SKOV3 cells in each group, after 24h of SKOV3 cells, the number of membranes in each group was counted under light microscope, and the number of membrane in the FOXC1 group was significantly increased compared with the control group in the 2 groups, the difference was statistically significant (P0.05). The number of cell transmissions compared with the 2 group was significantly higher than that of the control group. The difference was statistically significant (P0.001).3 FOXC1 cells MMP9, mRNA and protein expression of MCM2 3.1 interfered MMP9 in SKOV3 cells after FOXC1, mRNA and protein levels of MCM2 were detected by qRT-PCR detection. The difference was statistically significant (P0.05). The application of Western blot detection showed that the protein level expression of MCM2 was increased in the SKOV3 cells of the down-regulation group compared with the 2 groups, and the difference was statistically significant (P0.05).3.2 overexpressed FOXC1 in SKOV3 cells. 3 cells were compared with the control group of the 2 group MMP9, and the expression of mRNA in MCM2 decreased, the difference was statistically significant (P0.05). The results showed that the expression of MMP9 in SKOV3 cells of the FOXC1 group was higher than that of the control group of the 2 groups. The difference was statistically significant compared with the control group, and the difference was statistically significant (P0.05). The changes of p65 and p-p65 protein in SKOV3 cells after FOXC1 interference after FOXC1 were detected by Western blot. The results showed that the protein expression of p65 in SKOV3 cells of the down regulated FOXC1 group increased compared with those of the control group, but the difference was not statistically significant (P0.05). The protein expression of p-p65 in the FOXC1 group was significantly higher than that of the control group of 2 groups. The variation of p65 and p-p65 protein levels in SKOV3 cells after FOXC1 was statistically significant (P0.05).4.2 was detected by Western blot. The results showed that the protein expression of p65 in the up regulation of FOXC1 group in SKOV3 cells was lower than that of the control group, but the difference was not statistically significant (P0.05). The amount of protein expression in the up regulation group was compared with the control group of the 2 group. The difference was statistically significant (P0.05). Conclusion FOXC1 interference and overexpression of SKOV3 cell stable strain were successfully constructed. In the SKOV3 cells of epithelial ovarian cancer, the down regulation of FOXC1 could promote the proliferation and invasion of tumor cells, and the up regulation of FOX C1 to inhibit the proliferation and invasion of tumor cells may be by activating NF- kappa B. Signal transduction pathway can regulate the biological function of epithelial ovarian tumor cells and participate in the development of ovarian cancer to some extent.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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