小睑裂综合征相关基因Foxl2对Hela和Siha的生物学作用
发布时间：2018-07-25 08:45
【摘要】：小睑裂综合症(blephar-ophimosis-ptosis-epicanthus inversus syndrome,BPES)是常染色体显性遗传疾病,临床科室均有发现。其相关基因-Foxl2是一个单一的外显子编码的叉头转录因子,基因突变可导致小睑裂综合征的发生。在女性患者之中,BPES被分为两种类型:Ⅰ型伴有卵巢早衰,而II型卵巢功能及生育力正常[1,2]。Foxl2的转录m RNA先前主要被发现在小鼠胚胎的眼睑、胚胎及性成熟小鼠的卵巢中[1,3]。Foxl2一直表达在卵巢的颗粒细胞上[4]。在青少年群体,卵巢颗粒细胞肿瘤(OGCTs)中,其表达下降[5]。另外,Shah等在2009年发现体细胞Foxl2的突变即402C突变为G[5]。这个突变出现在97%的成人卵巢颗粒细胞肿瘤(OGCTs)中,且对这个肿瘤类型是特异的[6-10]。另发现在结直肠癌中存在Foxl2位点失活,是体细胞超甲基化导致的[11]。更有趣的是,Foxl2的这个突变在任何一种性-索基质细胞肿瘤及不相关的卵巢癌或乳腺癌中未发现[12,13]。另外,Wegman P等[14]在乳腺癌中发现Foxl2基因的表达,并且这可能与芳香化酶的表达及其临床预后相关。BPES相关基因Foxl2的功能越来越多的被发现。小睑裂综合征的相关基因Foxl2被相继被研究,如在眼睑、遗传学、妇产科卵巢功能及少量肿瘤等方面。肿瘤相关的研究仅仅局限于直结肠癌、其他性索间质细胞肿瘤、乳腺癌及卵巢癌之中。据我们所知,Foxl2尚未在宫颈癌中研究过,因此我们分析并探讨了Foxl2在宫颈癌中的表达及对Foxl2对宫颈癌细胞的生物学作用。第一部分小睑裂综合征相关基因Foxl2在宫颈癌中的表达目的检测小睑裂综合征相关基因Foxl2在宫颈癌组织中的表达,并检测Foxl2在宫颈腺癌及鳞癌这两种细胞系中的转录RNA的水平。方法收集的宫颈癌37例患者的癌变组织,分为两组:一组为腺癌,一组为鳞状细胞癌;取宫颈上皮内瘤变(CIN)的8例患者的病变组织,其中包括了低度鳞状内上皮瘤变(LSIL)患者5例,高度鳞状上皮内瘤变(HSIL)患者10例;取宫颈炎的10例患者的病变组织作为对照组。采用免疫组织化学染色技术,分别检测Foxl2在宫颈癌组织组、宫颈上皮内瘤变组和对照组中的表达;然后,培养人类宫颈癌的腺癌细胞系(Hela细胞)及鳞癌细胞系(Siha细胞),提取RNA,利用逆转录聚合酶链式反应(Reverse transcription polymerase chain reaction,RT-PCR)技术分别检测Foxl2基因在这这种癌症的两种细胞系中的表达水平。结果免疫组织化学染色结果显示,Foxl2在宫颈鳞癌组织的切片中表达的阳性率显著高于CIN组和对照组,Foxl2在宫颈鳞癌的组织中蛋白的表达水平相比宫颈腺癌及其他组,呈现为高表达状态。在宫颈腺癌组、CIN组及宫颈炎组中并不表达Foxl2蛋白,也不存在显著差异。RT-PCR结果显示Hela细胞和Siha细胞可转录Foxl2的m RNA。结论在宫颈鳞癌中Foxl2的表达量显著高于相对于宫颈腺癌、宫颈CIN组织及宫颈炎组织;Hela细胞及Siha细胞能转录Foxl2的m RNA。根据Foxl2在宫颈腺癌和鳞状细胞癌,这两种组织中的表达水平的不同,我们将进一步研究Foxl2基因在宫颈癌细胞中的生物学作用。第二部分小睑裂综合征相关基因Foxl2对宫颈癌细胞系的生物学作用目的构建两种细胞系的模型,过表达Foxl2的Hela细胞和沉默Foxl2的Siha细胞模型,研究Foxl2对这两种细胞的增殖、凋亡、粘附及侵袭能力的影响,从而分析Foxl2对宫颈癌的生物学作用。方法利用携带Foxl2的质粒转染Hela细胞,使其过表达Foxl2,同时沉默Foxl2基因在Siha细胞中的表达,利用蛋白免疫印迹法(Western Blotting)验证转染的效率,鉴定过表达及沉默模型;Foxl2过表达或者沉默后,Brdu法检测Hela和Siha的增殖能力;Annexin V/PI法检测Hela和Siha细胞的凋亡率;采用粘附试剂盒检测Hela和Siha细胞的粘附能力;采用侵袭实验分别检测过表达和沉默Foxl2的宫颈癌的两种细胞系的侵袭力;采用流式细胞术(FCM)检测过表达Foxl2的Hela细胞及沉默Foxl2的Siha细胞中Ki67、细胞增殖核抗原(proliferating cell nuclear antigen,PCNA)和Fas L的表达量,进一步检测和分析调节增殖和凋亡的相关因子的表达。结果Western Blotting结果,与对照组相比,过表达Hela细胞中Foxl2的表达量明显增加;沉默Siha细胞中Foxl2的表达量显著减少(P0.01),可见转染模型构建成功。Brdu及Annexin V/PI结果表明,与对照组相比,过表达Foxl2后,Hela的增殖能力明显被抑制(P0.01);沉默组的增殖能力增加,同时其凋亡显著降低(P0.001)。过表达Hela细胞的粘附无明显改变,但是沉默Foxl2可促进Siha的粘附。侵袭实验提示,过表达组细胞侵袭能力明显减低(P0.05);沉默组细胞侵袭能力提高(P0.05)。PCM结果显示,过表达的Hela中Ki67的表达水平明显降低(P0.001),Fas L的表达增加(P0.01)。沉默的Siha中Ki67的表达量显著增加(P0.001),并且Fas L的表达水平降低(P0.01)。但是,无论过表达还是沉默细胞组中PCNA的表达无明显变化。结论结果表明,小睑裂相关基因Foxl2抑制宫颈癌细胞Hela和Siha的增殖并促进凋亡;抑制Hela和Siha的侵袭能力;与此同时,促进Siha的粘附,但Foxl2对Hela细胞的粘附能力无显著作用。另外,Foxl2下调Ki67在Hela和Siha细胞的表达水平,其同时上调Fas L在Hela和Siha的表达。综上所述,我们的实验研究结果表明,Foxl2在宫颈鳞癌中高表达。一方面,Foxl2抑制宫颈癌细胞的增殖,并抑制其凋亡,这个生物学作用主要是Foxl2通过下调Ki67的表达和上调Fas L的表达水平发挥作用的;另一方面,Foxl2可能是一个新的肿瘤抑制因子,至少在宫颈癌里面。这可能对于评价宫颈癌的转移和复发是有价值的,但是仍需要进一步的研究。
[Abstract]:Blephar-ophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant hereditary disease, which is found in the clinical department. Its related gene -Foxl2 is a single exon encoded fork head transcription factor, and gene mutation can lead to the occurrence of small eyelid cleft syndrome. In female patients, BPES is divided into Two types: type I with premature ovarian failure, and II type ovarian function and normal [1,2].Foxl2 transcript of normal fertility, m RNA was previously found in the eyelids of mouse embryos, and in embryos and sexually mature mice, [1,3].Foxl2 has been expressed on the ovarian granulosa cells, [4]. in the adolescent population, ovarian granulosa cell tumor (OGCTs), and in the ovarian granulosa cells (OGCTs). To decrease [5]., Shah and so on in 2009 found that the mutation of the somatic cell Foxl2, that is, the mutation of 402C to G[5]., appears in 97% of the adult ovarian granulosa cell tumor (OGCTs), and that the tumor type is a specific [6-10]. and there is a Foxl2 site inactivation in the present colorectal cancer, which is more interesting in the hypermethylation of somatic cells. Yes, this mutation of Foxl2 has not been found in any of the sexual cord stromal cell tumors and unrelated ovarian and breast cancers, and the expression of the Foxl2 gene is found in Wegman P, such as Wegman P, and this may be associated with the increasing function of the.BPES related gene Foxl2 related to the expression of aromatase and its clinical prognosis. Now. Foxl2 related genes of the cleft palpebral syndrome have been studied, such as eyelids, genetics, ovarian function and a small number of tumors. Tumor related studies are limited to colorectal, other sexual stromal tumors, breast and ovarian cancers. As we know, Foxl2 has not been studied in cervical cancer. We analyzed and discussed the expression of Foxl2 in cervical cancer and the biological effect of Foxl2 on cervical cancer cells. Part 1 the expression of Foxl2 in cervical cancer was detected in cervical cancer, and the expression of Foxl2 in cervix cancer tissues was detected, and the two types of Foxl2 were detected in cervical adenocarcinoma and squamous cell carcinoma. The level of transcriptional RNA in the cell lines. Methods the cancerous tissues of 37 patients with cervical cancer were divided into two groups: a group of adenocarcinoma, a group of squamous cell carcinoma, and 8 cases of cervical intraepithelial neoplasia (CIN), including 5 cases of low degree squamous intraepithelial neoplasia (LSIL), and 10 cases of high squamous intraepithelial neoplasia (HSIL). The pathological tissue of 10 patients with cervicitis was used as the control group. Immunohistochemical staining was used to detect the expression of Foxl2 in cervical cancer tissue, cervical intraepithelial neoplasia and control group. Then, the adenocarcinoma cell lines (Hela cells) and squamous cell carcinoma cell lines (Siha cells) were cultured for human cervical cancer, and RNA was extracted, and reverse transcriptase was used. The expression of the Foxl2 gene in the two cell lines of this cancer was detected by the Reverse transcription polymerase chain reaction (RT-PCR) technique. The results of immunohistochemical staining showed that the positive rate of Foxl2 in the slices of cervical squamous cell carcinoma was significantly higher than that of the CIN group and the control group, and Foxl2 was in the uterus. The expression level of protein in the tissues of cervical squamous cell carcinoma was higher than that of cervical adenocarcinoma and other groups. In the cervical adenocarcinoma group, the Foxl2 protein was not expressed in the CIN group and the cervicitis group, and there was no significant difference between the.RT-PCR results and the m RNA. conclusion that Hela cells and Siha cells could transcribe Foxl2 in the squamous cell carcinoma of the cervix. Compared with cervical adenocarcinoma, cervical CIN tissue and cervicitis, Hela and Siha cells can transcribe Foxl2 m RNA. based on Foxl2 in cervical adenocarcinoma and squamous cell carcinoma, and the expression level of these two tissues is different. We will further study the biological role of Foxl2 gene in cervical cancer cells. Second part of the small lid cleft synthesis. The biological effect of the associated gene Foxl2 on cervical cancer cell lines, aim to construct a model of two cell lines, overexpress the Hela cells of Foxl2 and the Siha cell model of the silent Foxl2, and study the effects of Foxl2 on the proliferation, apoptosis, adhesion and invasion of these two cells, so as to analyze the biological effects of Foxl2 on cervical cancer. Hela cells were transfected with Foxl2 plasmid to express Foxl2, and the expression of Foxl2 gene in Siha cells was silenced. The transfection efficiency was verified by protein immunoblotting (Western Blotting), and the expression and silence model was identified. The proliferation ability of Hela and Siha was detected by Brdu method after the Foxl2 was overexpressed or silenced. The apoptosis rate of a and Siha cells, the adhesion ability of Hela and Siha cells was detected by the adhesive kit, and the invasiveness of two kinds of cell lines expressing and silent Foxl2 were detected by invasive assay, and FCM was used to detect Hela cells expressing Foxl2 and Ki67 in Siha cells with silent Foxl2, and cell proliferating nuclear antigen ( The expression of proliferating cell nuclear antigen, PCNA) and Fas L was used to further detect and analyze the expression of the related factors regulating proliferation and apoptosis. Results Western Blotting results, compared with the control group, the Foxl2 expression in the overexpressed Hela cells was significantly increased; the expression of the silent Siha cells decreased significantly, and the transfection was visible. The results of the successful model construction of.Brdu and Annexin V/PI showed that compared with the control group, the proliferation ability of Hela was obviously inhibited after overexpression of Foxl2 (P0.01), the proliferation ability of the silent group increased and its apoptosis decreased significantly (P0.001). The adhesion of over expressed Hela cells was not significantly altered, but the silence of Foxl2 could promote the adhesion of Siha. The invasion ability of the cells in the overexpressed group decreased significantly (P0.05), and the cell invasiveness in the silent group increased (P0.05).PCM results showed that the expression level of Ki67 in the overexpressed Hela was significantly decreased (P0.001), the expression of Fas L increased (P0.01). The expression of Ki67 in the silent Siha increased significantly (P0.001), and the expression level of Fas L decreased. There was no obvious change in the expression of PCNA in the over expression or silent cell group. Conclusion the results showed that the small lid fissure related gene Foxl2 inhibited the proliferation of Hela and Siha in cervical cancer cells and promoted apoptosis, and inhibited the invasion ability of Hela and Siha; at the same time, it promoted the adhesion of Siha, but Foxl2 had no significant effect on the adhesion ability of Hela cells. In addition, Foxl2 (Foxl2) had no significant effect on the adhesion of Hela cells. Down regulation of the expression level of Ki67 in Hela and Siha cells, and up regulation of the expression of Fas L in Hela and Siha. To sum up, our experimental results show that Foxl2 is highly expressed in cervical squamous cell carcinoma. On the one hand, Foxl2 inhibits the proliferation of cervical cancer cells and inhibits its apoptosis. This biological effect is mainly Foxl2 by down Ki67 expression and in the expression of Foxl2. Up - regulation the expression level of Fas L; on the other hand, Foxl2 may be a new tumor suppressor, at least in cervical cancer. This may be of value in the evaluation of metastasis and recurrence of cervical cancer, but further research is still needed.
【学位授予单位】：潍坊医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2143272
[Abstract]:Blephar-ophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant hereditary disease, which is found in the clinical department. Its related gene -Foxl2 is a single exon encoded fork head transcription factor, and gene mutation can lead to the occurrence of small eyelid cleft syndrome. In female patients, BPES is divided into Two types: type I with premature ovarian failure, and II type ovarian function and normal [1,2].Foxl2 transcript of normal fertility, m RNA was previously found in the eyelids of mouse embryos, and in embryos and sexually mature mice, [1,3].Foxl2 has been expressed on the ovarian granulosa cells, [4]. in the adolescent population, ovarian granulosa cell tumor (OGCTs), and in the ovarian granulosa cells (OGCTs). To decrease [5]., Shah and so on in 2009 found that the mutation of the somatic cell Foxl2, that is, the mutation of 402C to G[5]., appears in 97% of the adult ovarian granulosa cell tumor (OGCTs), and that the tumor type is a specific [6-10]. and there is a Foxl2 site inactivation in the present colorectal cancer, which is more interesting in the hypermethylation of somatic cells. Yes, this mutation of Foxl2 has not been found in any of the sexual cord stromal cell tumors and unrelated ovarian and breast cancers, and the expression of the Foxl2 gene is found in Wegman P, such as Wegman P, and this may be associated with the increasing function of the.BPES related gene Foxl2 related to the expression of aromatase and its clinical prognosis. Now. Foxl2 related genes of the cleft palpebral syndrome have been studied, such as eyelids, genetics, ovarian function and a small number of tumors. Tumor related studies are limited to colorectal, other sexual stromal tumors, breast and ovarian cancers. As we know, Foxl2 has not been studied in cervical cancer. We analyzed and discussed the expression of Foxl2 in cervical cancer and the biological effect of Foxl2 on cervical cancer cells. Part 1 the expression of Foxl2 in cervical cancer was detected in cervical cancer, and the expression of Foxl2 in cervix cancer tissues was detected, and the two types of Foxl2 were detected in cervical adenocarcinoma and squamous cell carcinoma. The level of transcriptional RNA in the cell lines. Methods the cancerous tissues of 37 patients with cervical cancer were divided into two groups: a group of adenocarcinoma, a group of squamous cell carcinoma, and 8 cases of cervical intraepithelial neoplasia (CIN), including 5 cases of low degree squamous intraepithelial neoplasia (LSIL), and 10 cases of high squamous intraepithelial neoplasia (HSIL). The pathological tissue of 10 patients with cervicitis was used as the control group. Immunohistochemical staining was used to detect the expression of Foxl2 in cervical cancer tissue, cervical intraepithelial neoplasia and control group. Then, the adenocarcinoma cell lines (Hela cells) and squamous cell carcinoma cell lines (Siha cells) were cultured for human cervical cancer, and RNA was extracted, and reverse transcriptase was used. The expression of the Foxl2 gene in the two cell lines of this cancer was detected by the Reverse transcription polymerase chain reaction (RT-PCR) technique. The results of immunohistochemical staining showed that the positive rate of Foxl2 in the slices of cervical squamous cell carcinoma was significantly higher than that of the CIN group and the control group, and Foxl2 was in the uterus. The expression level of protein in the tissues of cervical squamous cell carcinoma was higher than that of cervical adenocarcinoma and other groups. In the cervical adenocarcinoma group, the Foxl2 protein was not expressed in the CIN group and the cervicitis group, and there was no significant difference between the.RT-PCR results and the m RNA. conclusion that Hela cells and Siha cells could transcribe Foxl2 in the squamous cell carcinoma of the cervix. Compared with cervical adenocarcinoma, cervical CIN tissue and cervicitis, Hela and Siha cells can transcribe Foxl2 m RNA. based on Foxl2 in cervical adenocarcinoma and squamous cell carcinoma, and the expression level of these two tissues is different. We will further study the biological role of Foxl2 gene in cervical cancer cells. Second part of the small lid cleft synthesis. The biological effect of the associated gene Foxl2 on cervical cancer cell lines, aim to construct a model of two cell lines, overexpress the Hela cells of Foxl2 and the Siha cell model of the silent Foxl2, and study the effects of Foxl2 on the proliferation, apoptosis, adhesion and invasion of these two cells, so as to analyze the biological effects of Foxl2 on cervical cancer. Hela cells were transfected with Foxl2 plasmid to express Foxl2, and the expression of Foxl2 gene in Siha cells was silenced. The transfection efficiency was verified by protein immunoblotting (Western Blotting), and the expression and silence model was identified. The proliferation ability of Hela and Siha was detected by Brdu method after the Foxl2 was overexpressed or silenced. The apoptosis rate of a and Siha cells, the adhesion ability of Hela and Siha cells was detected by the adhesive kit, and the invasiveness of two kinds of cell lines expressing and silent Foxl2 were detected by invasive assay, and FCM was used to detect Hela cells expressing Foxl2 and Ki67 in Siha cells with silent Foxl2, and cell proliferating nuclear antigen ( The expression of proliferating cell nuclear antigen, PCNA) and Fas L was used to further detect and analyze the expression of the related factors regulating proliferation and apoptosis. Results Western Blotting results, compared with the control group, the Foxl2 expression in the overexpressed Hela cells was significantly increased; the expression of the silent Siha cells decreased significantly, and the transfection was visible. The results of the successful model construction of.Brdu and Annexin V/PI showed that compared with the control group, the proliferation ability of Hela was obviously inhibited after overexpression of Foxl2 (P0.01), the proliferation ability of the silent group increased and its apoptosis decreased significantly (P0.001). The adhesion of over expressed Hela cells was not significantly altered, but the silence of Foxl2 could promote the adhesion of Siha. The invasion ability of the cells in the overexpressed group decreased significantly (P0.05), and the cell invasiveness in the silent group increased (P0.05).PCM results showed that the expression level of Ki67 in the overexpressed Hela was significantly decreased (P0.001), the expression of Fas L increased (P0.01). The expression of Ki67 in the silent Siha increased significantly (P0.001), and the expression level of Fas L decreased. There was no obvious change in the expression of PCNA in the over expression or silent cell group. Conclusion the results showed that the small lid fissure related gene Foxl2 inhibited the proliferation of Hela and Siha in cervical cancer cells and promoted apoptosis, and inhibited the invasion ability of Hela and Siha; at the same time, it promoted the adhesion of Siha, but Foxl2 had no significant effect on the adhesion ability of Hela cells. In addition, Foxl2 (Foxl2) had no significant effect on the adhesion of Hela cells. Down regulation of the expression level of Ki67 in Hela and Siha cells, and up regulation of the expression of Fas L in Hela and Siha. To sum up, our experimental results show that Foxl2 is highly expressed in cervical squamous cell carcinoma. On the one hand, Foxl2 inhibits the proliferation of cervical cancer cells and inhibits its apoptosis. This biological effect is mainly Foxl2 by down Ki67 expression and in the expression of Foxl2. Up - regulation the expression level of Fas L; on the other hand, Foxl2 may be a new tumor suppressor, at least in cervical cancer. This may be of value in the evaluation of metastasis and recurrence of cervical cancer, but further research is still needed.
【学位授予单位】：潍坊医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
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