MACC1在宫颈癌中的表达及MACC1 siRNA对HeLa细胞生物学行为的影响
发布时间：2018-09-08 20:46
【摘要】：背景与目的 结肠癌转移相关基因1(metastasis-associated in colon cancer1, MACC1)是新近发现的与结肠癌转移相关的基因,其在多种肿瘤组织中存在异常高表达。MACC1是HGF/c-Met信号通路的关键调控点。目前,尚无MACC1与宫颈癌相关的报道,MACC1是否会参与宫颈癌的发生发展、其对宫颈癌发生发展的分子机制如何、其对宫颈癌的临床意义何在,这些问题一直困扰着我们。本研究主要探讨MACC1、肝细胞生长因子(hepatocyte growth factor, HGF)和c-Met在不同宫颈病变组织中的表达,分析了MACC1、HGF和c-Met的异常表达与宫颈癌临床病理因素的关系,评价了MACC1、HGF和c-Met在宫颈癌中表达的临床意义,进一步探讨了MACC1、HGF和c-Met的表达与人类乳头瘤病毒(human papillomavirus, HPV)感染在宫颈癌的发生发展中的关系。为揭示宫颈癌的发生发展机制,指导宫颈癌的临床诊断及治疗提供新的思路。 方法 1.采用免疫组织化学技术检测40例正常宫颈组织、20例宫颈上皮内瘤样变组织和100例宫颈癌组织中MACC1、HGF和c-Met的表达情况,比较不同宫颈组织中MACC1、HGF和c-Met表达的阳性率。 2.采用PCR技术检测40例正常宫颈组织、20例宫颈上皮内瘤样变组织和100例宫颈癌组织中HPV DNA表达,比较不同宫颈组织中HPV DNA表达的阳性率 3.分析MACC1、HGF和c-Met在宫颈癌中的异常表达与临床病理参数的关系。 4.探讨宫颈癌组织中MACC1、HGF和c-Met异常表达的相关性。 5.分析MACC1、HGF和c-Met在宫颈癌组织中的阳性表达与HPV持续感染的相关性。 结果 1. MACC1、HGF、c-Met和HPV DNA在宫颈癌、宫颈上皮内瘤样变组织中阳性表达率明显高于正常宫颈组织中的表达,差异有统计学意义(P0.05)。 2.在宫颈癌组织中MACC1、HGF和c-Met的阳性表达率与临床分期、组织学病理分级和淋巴结转移相关,差异有统计学意义(P0.05),三者在宫颈癌组织中的阳性表达率随着临床分期的变晚、组织病理分级变差和淋巴结转移的出现阳性率逐级上升；HPV DNA的阳性表达与宫颈癌的临床分期、组织学病理分级、淋巴结转移均无关(P0.05)。 3.在宫颈癌组织中MACC1的异常表达与HGF、c-Met的异常表达呈正相关(Spearman r=0.68, P=0.000; Spearman r=0.71, P=0.008), HGF蛋白与c-Met蛋白表达呈正相关(Spearman r=0.75, P＝0.000)。 4. MACC1、HGF和c-Met的阳性表达与HPV的持续感染密切相关(Spearman r=0.420, P=0.000)。 结论 1.在宫颈癌组织中MACC1、HGF和c-Met基因均过表达。 2.在宫颈癌组织中MACC1-HGF/c-Met-HPV形成了正反馈环,其对于宫颈癌的发生发展发挥了重要作用。 3. MACC1的过度表达促使致癌HPV感染的细胞异常增殖并恶变。 4. MACC1有望成为宫颈癌早期诊断及基因治疗的新靶点。 5.联合检测MACC1的表达和HPV的感染,将有助于癌前病变的诊断、病程进展的判断及患者预后的评估。 背景与目的 MACC1是新近报道的一种与调控肿瘤生长和转移的关键基因,在多种高转移性肿瘤中高表达。前期研究结果证实,MACC1在宫颈癌组织中呈高表达,并与肿瘤临床分期、组织病理分级、淋巴结转移密切相关。为了更好的了解MACC1在宫颈癌发病机制中的作用,分析MACC1与宫颈癌细胞的生物学行为的关系,本研究通过人工合成MACC1基因特异的siRNA(small interfering RNA)序列,并转染到宫颈癌HeLa细胞中,特异性抑制MACC1基因表达。最终研究并分析MACC1对宫颈癌HeLa细胞增殖、迁移和凋亡等的影响,初步阐明了MACC1参与宫颈癌发生发展的分子生物学机制,为宫颈癌的预防、早期诊断及RNA干扰技术治疗宫颈癌提供理论和实验依据。 方法 1.化学合成MACC1基因特异的siRNA序列,利用阳离子脂质体Lipofectamine2000转染到HeLa细胞。 2.采用免疫荧光染色观察MACC1siRNA转染HeLa细胞后细胞骨架形态的变化。 3.采用RT-PCR和Western blot方法检测转染前后HeLa细胞中MACC1mRNA和蛋白水平的变化情况。 4.采用Transwell迁移实验观察MACC1siRNA转染前后HeLa细胞迁移能力的改变。 5.采用MTT法检测MACC1siRNA转染前后HeLa细胞生长增殖能力的变化。 6.采用流式细胞仪检测MACC1siRNA转染前后HeLa细胞的凋亡能力和细胞周期的变化。 结果 1. MACC1siRNA在转染HeLa细胞后,细胞内整齐束状排列的骨架蛋白变成错综复杂的网状结构,且无方向性,部分断裂呈粗大的颗粒,散乱分布于胞浆；HeLa-siRNA细胞组、HeLa-NC细胞组和未转染的HeLa细胞组细胞骨架蛋白F-actin的积分荧光强度分别为：10.01±3.53、17.44±5.85和18.53±3.61. 2.在HeLa-siRNA细胞组MACC1mRNA的相对表达量是:0.47±0.06, HeLa-NC细胞组是：0.88+0.09,未转染的HeLa细胞组是：1.00+0.00,经统计学分析HeLa细胞在转染MACC1siRNA后,细胞MACC1mRNA表达水平明显降低,与HeLa-NC细胞组及未转染的HeLa细胞组相比,差异均有统计学意义(P0.05),而HeLa-NC细胞组和未转染的HeLa细胞组MACC1mRNA的相对表达量相比,差异无统计学意义(P0.05)。 3.在HeLa-siRNA细胞组、HeLa-NC细胞组及未转染的HeLa细胞组中MACC1蛋白的相对表达量分别为：0.46±0.05、0.96+0.05和1.01±0.27. MACC1蛋白在HeLa-siRNA细胞组中的相对表达量与HeLa-NC细胞组及未转染的HeLa细胞组的相比,差异均有统计学意义(P0.05),而HeLa-NC细胞组和未转染的HeLa细胞组MACC1蛋白的相对表达量相比,差异无统计学意义(P0.05)。 4. Transwell实验、MTT实验和流式细胞仪检测技术观察到转染MACC1siRNA后,HeLa细胞的迁移、增殖能力明显减弱,而凋亡明显增加,细胞周期G0/G1和G2/M期细胞比例也发生了明显的变化。 结论 1.应用RNA干扰技术能有效的抑制HeLa细胞内MACC1基因的表达。 2. MACC1基因的表达抑制能够改变HeLa细胞的生物学行为,如增殖和迁移能力受到抑制,凋亡率明显增加,同时将更多的细胞阻滞在G0/G1和G2/M期。 3.检测MACC1基因的表达,对于宫颈癌的预防、早期诊断和选择基因治疗的特异性靶点有非常重要的意义。 4.随着RNA干扰技术的发展,MACC1siRNA有望成为宫颈癌基因治疗的新策略。
[Abstract]:Background and purpose
Metastasis-associated in colon cancer1 (MACC1) is a recently discovered metastasis-related gene in colon cancer, which is highly expressed in a variety of tumor tissues. MACC1 is the key regulatory point of HGF/c-Met signaling pathway. These questions have been puzzling us all the time. This study mainly discussed the expression of MACC1, hepatocyte growth factor (HGF) and c-Met in different cervical lesions and analyzed the abnormalities of MACC1, HGF and c-Met. The relationship between the expression of MACC1, HGF and c-Met and the clinicopathological factors of cervical cancer was evaluated. The relationship between the expression of MACC1, HGF and c-Met and the occurrence and development of human papillomavirus (HPV) infection in cervical cancer was further discussed. Clinical diagnosis and treatment of cervical cancer provide new ideas.
Method
1. The expressions of MACC1, HGF and c-Met in 40 normal cervical tissues, 20 cervical intraepithelial neoplasia tissues and 100 cervical cancer tissues were detected by immunohistochemistry. The positive rates of MACC1, HGF and c-Met in different cervical tissues were compared.
2. HPV DNA expression in 40 normal cervical tissues, 20 cervical intraepithelial neoplasia tissues and 100 cervical cancer tissues was detected by PCR. The positive rate of HPV DNA expression in different cervical tissues was compared.
3. to analyze the relationship between abnormal expression of MACC1, HGF and c-Met in cervical cancer and clinicopathological parameters.
4. to explore the correlation between abnormal expression of MACC1, HGF and c-Met in cervical cancer.
5. to analyze the correlation between the positive expression of MACC1, HGF and c-Met in cervical cancer tissues and HPV persistent infection.
Result
1. The positive expression rates of MACC1, HGF, c-Met and HPV DNA in cervical carcinoma and cervical intraepithelial neoplasia were significantly higher than those in normal cervical tissues (P 0.05).
2. The positive expression rates of MACC1, HGF and c-Met in cervical cancer tissues were correlated with clinical stage, histopathological grade and lymph node metastasis. The difference was statistically significant (P 0.05). The positive expression rates of MACC1, HGF and c-Met in cervical cancer tissues increased gradually with the clinical stage. The positive expression of HPV DNA was not correlated with clinical stage, histopathological grade and lymph node metastasis (P 0.05).
3. The abnormal expression of MACC1 was positively correlated with the abnormal expression of HGF and c-Met (Spearman r = 0.68, P = 0.000; Spearman r = 0.71, P = 0.008), and HGF protein was positively correlated with the expression of c-Met protein (Spearman r = 0.75, P = 0.000).
4. the positive expression of MACC1, HGF and c-Met was closely related to the persistent infection of HPV (Spearman r=0.420, P=0.000).
conclusion
1. in cervical cancer tissues, MACC1, HGF and c-Met genes were over expressed.
2. MACC1-HGF/c-Met-HPV forms a positive feedback loop in cervical cancer tissues, which plays an important role in the occurrence and development of cervical cancer.
3. the over expression of MACC1 promotes the abnormal proliferation and malignant transformation of HPV infected cells.
4. MACC1 is expected to become a new target for early diagnosis and gene therapy of cervical cancer.
5. Joint detection of MACC1 expression and HPV infection will be helpful to the diagnosis of precancerous lesions, the judgment of disease progression and the prognosis of patients.
Background and purpose
MACC1 is a recently reported key gene that regulates tumor growth and metastasis, and is highly expressed in many highly metastatic tumors. Previous studies have confirmed that MACC1 is highly expressed in cervical cancer tissues, and is closely related to clinical stage, histopathological grade and lymph node metastasis. The purpose of this study is to analyze the relationship between MACC1 and the biological behavior of cervical cancer cells. In this study, we synthesized a small interfering RNA sequence of MACC1 gene and transfected it into cervical cancer HeLa cells to specifically inhibit the expression of MACC1 gene. The molecular biological mechanism of MACC1 involved in the occurrence and development of cervical cancer was elucidated preliminarily, which provided theoretical and experimental basis for the prevention, early diagnosis and RNA interference therapy of cervical cancer.
Method
1. The specific siRNA sequence of MACC1 gene was synthesized and transfected into HeLa cells by cationic liposome Lipofectamine 2000.
2. the expression of cytoskeleton in MACC1siRNA transfected HeLa cells was observed by immunofluorescence staining.
3. The changes of MACC1 mRNA and protein levels in HeLa cells before and after transfection were detected by RT-PCR and Western blot.
4. Transwell migration assay was used to observe the migration of HeLa cells before and after MACC1siRNA transfection.
5. MTT method was used to detect the growth and proliferation of HeLa cells before and after MACC1siRNA transfection.
6. The apoptosis ability and cell cycle of HeLa cells were detected by flow cytometry before and after MACC1siRNA transfection.
Result
1. After transfection of MACC 1siRNA into HeLa cells, the cytoskeletal proteins arranged in bundles became intricate network structure, and some of them were disrupted in coarse granules and scattered in cytoplasm. The integral fluorescence intensity of F-actin in HeLa-siRNA cell group, HeLa-NC cell group and non-transfected HeLa cell group were respectively different. The results were as follows: 10.01 + 3.53,17.44 + 5.85 and 18.53 + 3.61.
2. The relative expression of MACC1 mRNA in HeLa-siRNA cell group was 0.47+0.06, that in HeLa-NC cell group was 0.88+0.09, that in HeLa-NC cell group was 1.00+0.00, and that in HeLa-NC cell group was 1.00+0.00. There was no significant difference in the relative expression of MACC1 mRNA between HeLa-NC cells and non-transfected HeLa cells (P 0.05).
3. The relative expression levels of MACC1 protein in HeLa-siRNA cell group, HeLa-NC cell group and non-transfected HeLa cell group were 0.46+0.05, 0.96+0.05 and 1.01+0.27, respectively. There was no significant difference in the relative expression of MACC1 protein between La-NC cell group and non-transfected HeLa cell group (P 0.05).
4. Transwell assay, MTT assay and flow cytometry showed that after transfection of MACC1siRNA, the migration, proliferation and apoptosis of HeLa cells were significantly decreased, and the proportion of cells in G0/G1 and G2/M phases of cell cycle was also significantly changed.
conclusion
1. the application of RNA interference technology can effectively inhibit the expression of MACC1 gene in HeLa cells.
2. Inhibition of MACC1 gene expression can change the biological behavior of HeLa cells, such as inhibition of proliferation and migration, marked increase of apoptosis rate, and block more cells in G0/G1 and G2/M phases.
3. Detection of MACC1 gene expression is very important for the prevention of cervical cancer, early diagnosis and selection of specific targets of gene therapy.
4. with the development of RNA interference technology, MACC1siRNA is expected to become a new strategy for gene therapy of cervical cancer.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2231652
[Abstract]:Background and purpose
Metastasis-associated in colon cancer1 (MACC1) is a recently discovered metastasis-related gene in colon cancer, which is highly expressed in a variety of tumor tissues. MACC1 is the key regulatory point of HGF/c-Met signaling pathway. These questions have been puzzling us all the time. This study mainly discussed the expression of MACC1, hepatocyte growth factor (HGF) and c-Met in different cervical lesions and analyzed the abnormalities of MACC1, HGF and c-Met. The relationship between the expression of MACC1, HGF and c-Met and the clinicopathological factors of cervical cancer was evaluated. The relationship between the expression of MACC1, HGF and c-Met and the occurrence and development of human papillomavirus (HPV) infection in cervical cancer was further discussed. Clinical diagnosis and treatment of cervical cancer provide new ideas.
Method
1. The expressions of MACC1, HGF and c-Met in 40 normal cervical tissues, 20 cervical intraepithelial neoplasia tissues and 100 cervical cancer tissues were detected by immunohistochemistry. The positive rates of MACC1, HGF and c-Met in different cervical tissues were compared.
2. HPV DNA expression in 40 normal cervical tissues, 20 cervical intraepithelial neoplasia tissues and 100 cervical cancer tissues was detected by PCR. The positive rate of HPV DNA expression in different cervical tissues was compared.
3. to analyze the relationship between abnormal expression of MACC1, HGF and c-Met in cervical cancer and clinicopathological parameters.
4. to explore the correlation between abnormal expression of MACC1, HGF and c-Met in cervical cancer.
5. to analyze the correlation between the positive expression of MACC1, HGF and c-Met in cervical cancer tissues and HPV persistent infection.
Result
1. The positive expression rates of MACC1, HGF, c-Met and HPV DNA in cervical carcinoma and cervical intraepithelial neoplasia were significantly higher than those in normal cervical tissues (P 0.05).
2. The positive expression rates of MACC1, HGF and c-Met in cervical cancer tissues were correlated with clinical stage, histopathological grade and lymph node metastasis. The difference was statistically significant (P 0.05). The positive expression rates of MACC1, HGF and c-Met in cervical cancer tissues increased gradually with the clinical stage. The positive expression of HPV DNA was not correlated with clinical stage, histopathological grade and lymph node metastasis (P 0.05).
3. The abnormal expression of MACC1 was positively correlated with the abnormal expression of HGF and c-Met (Spearman r = 0.68, P = 0.000; Spearman r = 0.71, P = 0.008), and HGF protein was positively correlated with the expression of c-Met protein (Spearman r = 0.75, P = 0.000).
4. the positive expression of MACC1, HGF and c-Met was closely related to the persistent infection of HPV (Spearman r=0.420, P=0.000).
conclusion
1. in cervical cancer tissues, MACC1, HGF and c-Met genes were over expressed.
2. MACC1-HGF/c-Met-HPV forms a positive feedback loop in cervical cancer tissues, which plays an important role in the occurrence and development of cervical cancer.
3. the over expression of MACC1 promotes the abnormal proliferation and malignant transformation of HPV infected cells.
4. MACC1 is expected to become a new target for early diagnosis and gene therapy of cervical cancer.
5. Joint detection of MACC1 expression and HPV infection will be helpful to the diagnosis of precancerous lesions, the judgment of disease progression and the prognosis of patients.
Background and purpose
MACC1 is a recently reported key gene that regulates tumor growth and metastasis, and is highly expressed in many highly metastatic tumors. Previous studies have confirmed that MACC1 is highly expressed in cervical cancer tissues, and is closely related to clinical stage, histopathological grade and lymph node metastasis. The purpose of this study is to analyze the relationship between MACC1 and the biological behavior of cervical cancer cells. In this study, we synthesized a small interfering RNA sequence of MACC1 gene and transfected it into cervical cancer HeLa cells to specifically inhibit the expression of MACC1 gene. The molecular biological mechanism of MACC1 involved in the occurrence and development of cervical cancer was elucidated preliminarily, which provided theoretical and experimental basis for the prevention, early diagnosis and RNA interference therapy of cervical cancer.
Method
1. The specific siRNA sequence of MACC1 gene was synthesized and transfected into HeLa cells by cationic liposome Lipofectamine 2000.
2. the expression of cytoskeleton in MACC1siRNA transfected HeLa cells was observed by immunofluorescence staining.
3. The changes of MACC1 mRNA and protein levels in HeLa cells before and after transfection were detected by RT-PCR and Western blot.
4. Transwell migration assay was used to observe the migration of HeLa cells before and after MACC1siRNA transfection.
5. MTT method was used to detect the growth and proliferation of HeLa cells before and after MACC1siRNA transfection.
6. The apoptosis ability and cell cycle of HeLa cells were detected by flow cytometry before and after MACC1siRNA transfection.
Result
1. After transfection of MACC 1siRNA into HeLa cells, the cytoskeletal proteins arranged in bundles became intricate network structure, and some of them were disrupted in coarse granules and scattered in cytoplasm. The integral fluorescence intensity of F-actin in HeLa-siRNA cell group, HeLa-NC cell group and non-transfected HeLa cell group were respectively different. The results were as follows: 10.01 + 3.53,17.44 + 5.85 and 18.53 + 3.61.
2. The relative expression of MACC1 mRNA in HeLa-siRNA cell group was 0.47+0.06, that in HeLa-NC cell group was 0.88+0.09, that in HeLa-NC cell group was 1.00+0.00, and that in HeLa-NC cell group was 1.00+0.00. There was no significant difference in the relative expression of MACC1 mRNA between HeLa-NC cells and non-transfected HeLa cells (P 0.05).
3. The relative expression levels of MACC1 protein in HeLa-siRNA cell group, HeLa-NC cell group and non-transfected HeLa cell group were 0.46+0.05, 0.96+0.05 and 1.01+0.27, respectively. There was no significant difference in the relative expression of MACC1 protein between La-NC cell group and non-transfected HeLa cell group (P 0.05).
4. Transwell assay, MTT assay and flow cytometry showed that after transfection of MACC1siRNA, the migration, proliferation and apoptosis of HeLa cells were significantly decreased, and the proportion of cells in G0/G1 and G2/M phases of cell cycle was also significantly changed.
conclusion
1. the application of RNA interference technology can effectively inhibit the expression of MACC1 gene in HeLa cells.
2. Inhibition of MACC1 gene expression can change the biological behavior of HeLa cells, such as inhibition of proliferation and migration, marked increase of apoptosis rate, and block more cells in G0/G1 and G2/M phases.
3. Detection of MACC1 gene expression is very important for the prevention of cervical cancer, early diagnosis and selection of specific targets of gene therapy.
4. with the development of RNA interference technology, MACC1siRNA is expected to become a new strategy for gene therapy of cervical cancer.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
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