MACC1在宮頸癌中的表達及MACC1 siRNA對HeLa細胞生物學(xué)行為的影響
發(fā)布時間：2018-09-08 20:46
【摘要】：背景與目的 結(jié)腸癌轉(zhuǎn)移相關(guān)基因1(metastasis-associated in colon cancer1, MACC1)是新近發(fā)現(xiàn)的與結(jié)腸癌轉(zhuǎn)移相關(guān)的基因,其在多種腫瘤組織中存在異常高表達。MACC1是HGF/c-Met信號通路的關(guān)鍵調(diào)控點。目前,尚無MACC1與宮頸癌相關(guān)的報道,MACC1是否會參與宮頸癌的發(fā)生發(fā)展、其對宮頸癌發(fā)生發(fā)展的分子機制如何、其對宮頸癌的臨床意義何在,這些問題一直困擾著我們。本研究主要探討MACC1、肝細胞生長因子(hepatocyte growth factor, HGF)和c-Met在不同宮頸病變組織中的表達,分析了MACC1、HGF和c-Met的異常表達與宮頸癌臨床病理因素的關(guān)系,評價了MACC1、HGF和c-Met在宮頸癌中表達的臨床意義,進一步探討了MACC1、HGF和c-Met的表達與人類乳頭瘤病毒(human papillomavirus, HPV)感染在宮頸癌的發(fā)生發(fā)展中的關(guān)系。為揭示宮頸癌的發(fā)生發(fā)展機制,指導(dǎo)宮頸癌的臨床診斷及治療提供新的思路。 方法 1.采用免疫組織化學(xué)技術(shù)檢測40例正常宮頸組織、20例宮頸上皮內(nèi)瘤樣變組織和100例宮頸癌組織中MACC1、HGF和c-Met的表達情況,比較不同宮頸組織中MACC1、HGF和c-Met表達的陽性率。 2.采用PCR技術(shù)檢測40例正常宮頸組織、20例宮頸上皮內(nèi)瘤樣變組織和100例宮頸癌組織中HPV DNA表達,比較不同宮頸組織中HPV DNA表達的陽性率 3.分析MACC1、HGF和c-Met在宮頸癌中的異常表達與臨床病理參數(shù)的關(guān)系。 4.探討宮頸癌組織中MACC1、HGF和c-Met異常表達的相關(guān)性。 5.分析MACC1、HGF和c-Met在宮頸癌組織中的陽性表達與HPV持續(xù)感染的相關(guān)性。 結(jié)果 1. MACC1、HGF、c-Met和HPV DNA在宮頸癌、宮頸上皮內(nèi)瘤樣變組織中陽性表達率明顯高于正常宮頸組織中的表達,差異有統(tǒng)計學(xué)意義(P0.05)。 2.在宮頸癌組織中MACC1、HGF和c-Met的陽性表達率與臨床分期、組織學(xué)病理分級和淋巴結(jié)轉(zhuǎn)移相關(guān),差異有統(tǒng)計學(xué)意義(P0.05),三者在宮頸癌組織中的陽性表達率隨著臨床分期的變晚、組織病理分級變差和淋巴結(jié)轉(zhuǎn)移的出現(xiàn)陽性率逐級上升；HPV DNA的陽性表達與宮頸癌的臨床分期、組織學(xué)病理分級、淋巴結(jié)轉(zhuǎn)移均無關(guān)(P0.05)。 3.在宮頸癌組織中MACC1的異常表達與HGF、c-Met的異常表達呈正相關(guān)(Spearman r=0.68, P=0.000; Spearman r=0.71, P=0.008), HGF蛋白與c-Met蛋白表達呈正相關(guān)(Spearman r=0.75, P＝0.000)。 4. MACC1、HGF和c-Met的陽性表達與HPV的持續(xù)感染密切相關(guān)(Spearman r=0.420, P=0.000)。 結(jié)論 1.在宮頸癌組織中MACC1、HGF和c-Met基因均過表達。 2.在宮頸癌組織中MACC1-HGF/c-Met-HPV形成了正反饋環(huán),其對于宮頸癌的發(fā)生發(fā)展發(fā)揮了重要作用。 3. MACC1的過度表達促使致癌HPV感染的細胞異常增殖并惡變。 4. MACC1有望成為宮頸癌早期診斷及基因治療的新靶點。 5.聯(lián)合檢測MACC1的表達和HPV的感染,將有助于癌前病變的診斷、病程進展的判斷及患者預(yù)后的評估。 背景與目的 MACC1是新近報道的一種與調(diào)控腫瘤生長和轉(zhuǎn)移的關(guān)鍵基因,在多種高轉(zhuǎn)移性腫瘤中高表達。前期研究結(jié)果證實,MACC1在宮頸癌組織中呈高表達,并與腫瘤臨床分期、組織病理分級、淋巴結(jié)轉(zhuǎn)移密切相關(guān)。為了更好的了解MACC1在宮頸癌發(fā)病機制中的作用,分析MACC1與宮頸癌細胞的生物學(xué)行為的關(guān)系,本研究通過人工合成MACC1基因特異的siRNA(small interfering RNA)序列,并轉(zhuǎn)染到宮頸癌HeLa細胞中,特異性抑制MACC1基因表達。最終研究并分析MACC1對宮頸癌HeLa細胞增殖、遷移和凋亡等的影響,初步闡明了MACC1參與宮頸癌發(fā)生發(fā)展的分子生物學(xué)機制,為宮頸癌的預(yù)防、早期診斷及RNA干擾技術(shù)治療宮頸癌提供理論和實驗依據(jù)。 方法 1.化學(xué)合成MACC1基因特異的siRNA序列,利用陽離子脂質(zhì)體Lipofectamine2000轉(zhuǎn)染到HeLa細胞。 2.采用免疫熒光染色觀察MACC1siRNA轉(zhuǎn)染HeLa細胞后細胞骨架形態(tài)的變化。 3.采用RT-PCR和Western blot方法檢測轉(zhuǎn)染前后HeLa細胞中MACC1mRNA和蛋白水平的變化情況。 4.采用Transwell遷移實驗觀察MACC1siRNA轉(zhuǎn)染前后HeLa細胞遷移能力的改變。 5.采用MTT法檢測MACC1siRNA轉(zhuǎn)染前后HeLa細胞生長增殖能力的變化。 6.采用流式細胞儀檢測MACC1siRNA轉(zhuǎn)染前后HeLa細胞的凋亡能力和細胞周期的變化。 結(jié)果 1. MACC1siRNA在轉(zhuǎn)染HeLa細胞后,細胞內(nèi)整齊束狀排列的骨架蛋白變成錯綜復(fù)雜的網(wǎng)狀結(jié)構(gòu),且無方向性,部分斷裂呈粗大的顆粒,散亂分布于胞漿；HeLa-siRNA細胞組、HeLa-NC細胞組和未轉(zhuǎn)染的HeLa細胞組細胞骨架蛋白F-actin的積分熒光強度分別為：10.01±3.53、17.44±5.85和18.53±3.61. 2.在HeLa-siRNA細胞組MACC1mRNA的相對表達量是:0.47±0.06, HeLa-NC細胞組是：0.88+0.09,未轉(zhuǎn)染的HeLa細胞組是：1.00+0.00,經(jīng)統(tǒng)計學(xué)分析HeLa細胞在轉(zhuǎn)染MACC1siRNA后,細胞MACC1mRNA表達水平明顯降低,與HeLa-NC細胞組及未轉(zhuǎn)染的HeLa細胞組相比,差異均有統(tǒng)計學(xué)意義(P0.05),而HeLa-NC細胞組和未轉(zhuǎn)染的HeLa細胞組MACC1mRNA的相對表達量相比,差異無統(tǒng)計學(xué)意義(P0.05)。 3.在HeLa-siRNA細胞組、HeLa-NC細胞組及未轉(zhuǎn)染的HeLa細胞組中MACC1蛋白的相對表達量分別為：0.46±0.05、0.96+0.05和1.01±0.27. MACC1蛋白在HeLa-siRNA細胞組中的相對表達量與HeLa-NC細胞組及未轉(zhuǎn)染的HeLa細胞組的相比,差異均有統(tǒng)計學(xué)意義(P0.05),而HeLa-NC細胞組和未轉(zhuǎn)染的HeLa細胞組MACC1蛋白的相對表達量相比,差異無統(tǒng)計學(xué)意義(P0.05)。 4. Transwell實驗、MTT實驗和流式細胞儀檢測技術(shù)觀察到轉(zhuǎn)染MACC1siRNA后,HeLa細胞的遷移、增殖能力明顯減弱,而凋亡明顯增加,細胞周期G0/G1和G2/M期細胞比例也發(fā)生了明顯的變化。 結(jié)論 1.應(yīng)用RNA干擾技術(shù)能有效的抑制HeLa細胞內(nèi)MACC1基因的表達。 2. MACC1基因的表達抑制能夠改變HeLa細胞的生物學(xué)行為,如增殖和遷移能力受到抑制,凋亡率明顯增加,同時將更多的細胞阻滯在G0/G1和G2/M期。 3.檢測MACC1基因的表達,對于宮頸癌的預(yù)防、早期診斷和選擇基因治療的特異性靶點有非常重要的意義。 4.隨著RNA干擾技術(shù)的發(fā)展,MACC1siRNA有望成為宮頸癌基因治療的新策略。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.33
本文編號：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.33
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