miR-218与宫颈癌恶性程度的相关性及其作用机制的初步探讨
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摘要
宫颈癌是女性生殖系统最常见的恶性肿瘤之一,在世界范围内发病率居第二位,仅次于乳腺癌,每年约有超过50万新发病例。2007年有25万人因此丧生,其中80%的病例发生在发展中国家。中国每年约有新发病例13.15万,占世界新发病例的30%。近年来,宫颈癌的发病年龄趋向年轻化,成为严重威胁中青年妇女健康的重大妇科肿瘤之一。
通过大量的临床、分子生物学及流行病学研究,人乳头瘤病毒(human papillomavirus, HPV)已被证实是引起宫颈癌及宫颈上皮内瘤变(CIN)的主要病因。女性中HPV感染非常普遍,多数可自然清除,仅少数持续性感染,并可发展为CIN,最终进展为浸润性宫颈癌。宫颈癌主要的病理类型有宫颈鳞癌和腺癌,HPV 16与宫颈鳞癌有关,HPV 18与宫颈腺癌有关,但宫颈癌发生的具体分子机制尚不明确。
微小RNA(microRNA, miRNA)是一类长约22个核苷酸的单链非编码RNA,通过与靶mRNA完全或不完全互补配对,导致靶基因降解或抑制其翻译,从而在基因的表达调控中发挥着重要作用。miRNA参与了动植物许多复杂的生命过程,包括发育、器官形成、凋亡和细胞增殖等。研究表明,miRNAs表达异常与恶性肿瘤发生关系密切,miRNAs很可能是一类潜在的癌基因或抑癌基因。最近有研究(1)发现HPV 16阳性的细胞系、宫颈损伤和宫颈癌组织与正常宫颈组织、HPV阴性的宫颈癌细胞系相比,miR-218的表达存在差异,提示miR-218的表达失衡可能参与了宫颈癌的发病过程。目前国内外尚无miR-218与宫颈癌恶性程度相关性的报道,而且文献报道的miR-218在宫颈癌中的表达只检测了HPV16阳性细胞株和少数几例宫颈癌标本,并且这种检测只是局限于组织中。而血清作为生物检测样本,具有取材方便,无创伤性,可连续体外检测的优点,且血清miRNA的高度稳定性也使得病人的实际值与实验的测试值相当吻合。因此有必要深入开展miR-218在宫颈癌中的表达和功能研究,以及对血清中的表达进行检测,为深入研究宫颈癌的发病机理、早期诊断和治疗提供一种新的理论与实验基础。
基于此,本课题的研究内容包括以下两部分：
第一部分miR-218的表达水平与宫颈癌恶性程度的相关性
背景和目的：宫颈癌是女性生殖系统最常见的恶性肿瘤之一。在15种致癌性HPV病毒中,HPV 16最为常见,HPV 16阳性宫颈癌在所有宫颈癌中所占比例超过了50%。与正常宫颈组织、HPV阴性的宫颈癌细胞系相比,HPV 16阳性的宫颈癌细胞系、宫颈损伤和宫颈癌组织中miR-218特异性低表达。在本研究中,我们检测了正常宫颈组织、宫颈癌细胞系、癌前病变和宫颈癌中miR-218的表达状况,检测了宫颈癌患者血清中miR-218的表达水平,旨在探讨miR-218的降低程度与感染的HPV型别、宫颈癌恶性程度的关系,探讨宫颈癌组织和血清中miR-218表达水平的相关性,以及探讨血清中miR-218的表达水平能否反映宫颈癌的恶性程度,为以后的早期诊断提供一种实验基础。
材料和方法：本研究收集了22例子宫肌瘤患者的正常宫颈组织作为对照组,收集了78例宫颈癌患者手术标本、22例石蜡包埋标本和18例宫颈癌患者的血清标本作为实验组,检测了miR-218在正常宫颈组织、宫颈癌细胞系、癌前病变和宫颈癌中的表达,采用导流杂交法检测了感染的HPV的型别,探讨了miR-218的表达水平与HPV型别、宫颈癌恶性程度的相关性。对于收集到的部分宫颈癌患者的血清标本,我们分离得到了小分子RNA,检测了miR-218的表达,使用Pearson's相关系数描述了组织和血清中miR-218表达水平的相关性。
结果：
1.我们成功建立了检测miR-218表达的实时定量RT-PCR方法(UPL法),确定了其检测的线性范围。
2.验证了HPV 16阳性细胞系SiHa中miR-218的低表达。检测到在HPV 18阳性的细胞系HeLa中,miR-218也呈低水平表达。
3.高危型HPV感染的组织标本中miR-218表达低于低危型组织标本和对照组标本,低危型感染组与对照组中miR-218表达水平的差异无统计学意义。
4.在正常组织、CIN和宫颈癌中,miR-218由高到低依次是：正常组织>CINⅠ>CINⅡ～Ⅲ>宫颈癌；而且,miR-218在宫颈癌患者血清中也呈低水平表达,与组织中的低表达存在相关性。
结论：
1.HPV 18(+)的宫颈癌细胞系HeLa中miR-218呈低水平表达。
2.宫颈癌和癌前病变中,高危型HPV感染组织中miR-218呈低水平表达。
3.随着宫颈癌恶性程度的升高,miR-218的表达水平呈进行性降低,这种降低可以反映在血清中。
第二部分miR-218在宫颈癌发病机制中作用的初步探讨
背景和目的：miR-218与宫颈癌的恶性程度密切相关,但在宫颈癌发病机制中所起的作用尚不清楚。本研究在体外人为改变宫颈癌细胞株中miR-218的表达,检测其对细胞生物学活性的影响,并初步探讨其影响机制。
材料和方法：在脂质体siPORT NeoFX的介导下,将Pre-miR miRNA Precursor、Anti-miR miRNA Inhibitors以及对应的阴性对照分别转染入C-33a、SiHa和HeLa细胞株中,检测不同时间点miR-218表达水平的改变,用MTT、流式细胞术检测其对各组细胞的增殖、凋亡、细胞周期的影响,用RT-PCR方法检测了P13K调节亚单位p85 mRNA的表达,并用Western blotting检测了转染前后p85,Akt及p-Akt的表达。
结果：
1.我们成功地将miR-218转染入SiHa细胞,转染后24小时,miR-218水平改变最为显著,随时间的延长,逐渐趋向正常水平。
2.在转染后的24小时,miR-218水平升高可促进SiHa, HeLa细胞增殖,抑制细胞凋亡；miR-218水平降低可抑制SiHa、HeLa的增殖,对细胞凋亡有促进倾向,但无统计学意义。在转染后的48,72小时,这种促进或抑制作用消失。miR-218表达水平的改变对SiHa、HeLa细胞的细胞周期均无影响。对于C-33a,miR-218的表达水平改变对其增殖、凋亡和细胞周期均无影响。
3.miR-218水平升高可促进P13K调节亚单位p85a蛋白的表达和下游效应蛋白Akt的磷酸化,反之抑制p85蛋白的表达和Akt的磷酸化,对p85a mRNA的表达无影响。
结论：虽然miR-218随着宫颈癌恶性程度的升高而表达降低,但是体外实验表明升高miR-218的表达水平对肿瘤细胞系SiHa、HeLa的生长表现出促进作用,降低miR-218的表达对SiHa、HeLa的生长表现出抑制作用,而且只有当miR-218处于高浓度时才表现出这些作用,这说明miR-218对肿瘤细胞作用的复杂性,需要进一步进行体内实验才能充分体现出它的作用。
Objects:
The purpose of this study was to measure the expression level changes of microRNA-218 (miR-218) in normal cervical tissues, cervical intraepithelial neoplasm (CIN) and cervical cancer, and investigate the relationship between miR-218 expression levels with malignancy of cervical cancer. And miR-218 expression levels in serum were measured to study whether it could represent the changes of miR-218 in cervical cancer tissues. Then miR-218 concentrations were changed in cervical cell lines to study the effect of miR-218 on proliferation, apoptosis and cell cycle. Also the signal pathway of PI3K-Akt was determined to investigate whether this pathway was involved in the regulatory roles.
Methods:
1. We set up a method measuring the expression level of miR-218 using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), and detected the expression levels of miR-218 in normal tissues, cervical cell line, cervical intraepithelial neoplasm (CIN) and cervical tissues.
2. HPV types in tissues were measured using Hybrimax to study the effect of high risk HPV types on expression levels of miR-218.
3. Small molecular RNA was extracted using extracting kit of Norgen company from serum and expression levels of miR-218 in serum were detected.
4. Using siPORT NeoFX, we transfected Pre-miR miRNA Precursor, Anti-miR miRNA Inhibitors with their negative control into cervical cell lines, and changes of mature miR-218 expression levels were measured in different time points,
5. Proliferation, apoptosis and cell cycle of cervical cell lines were detected using MTT, flow cytometry, respectively.
6. Changes of protein expression of PI3K regulatory subunit p85, p-Akt and Akt were determined using Western blotting and expression changes of p85a mRNA were measured by RT-PCR.
Results:
1. We developed a method successfully to determine the expression of miR-218 using universal probe library (UPL, Roche).
2. We determined the expression of miR-218 in cervical cell lines and cervical tissues. In cell line, the expression levels from high to low were C-33A>Hela>SiHa. In cervical tissues, the expression levels were normal cervical tissues>CIN I>CINⅡ-Ⅲ>cervical cancer.
3. Infection with high risk HPV types could reduce the expression levels of miR-218 in cervical tissues, and infection with low risk HPV types could not affect the expression levels in cervical tissues.
4. The changes of miR-218 expression levels in serum had a close relationship with the changes of miR-218 in cervical tissues.
5. After reverse transfection of miR-218 precursor/inhibitor into cervical cell, mature miR-218 expression levels were measured at three time points. The highest expression level of miR-218 was at 24 hour, and then gradually returned to normal.
6. At 24 hour after reverse transfection, promoting the expression of miR-218 could enhance proliferation, decrease apoptosis of SiHa and HeLa cells. Otherwise, inhibiting the expression of miR-218 could reduce proliferation. And at 48 hour and 72 hour, proliferation and apoptosis were not affected by miR-218. Cell cycle of SiHa and HeLa cells were not affected by miR-218 at the three time points. Changes of miR-218 showed no effect on cell proliferation, apoptosis and cell cycle of C-33a cell.
7. The up-regulation of miR-218 could increase the expression of p85a protein and phosphorylation of Akt, and down-regulation of miR-218 showed inverse effects. The mRNA of p85αwas not affected in this process.
Conclusions:
1. High risk HPV types could reduce the expression levels of miR-218, and low risk HPV types could not affect the expression of miR-218.
2. The expression levels of miR-218 were reduced with the malignancy of cervical cancer, and this relationship could be represented in serum.
3. miR-218 could regulate the proliferation and apoptosis of SiHa and HeLa, and these effects only existed when miR-218 was in the state of high concentrations.
通过大量的临床、分子生物学及流行病学研究,人乳头瘤病毒(human papillomavirus, HPV)已被证实是引起宫颈癌及宫颈上皮内瘤变(CIN)的主要病因。女性中HPV感染非常普遍,多数可自然清除,仅少数持续性感染,并可发展为CIN,最终进展为浸润性宫颈癌。宫颈癌主要的病理类型有宫颈鳞癌和腺癌,HPV 16与宫颈鳞癌有关,HPV 18与宫颈腺癌有关,但宫颈癌发生的具体分子机制尚不明确。
微小RNA(microRNA, miRNA)是一类长约22个核苷酸的单链非编码RNA,通过与靶mRNA完全或不完全互补配对,导致靶基因降解或抑制其翻译,从而在基因的表达调控中发挥着重要作用。miRNA参与了动植物许多复杂的生命过程,包括发育、器官形成、凋亡和细胞增殖等。研究表明,miRNAs表达异常与恶性肿瘤发生关系密切,miRNAs很可能是一类潜在的癌基因或抑癌基因。最近有研究(1)发现HPV 16阳性的细胞系、宫颈损伤和宫颈癌组织与正常宫颈组织、HPV阴性的宫颈癌细胞系相比,miR-218的表达存在差异,提示miR-218的表达失衡可能参与了宫颈癌的发病过程。目前国内外尚无miR-218与宫颈癌恶性程度相关性的报道,而且文献报道的miR-218在宫颈癌中的表达只检测了HPV16阳性细胞株和少数几例宫颈癌标本,并且这种检测只是局限于组织中。而血清作为生物检测样本,具有取材方便,无创伤性,可连续体外检测的优点,且血清miRNA的高度稳定性也使得病人的实际值与实验的测试值相当吻合。因此有必要深入开展miR-218在宫颈癌中的表达和功能研究,以及对血清中的表达进行检测,为深入研究宫颈癌的发病机理、早期诊断和治疗提供一种新的理论与实验基础。
基于此,本课题的研究内容包括以下两部分：
第一部分miR-218的表达水平与宫颈癌恶性程度的相关性
背景和目的：宫颈癌是女性生殖系统最常见的恶性肿瘤之一。在15种致癌性HPV病毒中,HPV 16最为常见,HPV 16阳性宫颈癌在所有宫颈癌中所占比例超过了50%。与正常宫颈组织、HPV阴性的宫颈癌细胞系相比,HPV 16阳性的宫颈癌细胞系、宫颈损伤和宫颈癌组织中miR-218特异性低表达。在本研究中,我们检测了正常宫颈组织、宫颈癌细胞系、癌前病变和宫颈癌中miR-218的表达状况,检测了宫颈癌患者血清中miR-218的表达水平,旨在探讨miR-218的降低程度与感染的HPV型别、宫颈癌恶性程度的关系,探讨宫颈癌组织和血清中miR-218表达水平的相关性,以及探讨血清中miR-218的表达水平能否反映宫颈癌的恶性程度,为以后的早期诊断提供一种实验基础。
材料和方法：本研究收集了22例子宫肌瘤患者的正常宫颈组织作为对照组,收集了78例宫颈癌患者手术标本、22例石蜡包埋标本和18例宫颈癌患者的血清标本作为实验组,检测了miR-218在正常宫颈组织、宫颈癌细胞系、癌前病变和宫颈癌中的表达,采用导流杂交法检测了感染的HPV的型别,探讨了miR-218的表达水平与HPV型别、宫颈癌恶性程度的相关性。对于收集到的部分宫颈癌患者的血清标本,我们分离得到了小分子RNA,检测了miR-218的表达,使用Pearson's相关系数描述了组织和血清中miR-218表达水平的相关性。
结果：
1.我们成功建立了检测miR-218表达的实时定量RT-PCR方法(UPL法),确定了其检测的线性范围。
2.验证了HPV 16阳性细胞系SiHa中miR-218的低表达。检测到在HPV 18阳性的细胞系HeLa中,miR-218也呈低水平表达。
3.高危型HPV感染的组织标本中miR-218表达低于低危型组织标本和对照组标本,低危型感染组与对照组中miR-218表达水平的差异无统计学意义。
4.在正常组织、CIN和宫颈癌中,miR-218由高到低依次是：正常组织>CINⅠ>CINⅡ～Ⅲ>宫颈癌；而且,miR-218在宫颈癌患者血清中也呈低水平表达,与组织中的低表达存在相关性。
结论：
1.HPV 18(+)的宫颈癌细胞系HeLa中miR-218呈低水平表达。
2.宫颈癌和癌前病变中,高危型HPV感染组织中miR-218呈低水平表达。
3.随着宫颈癌恶性程度的升高,miR-218的表达水平呈进行性降低,这种降低可以反映在血清中。
第二部分miR-218在宫颈癌发病机制中作用的初步探讨
背景和目的：miR-218与宫颈癌的恶性程度密切相关,但在宫颈癌发病机制中所起的作用尚不清楚。本研究在体外人为改变宫颈癌细胞株中miR-218的表达,检测其对细胞生物学活性的影响,并初步探讨其影响机制。
材料和方法：在脂质体siPORT NeoFX的介导下,将Pre-miR miRNA Precursor、Anti-miR miRNA Inhibitors以及对应的阴性对照分别转染入C-33a、SiHa和HeLa细胞株中,检测不同时间点miR-218表达水平的改变,用MTT、流式细胞术检测其对各组细胞的增殖、凋亡、细胞周期的影响,用RT-PCR方法检测了P13K调节亚单位p85 mRNA的表达,并用Western blotting检测了转染前后p85,Akt及p-Akt的表达。
结果：
1.我们成功地将miR-218转染入SiHa细胞,转染后24小时,miR-218水平改变最为显著,随时间的延长,逐渐趋向正常水平。
2.在转染后的24小时,miR-218水平升高可促进SiHa, HeLa细胞增殖,抑制细胞凋亡；miR-218水平降低可抑制SiHa、HeLa的增殖,对细胞凋亡有促进倾向,但无统计学意义。在转染后的48,72小时,这种促进或抑制作用消失。miR-218表达水平的改变对SiHa、HeLa细胞的细胞周期均无影响。对于C-33a,miR-218的表达水平改变对其增殖、凋亡和细胞周期均无影响。
3.miR-218水平升高可促进P13K调节亚单位p85a蛋白的表达和下游效应蛋白Akt的磷酸化,反之抑制p85蛋白的表达和Akt的磷酸化,对p85a mRNA的表达无影响。
结论：虽然miR-218随着宫颈癌恶性程度的升高而表达降低,但是体外实验表明升高miR-218的表达水平对肿瘤细胞系SiHa、HeLa的生长表现出促进作用,降低miR-218的表达对SiHa、HeLa的生长表现出抑制作用,而且只有当miR-218处于高浓度时才表现出这些作用,这说明miR-218对肿瘤细胞作用的复杂性,需要进一步进行体内实验才能充分体现出它的作用。
Objects:
The purpose of this study was to measure the expression level changes of microRNA-218 (miR-218) in normal cervical tissues, cervical intraepithelial neoplasm (CIN) and cervical cancer, and investigate the relationship between miR-218 expression levels with malignancy of cervical cancer. And miR-218 expression levels in serum were measured to study whether it could represent the changes of miR-218 in cervical cancer tissues. Then miR-218 concentrations were changed in cervical cell lines to study the effect of miR-218 on proliferation, apoptosis and cell cycle. Also the signal pathway of PI3K-Akt was determined to investigate whether this pathway was involved in the regulatory roles.
Methods:
1. We set up a method measuring the expression level of miR-218 using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), and detected the expression levels of miR-218 in normal tissues, cervical cell line, cervical intraepithelial neoplasm (CIN) and cervical tissues.
2. HPV types in tissues were measured using Hybrimax to study the effect of high risk HPV types on expression levels of miR-218.
3. Small molecular RNA was extracted using extracting kit of Norgen company from serum and expression levels of miR-218 in serum were detected.
4. Using siPORT NeoFX, we transfected Pre-miR miRNA Precursor, Anti-miR miRNA Inhibitors with their negative control into cervical cell lines, and changes of mature miR-218 expression levels were measured in different time points,
5. Proliferation, apoptosis and cell cycle of cervical cell lines were detected using MTT, flow cytometry, respectively.
6. Changes of protein expression of PI3K regulatory subunit p85, p-Akt and Akt were determined using Western blotting and expression changes of p85a mRNA were measured by RT-PCR.
Results:
1. We developed a method successfully to determine the expression of miR-218 using universal probe library (UPL, Roche).
2. We determined the expression of miR-218 in cervical cell lines and cervical tissues. In cell line, the expression levels from high to low were C-33A>Hela>SiHa. In cervical tissues, the expression levels were normal cervical tissues>CIN I>CINⅡ-Ⅲ>cervical cancer.
3. Infection with high risk HPV types could reduce the expression levels of miR-218 in cervical tissues, and infection with low risk HPV types could not affect the expression levels in cervical tissues.
4. The changes of miR-218 expression levels in serum had a close relationship with the changes of miR-218 in cervical tissues.
5. After reverse transfection of miR-218 precursor/inhibitor into cervical cell, mature miR-218 expression levels were measured at three time points. The highest expression level of miR-218 was at 24 hour, and then gradually returned to normal.
6. At 24 hour after reverse transfection, promoting the expression of miR-218 could enhance proliferation, decrease apoptosis of SiHa and HeLa cells. Otherwise, inhibiting the expression of miR-218 could reduce proliferation. And at 48 hour and 72 hour, proliferation and apoptosis were not affected by miR-218. Cell cycle of SiHa and HeLa cells were not affected by miR-218 at the three time points. Changes of miR-218 showed no effect on cell proliferation, apoptosis and cell cycle of C-33a cell.
7. The up-regulation of miR-218 could increase the expression of p85a protein and phosphorylation of Akt, and down-regulation of miR-218 showed inverse effects. The mRNA of p85αwas not affected in this process.
Conclusions:
1. High risk HPV types could reduce the expression levels of miR-218, and low risk HPV types could not affect the expression of miR-218.
2. The expression levels of miR-218 were reduced with the malignancy of cervical cancer, and this relationship could be represented in serum.
3. miR-218 could regulate the proliferation and apoptosis of SiHa and HeLa, and these effects only existed when miR-218 was in the state of high concentrations.
引文
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