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.
引文
1. Castellsague X, Diaz M, de Sanjose S, et al. Worldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactors:implications for screening and prevention. J Natl Cancer Inst 2006; 98:303-15.
2. Butler D, Collins C, Mabruk M, Walsh C, Leader M, Kay E. Deletion of the FHIT gene in neoplastic and invasive cervical lesions is related to high-risk HPV infection but is independent of histopathological features. The Journal of Pathology 2000; 192: 502-10.
3. Ferber M, Montoya D, Yu C, et al. Integrations of the hepatitis B virus (HBV) and human papillomavirus (HPV) into the human telomerase reverse transcriptase (hTERT) gene in liver and cervical cancers. Oncogene 2003; 22:3813-20.
4. Hopman A, Smedts F, Dignef W, et al. Transition of high-grade cervical intraepithelial neoplasia to micro-invasive carcinoma is characterized by integration of HPV 1618 and numerical chromosome abnormalities. The Journal of Pathology 2004; 202:23-33.
5. Mougin C, Humbey O, Gay C, Riethmuller D. [Human papillomaviruses, cell cycle and cervical cancer]. J Gynecol Obstet Biol Reprod (Paris) 2000; 29:13-20.
6. Song S, Pitot H, Lambert P. The human papillomavirus type 16 E6 gene alone is sufficient to induce carcinomas in transgenic animals. Journal of virology 1999; 73: 5887.
7. Zhang D, Ngan H, Cheng R, Cheung A, Liu S, Tsao S. Clinical significance of telomerase activation and telomeric restriction fragment (TRF) in cervical cancer. European Journal of Cancer 1999; 35:154-60.
8.张正军.端粒,端粒酶与肿瘤.中国组织化学与细胞化学杂志2001;10:228-32.
9. Martin CM, Astbury K, O'Leary JJ. Molecular profiling of cervical neoplasia. Expert Rev Mol Diagn 2006; 6:217-29.
10. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2002; 99:15524-9.
11. Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005; 65:7065-70.
12. Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A 2006; 103: 2257-61.
13. Lu L, Katsaros D, Rigault de la Longrais I, Sochirca O, Yu H. Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis. Cancer research 2007; 67:10117.
14. Murakami Y, Yasuda T, Saigo K, et al. Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2005; 25:2537-45.
15. Kutay H, Bai S, Datta J, et al. Downregulation of miR-122 in the rodent and human hepatocellular carcinomas. Journal of cellular biochemistry 2006; 99:671-8.
16. Roldo C, Missiaglia E, Hagan J, et al. MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. Journal of Clinical Oncology 2006; 24:4677.
17. He H, Jazdzewski K, Li W, et al. The role of microRNA genes in papillary thyroid carcinoma. Proceedings of the National Academy of Sciences of the United States of America 2005; 102:19075.
18. Mitchell P, Parkin R, Kroh E, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences 2008; 105:10513.
19. Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Research 2008; 18: 997-1006.
20. Leite KR, Sousa-Canavez JM, Reis ST, et al. Change in expression of miR-let7c, miR-100, and miR-218 from high grade localized prostate cancer to metastasis. Urol Oncol 2009.
21. Liu W, Gong YH, Chao TF, et al. Identification of differentially expressed microRNAs by microarray:a possible role for microRNAs gene in medulloblastomas. Chin Med J (Engl) 2009; 122:2405-11.
22. Kagawa N, Maruno M, Suzuki T, et al. Detection of genetic and chromosomal aberrations in medulloblastomas and primitive neuroectodermal tumors with DNA microarrays. Brain Tumor Pathol 2006; 23:41-7.
23. Schembri F, Sridhar S, Perdomo C, et al. MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium. Proc Natl Acad Sci U S A 2009; 106:2319-24.
24. Zanette DL, Rivadavia F, Molfetta GA, et al. miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia. Braz J Med Biol Res 2007; 40: 1435-40.
25. Martinez I, Gardiner AS, Board KF, Monzon FA, Edwards RP, Khan SA. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells. Oncogene 2008; 27:2575-82.
26. Lee JW, Choi CH, Choi JJ,et al. Altered MicroRNA expression in cervical carcinomas. Clin Cancer Res 2008; 14:2535-42.
27.曹利平.改良一步法从甲醛固定-石蜡包埋组织中提取RNA.中华医学杂志2000;80:2.
28. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005; 33:e179.
29. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP. Protocol:a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 2007; 3:12.
30. Schefe J, Lehmann K, Buschmann I, Unger T, Funke-Kaiser H. Quantitative real-time RT-PCR data analysis:current concepts and the novel "gene expression's C T difference" formula. Journal of Molecular Medicine 2006; 84:901-10.
31.刘敏,王传新,邓小梅,et al.应用液相基因芯片技术筛查山东地区高危人群人乳头瘤病毒基因型.中华流行病学杂志2007;28:487-90.
32. He L, Hannon GJ. MicroRNAs:small RNAs with a big role in gene regulation. Nat Rev Genet 2004; 5:522-31.
33. Kim VN. Small RNAs:classification, biogenesis, and function. Mol Cells 2005; 19:1-15.
34. Duncan DD, Eshoo M, Esau C, Freier SM, Lollo BA. Absolute quantitation of microRNAs with a PCR-based assay. Anal Biochem 2006; 359:268-70.
35. Clavel C, Masure M, Bory J, et al. Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions:a study of 7932 women. British journal of cancer 2001; 84:1616.
36. Negri G, Gampenrieder J, Vigl E, Haitel A, Menia E, Mian C. Human papilloma virus typing at large loop excision of the transformation zone of the cervix uteri. Anticancer research 2003; 23:4289-92.
37.陶萍萍,卞美璐,梁静,et al.导流杂交与杂交捕获二代及原位杂交在女性生殖道人乳状瘤病毒检测中的比较.中日友好医院学报2005;19:325-7.
38. Lebanony D, Benjamin H, Gilad S, et al. Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol 2009; 27:2030-7.
39. Li J, Smyth P, Flavin R, et al. Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded(FFPE) cells and snap frozen cells. BMC biotechnology 2007; 7: 36.
40. Ng E, Chong W. Differential expression of microRNAs in plasma of colorectal cancer patients:a potential marker for colorectal cancer screening. British Medical Journal 2009.
41. Xie Y, Todd N, Liu Z, et al. Altered miRNA expression in sputum for diagnosis of non-small cell lung cancer. Lung Cancer; 67:170-6.
1. Volinia S, Calin Q Liu C, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proceedings of the National Academy of Sciences 2006; 103:2257.
2. Pasquinelli AE, Ruvkun G Control of developmental timing by micrornas and their targets. Annu Rev Cell Dev Biol 2002; 18:495-513.
3. Lee Y, Ahn C, Han J, et al. The nuclear RNase Ⅲ Drosha initiates microRNA processing. Nature 2003; 425:415-9.
4. Lund E, Guttinger S, Calado A, Dahlberg J, Kutay U. Nuclear export of microRNA precursors. Science 2004; 303:95.
5. Bernstein E, Caudy A, Hammond S, Hannon G. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409:363-6.
6. Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell 2004; 116:281-97.
7. Bartel D. MicroRNAs::Genomics, Biogenesis, Mechanism, and Function. Cell 2004; 116:281-97.
8. Ambros V. The functions of animal microRNAs. Nature 2004; 431:350-5.
9. Du T, Zamore P. microPrimer:the biogenesis and function of microRNA. Development 2005; 132:4645.
10. Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 2005; 438:685-9.
11. Zhang B, Pan X, Cobb G, Anderson T. MicroRNAs as oncogenes and tumor suppressors. Developmental biology 2007; 302:1-12.
12. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001; 294:853.
13. Sethupathy P, Megraw M, Hatzigeorgiou A. A guide through present computational approaches for the identification of mammalian microRNA targets. Nature methods 2006; 3:881-6.
14. Mourelatos Z, Dostie J, Paushkin S, et al. miRNPs:a novel class of ribonucleoproteins containing numerous microRNAs. Genes & development 2002; 16: 720.
15. Lee R, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001;294:862.
16. Lau N, Lim L, Weinstein E, Bartel D. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001; 294:858.
17. Brennecke J, Stark A, Russell R, Cohen S. Principles of microRNA-target recognition. PLoS Biol 2005; 3:e85.
18. Lewis B, Shih I. Prediction of mammalian microRNA targets. Cell 2003; 115: 787-98.
19. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield M. Cellular function of phosphoinositide 3-kinase:implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 2001; 17:615-75.
20. Altomare D, Testa J. Perturbations of the AKT signaling pathway in human cancer. Oncogene 2005; 24:7455-64.
21. Bellacosa A, Kumar C, Cristofano A, Testa J. Activation of AKT kinases in cancer:implications for therapeutic targeting. Advances in Cancer Research 2005: 29-86.
22. Vivanco I, Sawyers C. The phosphatidylinositol 3-kinase-Akt pathway in human cancer. Nature Reviews Cancer 2002; 2:489-501.
23. Ward S, Finan P. Isoform-specific phosphoinositide 3-kinase inhibitors as therapeutic agents. Current opinion in pharmacology 2003; 3:426-34.
24. Solit D, Basso A, Olshen A, Scher H, Rosen N. Inhibition of heat shock protein 90 function down-regulates Akt kinase and sensitizes tumors to Taxol. Cancer research 2003; 63:2139.
25. Samuels Y, Diaz Jr L, Schmidt-Kittler O, et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005; 7:561-73.
26. Kang S, Bader A, Vogt P. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proceedings of the National Academy of Sciences 2005; 102:802.
27. Philp A, Campbell I, Leet C, et al. The phosphatidylinositol 3'-kinase p85{alpha} gene is an oncogene in human ovarian and colon tumors. Cancer research 2001; 61: 7426.
28. Shayesteh L, Lu Y, Kuo W, et al. PIK3CA is implicated as an oncogene in ovarian cancer. Nature genetics 1999; 21:99-102.
29. Ma Y, Wei S, Lin Y, et al. PIK3CA as an oncogene in cervical cancer. Oncogene 2000; 19:2739.
30. Woenckhaus J, Steger K, Werner E, et al. Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma. The Journal of Pathology 2002; 198:335-42.
31. Byun D, Cho K, Ryu B, et al. Frequent monoallelic deletion of PTEN and its reciprocal associatioin with PIK3CA amplification in gastric carcinoma. International journal of cancer 2003; 104:318-27.
32. Knobbe CB, Reifenberger G Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol 2003; 13:507-18.
33. Calin G, Sevignani C, Dumitru C, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proceedings of the National Academy of Sciences 2004; 101:2999.
34. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A. Identification of mammalian microRNA host genes and transcription units. Genome Res 2004; 14: 1902-10.
35. Narayan G, Goparaju C, Arias-Pulido H, et al. Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression. Molecular cancer 2006; 5:16.
36. Cheng AM, Byrom MW, Shelton J, Ford LP. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res 2005; 33:1290-7.
37. Gao C, Zhang Z, Liu W, Xiao S, Gu W, Lu H. Reduced microRNA-218 expression is associated with high nuclear factor kappa B activation in gastric cancer. Cancer 2009.
38. Leite KR, Sousa-Canavez JM, Reis ST, et al. Change in expression of miR-let7c, miR-100, and miR-218 from high grade localized prostate cancer to metastasis. Urol Oncol 2009.
1. WHO. Initiative for Vaccine Research (IVR). Human papillomavirus. http://wwwwhoint/vaccine_research/diseases/viral_cancers/en/index3html (accessed April 1,2009) 2007.
2. Smith JS, Lindsay L, Hoots B, et al. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions:a meta-analysis update. Int J Cancer 2007; 121:621-32.
3. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 1993; 75:495-505.
4. La Torre G, de Waure C, Chiaradia G, Mannocci A, Ricciardi W. HPV vaccine efficacy in preventing persistent cervical HPV infection:a systematic review and meta-analysis. Vaccine 2007; 25:8352-8.
5. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120:15-20.
6. Ambros V. MicroRNA pathways in flies and worms:growth, death, fat, stress, and timing. Cell 2003; 113:673-6.
7. Culp TD, Budgeon LR, Marinkovich MP, Meneguzzi G, Christensen ND. Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells. J Virol 2006; 80:8940-50.
8. Martinez I, Gardiner AS, Board KF, Monzon FA, Edwards RP, Khan SA. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells. Oncogene 2008; 27:2575-82.
9. Roymans D, Slegers H. Phosphatidylinositol 3-kinases in tumor progression. Eur J Biochem 2001; 268:487-98.
10. Cui B, Zheng B, Zhang X, Stendahl U, Andersson S, Wallin KL. Mutation of PIK3CA:possible risk factor for cervical carcinogenesis in older women. Int J Oncol 2009; 34:409-16.
11. Zhang XY, Zhang HY, Zhang PN, Lu X, Sun H. Elevated phosphatidylinositol 3-kinase activation and its clinicopathological significance in cervical cancer. Eur J Obstet Gynecol Reprod Biol 2008; 139:237-44.
12. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP. Done-Protocol:a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 2007; 3:12.
13. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75:843-54.
14. Ruvkun G Molecular biology. Glimpses of a tiny RNA world. Science 2001; 294: 797-9.
15. Leite KR, Sousa-Canavez JM, Reis ST, et al. Change in expression of miR-let7c, miR-100, and miR-218 from high grade localized prostate cancer to metastasis. Urol Oncol 2009.
16. Gao C, Zhang Z, Liu W, Xiao S, Gu W, Lu H. Reduced microRNA-218 expression is associated with high nuclear factor kappa B activation in gastric cancer. Cancer 2009.
17. Schembri F, Sridhar S, Perdomo C, et al. MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium. Proc Natl Acad Sci U S A 2009; 106:2319-24.
18. Zanette DL, Rivadavia F, Molfetta GA, et al. miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia. Braz J Med Biol Res 2007; 40: 1435-40.
19. Cheng AM, Byrom MW, Shelton J, Ford LP. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res 2005; 33:1290-7.
20. Kang S, Bader AG, Vogt PK. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci U S A 2005; 102:802-7.
21. Samuels Y, Diaz LA, Jr., Schmidt-Kittler O, et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005; 7:561-73.
22. Philp AJ, Campbell IG, Leet C, et al. The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors. Cancer Res 2001; 61: 7426-9.
23. Goto T, Takano M, Sasa H, Tsuda H, Yamauchi K, Kikuchi Y. Clinical significance of immunocytochemistry for PIK3CA as a carcinogenesis-related marker on liquid-based cytology in cervical intraepithelial neoplasia. Oncol Rep 2006; 15: 387-91.
24. Ma YY, Wei SJ, Lin YC, et al. PIK3CA as an oncogene in cervical cancer. Oncogene 2000; 19:2739-44.
25. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002; 2:489-501.
26. Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations:the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 2006; 6:184-92.
27. Datta SR, Brunet A, Greenberg ME. Cellular survival:a play in three Akts. Genes Dev 1999; 13:2905-27.
1. Lee R, Feinbaum R, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75:843-54.
2. Wightman B, Ha I, Ruvkun G Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993; 75: 855-62.
3. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001; 294:853.
4. Lee R, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001; 294:862.
5. Lau N, Lim L, Weinstein E, Bartel D. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001; 294:858.
6. Griffiths-Jones S. The microRNA registry. Nucleic Acids Research 2004; 32: D109.
7. Lewis B, Burge C, Bartel D. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120: 15-20.
8. Bushati N, Cohen S. microRNA functions.2007.
9. Bartel D. MicroRNAs::Genomics, Biogenesis, Mechanism, and Function. Cell 2004; 116:281-97.
10. Kim V. MicroRNA biogenesis:coordinated cropping and dicing. Nature Reviews Molecular Cell Biology 2005; 6:376-85.
11. Filipowicz W, Bhattacharyya S, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight? Nature Reviews Genetics 2008; 9: 102-14.
12. hman M. A-to-I editing challenger or ally to the microRNA process. Biochimie 2007; 89:1171-6.
13. Matzke M, Matzke A, Kooter J. RNA:guiding gene silencing. Science 2001; 293: 1080-3.
14. Calin G, Sevignani C, Dumitru C, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proceedings of the National Academy of Sciences 2004; 101:2999.
15. He L, Thomson J, Hemann M, et al. A microRNA polycistron as a potential human oncogene. Nature 2005; 435:828-33.
16. Ying S, Lin S. Intronic microRNAs. Biochemical and biophysical research communications 2005; 326:515-20.
17. CAI X, HAGEDORN C, CULLEN B. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 2004; 10: 1957.
18. Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase 11. The EMBO journal 2004; 23:4051-60.
19. Gregory R, Yan K, Amuthan G, et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 2004; 432:235-40.
20. Yi R, Qin Y, Macara I, Cullen B. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes & development 2003; 17:3011.
21. Hutvagner G, Zamore P. A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002; 297:2056.
22. Hammond S, Bernstein E, Beach D, Hannon G. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000; 404: 293-6.
23. Martinez J, Tuschl T. RISC is a 5'phosphomonoester-producing RNA endonuclease. Genes & development 2004; 18:975.
24. Zeng Y, Yi R, Cullen B. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proceedings of the National Academy of Sciences of the United States of America 2003; 100:9779.
25. Schwarz D, Tomari Y, Zamore P. The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Current Biology 2004; 14:787-91.
26. Kern W, Kohlmann A, Wuchter C, et al. Correlation of protein expression and gene expression in acute leukemia. Cytometry Part B:Clinical Cytometry 2003; 55: 29-36.
27. Llave C, Kasschau K, Rector M, Carrington J. Endogenous and silencing-associated small RNAs in plants. The Plant Cell Online 2002; 14:1605.
28. LAI E. Micro RNAs are complementary to 3'UTR sequence motifs that mediate negative post-transcriptional regulation. Nature genetics 2002; 30:363-4.
29. Stark A, Brennecke J, Russell R, Cohen S. Identification of Drosophila microRNA targets. PLoS Biol 2003; 1:E60.
30. Wang X, Reyes J, Chua N, Gaasterland T. Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets. Genome Biology 2004; 5: R65.
31. Pillai R, Bhattacharyya S, Artus C, et al. Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 2005; 309:1573.
32. Doench J, Petersen C, Sharp P. siRNAs can function as miRNAs. Genes & development 2003; 17:438.
33. Calin G, Dumitru C, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002; 99:15524-9.
34. Michael M, O Connor S, Van Holst Pellekaan N, Young G, James R. Reduced accumulation of specific microRNAs in colorectal neoplasia. Molecular Cancer Research 2003; 1:882-91.
35. Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Research 2004; 64:3753.
36. Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A. High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes, Chromosomes and Cancer 2003; 39:167-9.
37. Ryther R, Flynt A, Phillips J, Patton J. siRNA therapeutics:big potential from small RNAs. Gene Therapy 2004; 12:5-11.
38. Stevenson M. Therapeutic potential of RNA interference. The New England journal of medicine 2004; 351:1772.
1. Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nat Rev Mol Cell Biol 2005; 6:376-85.
2. Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell 2004; 116:281-97.
3. He L, Hannon GJ. MicroRNAs:small RNAs with a big role in gene regulation. Nat Rev Genet 2004; 5:522-31.
4. Kim VN. Small RNAs:classification, biogenesis, and function. Mol Cells 2005; 19:1-15.
5. Ambros V. microRNAs:tiny regulators with great potential. Cell 2001; 107: 823-6.
6. Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS. Post-transcriptional gene silencing by siRNAs and miRNAs. Curr Opin Struct Biol 2005; 15:331-41.
7. Duncan DD, Eshoo M, Esau C, Freier SM, Lollo BA. Absolute quantitation of microRNAs with a PCR-based assay. Anal Biochem 2006; 359:268-70.
8. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP. Done-Protocol:a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 2007; 3:12.
9. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005;33:e179.
10. Tang F, Hajkova P, Barton SC, Lao K, Surani MA. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res 2006; 34:e9.
11. Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 2005; 11:1737-44.
12. Shi R, Chiang VL. Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 2005; 39:519-25.
13.张旗,何湘君,潘秀英.RNA加尾和引物延伸RT-PCR法实时定量检测microRNA.北京大学学报(医学版)2007.
14. Lu D, Read RL, Humphreys DT, Battah FM, Martin DIK, Rasko JEJ. PCR-based expression analysis and identification of microRNAs. Journal of RNAi and Gene Silencing 2005; 1:6.