microRNA-221和microRNA-222与恶性肿瘤的研究进展

微RNA(micmRNA)-221和microRNA-222是成簇的microRNA,在恶性肿瘤中起促癌作用,在胶质母细胞瘤、前列腺癌、乳头状甲状腺癌等恶性肿瘤中高表达.microRNA-221和microRNA-222通过调控其特定的靶基因行使促癌作用.在所有预测的靶基因中,p27kip1和p57kip2是已被验证的靶基因,microRNA-221和microRNA-222通过下调p27kip1和p57kip2的表达来促进肿瘤的形成和生长.     

反义miR-221/222上调p27kip1对MCF-7乳腺癌细胞系的放射增敏作用

目的探讨敲低miR-221／222表达上调p27^kip1对MCF-7人乳腺癌细胞系放射敏感性的影响。方法经生物信息学分析查询miR-221／222成熟体序列和它们与p27^kip1的关系。脂质体共转染反义寡聚核苷酸（反义miR-221／222）后,用Northern blot法检测转染后MCF-7细胞miR-221、miR-222表达水平;将实验细胞分为6组：对照组、对照照射组、无义序列组、无义序列照射组、反义miR-221／222共转染组和反义miR-221／222共转染照射组。用MTT法检测细胞增殖及放射协同作用,流式细胞仪分析细胞周期,克隆形成实验检测细胞增殖,Western blot分析p27^kip1蛋白的表达变化。数据间的方差分析采用F检验;两两比较采用LSD-t检验。结果经生物信息学分析显示miR-221／222成熟体序列的种子序列几乎一致,p27^kip1是miR-221／222的靶基因。Northern blot显示反义miR-221／222共转染组miR-221、miR-222的表达水平明显下降（miR-221：P=0．000;miR-222：P=0．000）。对照组及无义序列组之间miR-221、miR-222表达水平的差异无统计学意义（miR-221：P=0．371;miR-222：P=0．284）。MTT结果显示转染后第4天共转染组肿瘤细胞生长抑制效果最好,细胞增殖率明显低于对照组和无义序列组（P=0．000）,但与放射治疗无协同作用（P=0．091）。流式细胞术检测可见共转染组细胞周期存在G0／G1期阻滞（P=0．000）。经放射治疗后,可明显降低S期比例（P=0．002）。克隆形成实验表明反义miR-221／222可增加MCF-7细胞的放射敏感性。Western blot显示反义miR-221／222共转染组的p27^kip1蛋白表达明显上调（P=0．000）。结论反义miR-221／222通过上调p27^kip1蛋白表达可以增加MCF-7人乳腺癌细胞系放射敏感性。     

p27kip1真核表达载体的构建及其对人胆管癌细胞生长的影响

目的：构建p27^kip1真核表达载体并进行基因转染,研究其对人胆管癌细胞生长特性的影响。方法：将人p27^kip1 cNDA克隆至真核表达载体pClneo的No1 1酶切位点中,然后应用DOTAP脂质体将重组质粒转染人胆管癌QBC939细胞中,用G418筛选抗性细胞克隆,采用PCR、RT-PCR和Western印迹法检测外源性目的基因在靶细胞基因组中的整合及表达,以及转基因细胞对裸鼠致瘤性能力的改变。结果：成功构建p27^kip1基因真核表达载体,并将其导入QBC939细胞中。经G418筛选得到稳定表达p27^kip1基因的细胞株,p27^kip1基因转染细胞生长速度明显减慢,并出现分化的迹象,其裸鼠致瘤性能力明显降低。结论：p27^kip1基因具有抑癌基因的作用,在未来胆管癌的基因治疗中可能具有应用前景。     

microRNA-613在恶性肿瘤中的研究进展

微小RNA（microRNA，miRNA）作为一种在恶性肿瘤发生中起到非常重要作用的短链非编码RNA，通过与其靶基因的特异性结合从转录后水平调控肿瘤相关基因的表达。微小RNA-613（microRNA-613，miR-613）定位在人染色体12p13.1，通过对靶基因的调控，参与肿瘤细胞的增殖、分化、凋亡、癌周浸润等恶性进程的调控。近年来研究表明，miR-613在多种肿瘤中异常表达并与肿瘤的临床特征及预后密切相关。鉴于其在恶性肿瘤中的重要作用，miR-613或可成为分子靶向治疗的新靶点。     

放射线对鼻咽癌细胞周期阻滞、凋亡和抑癌基因p57^kip2表达的影响

目的：研究放射线对鼻咽癌细胞（CNE）周期阻滞、凋亡和抑癌基因p57^kip2蛋白表达的影响。方法：运用流式细胞术检测放射线诱导的细胞周期阻滞、凋亡；运用免疫组织化学法和Westernblot法检测抑癌基因p57^kip2蛋白的表达。结果：照射后，CNE细胞G1期无明显阻滞，S期出现短暂堆积，G2／M期阻滞强度具剂量和时间依赖性，随照射剂量增加，其阻滞程度增强，阻滞时间延长，G2／M期阻滞在12、24h与照射剂量呈正相关（P〈0．01），随照射后时间延长而增强，峰值出现于12Gy24h；凋亡发生率与照射剂量和照射后时间均呈正相关（P〈0．01）。p57^kip2蛋白表达在照射后随照射剂量的增加和照射后时间的延长均呈现上调（P〈0．01）。结论：放射线对鼻咽癌细胞G2／M期阻滞、凋亡率和抑癌基因p57^kip2蛋白表达均有明显的增强作用。     

乳腺癌组织p27^kip1的表达及其与细胞增殖的关系

目的：研究乳腺癌组织中细胞周期抑制剂 p27^kip1的表达及其意义,并探讨其与细胞增殖的关系。方法：应用免疫组化SP法检测80例乳腺癌和20例癌旁正常组织中 p27^kip1和增殖细胞核抗原（proliferative cell nuclear antigen,PCNA）的表达。结果：乳腺癌组织中 p27^kip1高表达率为53．75％（43／80）,明显低于癌旁正常组织（P〈0．01）。 p27^kip1表达与组织学分级、TNM分期及淋巴结转移均相关,P〈0．05。乳腺癌组织中 p27^kip1的表达与PCNALI呈负相关,r=0．372,P〈0．05。 p27^kip1高表达的乳腺癌术后5年无病生存率（DFS）明显高于 p27^kip1低表达者（P〈0．05）。结论： p27^kip1表达缺失可促进肿瘤细胞增殖,是判断乳腺癌发生、发展及预后的有效生物学指标。     

肝细胞癌组织中endoglin（CD105）和VEGF及p57^kip2表达与预后的关系

目的：探讨肝细胞癌(hepatocellular carcinoma，HCC)endoglin(CD127)、血管内皮生长因子(vascular endothelial growth factor，VEGF)和有丝分裂抑制因子(p57^kip2)的表达与组织形态学和自然病程的关系。方法：SP法检测143例资料完整的HCC中63例获访者肝癌组织和20例非肝癌肝组织中endoglin(CD127)、VEGF和p57^kip2的表达。结果：肝癌组织endoglin(CD127)、VEGF和p57^kip2免疫组化阳性率分别为68．3％(43/63)、73．0％(46/63)和68．3％(43/63)，显著高于癌旁组织及非癌肝组织．P=0．0000。癌组织分化与VEGF、endoglin(CD127)和p57^kip2表达差异无统计学意义(P值分别为0．0978、0．1393和0．8397)。endoglin(CD27)、VEGF和p57^kip2表达不同，患者的生存时间有差别(P值分别为0．0013、0．0039和0．0008)．endoglin(CD127)和VEGF高表达。可能预示预后较差，而p57^kip2高表达可能预示预后较好。p57^kip2表达与endoglin(CD127)和VEGF表达呈负相关，r^*值分别为-0．3571和-0．1725。结论：endoglin(CD127)、VEGF蛋白的过度表达和p57^kip2丢失可能与肝癌的发展及预后有关。     

乳腺癌中RUNX3、cyclin D1和p27kip1蛋白的表达及其意义

[目的]探讨乳腺癌组织中RUNX3、cyclinD1和p27^kip1基因的表达及其临床意义。[方法]采用免疫组织化学SP法检测88例乳腺癌、40例乳腺纤维腺瘤、40例乳腺增生病中RUNX3、cyclinD1和p27^kip1的表达。[结果]（1）RUNX3和p27^kip1在乳腺癌中的阳性表达率分别为35．23％和51．14％，明显低于在乳腺纤维腺瘤（85％和82．5％）及乳腺增生病（87．5％和85％）中的表达率（P〈0．05）。乳腺癌中cyclinD1的表达率（63．63％）明显高于良性病变中表达。（2）RUNX3和p27^kip1蛋白表达与乳腺癌的临床分期、淋巴结转移呈负相关。p27^kip1蛋白表达与病理分级呈负相关。cyclinD1蛋白表达与病理分级呈正相关。（3）乳腺癌中RUNX3与p27^kip1蛋白的表达呈正相关性（P〈0．05）．与cyclinD1的表达呈负相关性（P〉0．05）。[结论]RUNX3、cyclinD1和p27^kip1在乳腺癌的发生发展中起重要作用。检测乳腺癌组织中RUNX3、cyclinD1和p27^kip1基因的表达对于评价患者的预后有一定价值。     

应用组织芯片技术研究p27^kip1和Skp2在星形胶质细胞瘤及增生中的表达和意义术

目的：探讨p27^kip1和Skp2在星形胶质细胞增生及星形肢质细胞瘤中的表达及其与肿瘤发生发展的关系。方法：利用组织芯片技术及PV6000通用型二步法免疫组化方法检测p27^kip1和Skp2在正常脑组织，星形胶质细胞增生，低和高级别星形胶质细胞瘤中的表达、结果：正常脑组织中p27^kip1阳性表达率91．7％。Skp2表达阴性：增生组中p27^kip1和Skp2阳性表达率分别为86．4％、28．6％．与正常组比较，p27^kip1无统计学意义；Skp2有统计学意义；低级别肿瘤组中二者阳性表达率分别为64．3％、46．7％．与增生组比较，p27^kip1有统计学意义；Skp2无统计学意义：高级别肿瘤组中二者阳性表达率分别为42．6％、69．6％．与低级别肿瘤组比较，差异均有统计学意义；p27^kip1和Skp2与组织学分级密切相关。结论：p27^kip1有可能成为鉴别星形胶质细胞增生与低级别星形胶质细胞瘤的客观指标，且p27^kip1和Skp2在星形腔质细胞瘤的发生发展过程中有重要作用。     

p27kip1在膀胱移行细胞癌中的表达及其与肿瘤细胞增殖的关系

目的：探讨p27^kip1在膀胱移行细胞癌（BTCC）中的表达及其与肿瘤细胞增殖之间的关系。方法：应用鼠抗人p27^kip1和Ki-67单克隆抗体对65例BTCC和12例正常黏膜进行免疫组化S－P法染色。结果：p27^kip1在BTCC中表达随着临床分期和病理分级的增高而降低（P＜0．05），有淋巴结转移组明显低于无淋巴结转移组（P＜0．05）。p27^kip1阳性表达率与Ki-67呈负相关。结论：p27^kip1对BTCC的分化、增殖、浸润和转移可能起抑制作用。检测BTCC组织中p27^kip1和Ki-67蛋白表达对评价BTCC的生物学行为和判断预后具有重要意义。     

MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer

To define novel pathways that regulate susceptibility to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in non-small cell lung cancer (NSCLC), we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant NSCLC cells, levels of different miRs are increased, and in particular, miR-221 and -222. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. Indeed, transfection with anti-miR-221 and -222 rendered CALU-1-resistant cells sensitive to TRAIL. Conversely, H460-sensitive cells treated with -221 and -222 pre-miRs become resistant to TRAIL. miR-221 and -222 target the 3'-UTR of Kit and p27(kip1) mRNAs, but interfere with TRAIL signaling mainly through p27(kip1). In conclusion, we show that high expression levels of miR-221 and -222 are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets or diagnostic tool for TRAIL resistance in NSCLC.     

MicroRNA-221 and microRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN

Background  

MicroRNAs (miRNAs) can function as either oncogenes or tumor suppressor genes via regulation of cell proliferation and/or apoptosis. MiR-221 and miR-222 were discovered to induce cell growth and cell cycle progression via direct targeting of p27 and p57 in various human malignancies. However, the roles of miR-221 and miR-222 have not been reported in human gastric cancer. In this study, we examined the impact of miR-221 and miR-222 on human gastric cancer cells, and identified target genes for miR-221 and miR-222 that might mediate their biology.     

X线辐射剂量对结直肠癌细胞中microRNA-221和p57kip2表达的影响

目的 探讨不同剂量X线辐射对人结直肠癌Caco2细胞系中microRNA-221（miR-221）和p57^kip2表达的影响。方法 常规培养Caco2细胞系并分为5组,分别给予不同剂量X线（0、2、4、6和8 Gy）照射,24 h后提取细胞总RNA和蛋白质,应用real-time Q-PCR检测细胞中miR-221和p57^kip2 mRNA的表达水平,Western blot检测p57kip2蛋白的表达变化。结果 Caco2细胞系在不同照射剂量下,miR-221表达水平随着照射剂量的增加而增加,而p57kip2蛋白表达水平则随着照射剂量的增加而逐步降低,且呈剂量依赖效应,两者差异均具有统计学意义（P<0.05）。结论 放射线辐射剂量可影响结直肠癌细胞中miR-221/ p57^kip2调控通路,抑制miR-221表达可能会提高结直肠癌细胞的放射敏感度。     

miR-221和miR-222在肿瘤中的研究进展

 由于微小RNA(miRNA)在肿瘤中发挥重要调控作用及其自身特点,使其可能成为基因靶向治疗的有效工具。因此,人们致力于寻找调节关键癌基因或抑癌基因的miRNA。研究发现miR-221及miR-222在多种人类肿瘤中不仅表达异常,而且在不同类型的肿瘤中也发挥着不同的癌基因或抑癌基因样作用,通过调控miR-221和miR-222的表达可能成为肿瘤治疗的有效方法。     

MicroRNAs 221 and 222 bypass quiescence and compromise cell survival

MicroRNAs (miRNA) have tumor suppressive and oncogenic potential in human cancer, but whether and how miRNAs control cell cycle progression is not understood. To address this question, we carried out a comprehensive analysis of miRNA expression during serum stimulation of quiescent human cells. Time course analyses revealed that four miRNAs are up-regulated and >100 miRNAs are down-regulated, as cells progress beyond the G(1)-S phase transition. We analyzed the function of two up-regulated miRNAs (miR-221 and miR-222) that are both predicted to target the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57. Our results show that miR-221 and miR-222 both directly target the 3' untranslated regions of p27 and p57 mRNAs to reduce reporter gene expression, as well as diminish p27 and p57 protein levels. Functional studies show that miR-221 and miR-222 prevent quiescence when elevated during growth factor deprivation and induce precocious S-phase entry, thereby triggering cell death. Thus, the physiologic up-regulation of miR-221 and miR-222 is tightly linked to a cell cycle checkpoint that ensures cell survival by coordinating competency for initiation of S phase with growth factor signaling pathways that stimulate cell proliferation.     

Adenovirus-mediated shRNAs for co-repression of miR-221 and miR-222 expression and function in glioblastoma cells

Aberrantly expressed miRNAs are linked to the regulation of oncogenes and/or tumor suppression genes within the cell signal transduction pathway network, thereby contributing to carcinogenesis. miRNA function can be antagonized, thus representing a novel anti-tumor approach for integrated cancer therapy. In this study, we designed adenovirally-expressed shRNAs that functionally co-repressed the expression of miR-221 and miR-222, which are related to glioblastoma, to overcome the low efficiency of gene therapy. In addition, we generated novel shRNAs whose 3' ends were mutated in the region complementary to the target miRNA's 5' seed region to reduce the stability of binding with the miRNA. Various inhibition levels of miRNA were achieved: classic shRNAs yielded the greatest reduction in miRNA levels, followed by mutated shRNAs and the blank control, as determined by qRT-PCR. These results were confirmed by the protein expression of p27kip1, the validated target of miR-221/222, the effect on cell cycle arrest in G1 phase, and the impact on cell apoptosis. These results suggested that we could produce shRNAs encoded by adenovirus that co-repressed multiple tumor-related miRNAs simultaneously, and that the level of repression and the effect on the function of a specific miRNA could be achieved in a semi-quantitative manner.     

MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.     

microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma

microRNAs are noncoding RNAs inhibiting expression of numerous target genes, and a few have been shown to act as oncogenes or tumor suppressors. We show that microRNA-7 (miR-7) is a potential tumor suppressor in glioblastoma targeting critical cancer pathways. miR-7 potently suppressed epidermal growth factor receptor expression, and furthermore it independently inhibited the Akt pathway via targeting upstream regulators. miR-7 expression was down-regulated in glioblastoma versus surrounding brain, with a mechanism involving impaired processing. Importantly, transfection with miR-7 decreased viability and invasiveness of primary glioblastoma lines. This study establishes miR-7 as a regulator of major cancer pathways and suggests that it has therapeutic potential for glioblastoma.     

MicroRNA-34 a对多种肿瘤细胞迁移的影响及其作用机制探讨

MicroRNA-34a是p53信号通路的核心成分,在多种肿瘤中表达下调,被认为是一种抑癌的microRNA,并且与肿瘤干细胞有着密切的联系。MicroRNA-34a可直接抑制多种信息调控因子、细胞周期蛋白的表达,进而抑制细胞增殖、抑制细胞迁移和侵袭、促进细胞衰老和凋亡,从而达到抑癌效果。细胞迁移是肿瘤细胞侵袭和转移的先决条件,是肿瘤发生发展的重要过程。MicroRNA-34a的低表达常见于乳腺癌、前列腺癌、膀胱癌、胃癌、结肠癌、骨癌等,这种低表达往往与细胞迁移能力增强有关,最终加剧肿瘤转移扩散。本文总结了近期关于microRNA-34a对不同种类肿瘤细胞迁移的影响及其作用机制,并进行了分析与归纳。