miR-34s增强结直肠癌细胞对放射敏感性的作用研究

目的:探讨miR-34s对结直肠癌细胞放射敏感性的影响以及在电离辐射致DNA损伤中的作用。方法:利用实时荧光定量PCR（real-time quantitative fluorescence PCR,qRT-PCR）检测miR-34a/b/c-5p在结直肠癌细胞中的表达水平,以及电离辐射后miR-34a/b/c-5p表达水平变化趋势。基于克隆形成法和单击多靶模型建立细胞存活曲线,分析miR-34a/b/c-5p对结直肠癌细胞放射敏感性的影响。过表达miR-34a/b/c-5p并进行照射,利用免疫荧光法检测照射后γH2AX焦点形成情况,进而分析miR-34a/b/c-5p对电离辐射致DNA损伤的作用。结果:miR-34a/b/c-5p在HCT116细胞中的表达水平明显高于HT29细胞（P＜0.05）。HCT116细胞经4 Gy照射后24 h内,miR-34a/b/c-5p表达水平呈双峰变化趋势。与miR-NC组相比,过表达miR-34a/b/c-5p可显著增加结直肠癌细胞的放射敏感性,miR-34a/b/c-5p组细胞平均致死剂量（D0）和准阈值剂量（Dq）均明显降低,且辐射增敏比（SER）明显增加。过表达miR-34a/b/c-5p可显著增加电离辐射诱导的DNA双链断裂（double strand breaks,DSBs）水平,照射后1 h和8 h γH2AX焦点数明显高于miR-NC组（P＜0.05）。结论:miR-34s为放射响应miRNA分子,过表达miR-34s可增加电离辐射诱导的DNA损伤水平并增强结直肠癌细胞的放射敏感性。     

X射线对肺癌细胞系p57kip2转录表达的影响

目的:研究X射线对肺癌细胞系NCI-H226和A549的印记基因p57kip2转录表达的影响。方法:对数生长期的肺癌细胞系NCI-H226和A549,分别予以2、4、8和12GyX射线照射,并于照射前和照射后6、12、24、36和48h分别提取RNA,采用RT-PCR技术检测各时相p57kip2mRNA的含量,分析不同剂量X射线照射后对其转录水平的影响。结果:两组肺癌细胞p57的转录表达在不同照射剂量和不同时间水平间差异有统计学意义,P<0·05,A549经过X射线照射后,p57kip2的mRNA含量明显升高,且与照射剂量呈线性依赖关系;NCI-H226虽然未见明显规律,但在X射线照射后的峰值mR-NA含量均明显升高。结论:X射线照射后能够上调肺癌细胞p57kip2的mRNA的表达,预示着p57可能参与了放射线所致细胞周期重新分布和细胞凋亡的过程,并且可能预测肺癌细胞的放射敏感性,但其表达与时间剂量的关系尚需要进一步研究。     

miR-145-5p靶向TAGLN2调控结直肠癌SW620细胞侵袭和迁移的机制探讨

目的:探究微小RNA-145-5p（miR-145-5p）在结直肠癌组织和细胞中的表达情况及其靶向调控肌动蛋白凝胶蛋白2（TAGLN2）对结直肠癌细胞侵袭和迁移能力的影响。方法:采用实时荧光定量PCR（qPCR）技术对48例结直肠癌患者癌组织、配对癌旁组织、结直肠癌细胞株（HCT8、SW620、HCT116、HT-29）及结直肠黏膜细胞FHC中的miR-145-5p和TAGLN2 mRNA表达进行定量分析。将SW620细胞设为空白对照组、miR-145-5p mimics组、mimics-NC组、pcDNA3.1-TAGLN2组、pcDNA3.1-Vector组和miR-145-5p mimics+pcDNA3.1-TAGLN2组,采用qPCR检测miR-145-5p和TAGLN2 mRNA表达,采用Transwell法检测细胞侵袭及迁移能力,采用免疫印迹法（Western blot）检测TAGLN2蛋白及EMT相关蛋白表达,采用双荧光素酶报告实验检测miR-145-5p和TAGLN2间的靶向关系。结果:miR-145-5p在结直肠癌组织中的表达水平显著低于癌旁组织（P<0.05）,并与结直肠癌患者的TNM分期和淋巴结转移相关（均P<0.05）;TAGLN2在结直肠癌组织中的表达水平显著高于癌旁组织,并与miR-145-5p表达呈负相关（P<0.05）;miR-145-5p和TAGLN2在结直肠癌HCT8、SW620、HCT116和HT-29细胞中的表达水平显著低于或高于FHC细胞（均P<0.05）。miR-145-5p过表达可降低SW620细胞的侵袭和迁移能力。miR-145-5p靶向调控TAGLN2表达,单独转染TAGLN2阳性质粒可增加SW620细胞的侵袭和迁移能力,与miR-145-5p mimics同时转染后,TAGLN2蛋白、波形蛋白（Vimentin）和神经钙黏素（N-cadherin）表达降低,上皮钙黏素（E-cadherin）表达升高,TAGLN2对SW620细胞侵袭和迁移能力的增强作用被显著抑制。结论:miR-145-5p在结直肠癌中呈低表达状态,其表达水平与结直肠癌患者的TNM分期和淋巴结转移密切相关,miR-145-5p靶向调控TAGLN2抑制结直肠癌细胞的侵袭和迁移能力。     

lncRNA LINC00909靶向miR-548-3p对结直肠癌细胞放射敏感性的研究

目的 探讨lncRNA LINC00909是否通过靶向miR-548-3p而影响结直肠癌细胞放射敏感性。方法 采用qRT-PCR检测结直肠癌组织、癌旁组织中LINC00909、miR-584-3p的表达量;体外培养结直肠癌细胞SW480、SW620,分别将si-NC、si-LINC00909、miR-NC、miR-584-3p mimics、si-LINC00909与anti-miR-NC、si-LINC00909与anti-miR-584-3p转染至SW480、SW620细胞,用4 Gy照射细胞;克隆形成实验检测细胞存活分数及放射增敏比;MTT检测细胞增殖;Transwell小室实验检测细胞迁移及侵袭;双荧光素酶报告实验验证LINC00909、miR-584-3p的靶向关系。裸鼠皮下移植瘤实验检测干扰LINC00909表达或抑制miR-584-3p表达对照射后移植瘤重量的影响。结果 结直肠癌组织中LINC00909的表达水平显著升高(P<0.05),miR-584-3p的表达水平显著降低(P<0.05);干扰LINC00909表达或miR-584-3p过表达后细胞存活分数明显降低(P<0.05),放射增敏比分别为2.017、1.762,并可抑制增殖、迁移及侵袭(P<0.05);双荧光素酶报告实验证实LINC00909可靶向结合miR-584-3p;干扰LINC00909表达后移植瘤重量显著降低(P<0.05)。共转染anti-miR-584-3p后移植瘤重量显著升高(P<0.05)。结论 干扰LINC00909表达可通过上调miR-548-3p的表达而减弱结直肠癌细胞增殖、迁移及侵袭能力从而增强细胞放射敏感性。     

MiR-195-5p对食管鳞癌细胞放射敏感性的影响及作用机制研究

目的：探讨miR-195-5p对食管鳞癌细胞放射敏感性的影响及作用机制。方法：采用4和8 Gy X射线照射KYSE510和KYSE150细胞,以未照射细胞为对照。采用CCK-8法检测细胞生长情况,qPCR检测细胞内miR-195-5p及survivin mRNA的表达水平,Western blot检测细胞内survivin蛋白的表达水平。KYSE510细胞分为转染miR-195-5p类似物组、转染miR-195-5p拮抗物组和相应的对照组,并采用X射线照射后,Incucyte实时动态活细胞监测和EdU掺入实验测定细胞增殖情况,克隆形成实验和流式细胞术分别测定细胞克隆形成率和凋亡率,双荧光素酶报告基因实验验证各组KYSE150细胞的荧光素酶活性。结果：经X射线照射后的KESE510和KYSE150细胞生长均受到明显抑制,与未照射组相比,照射组细胞miR-195-5p表达降低,survivin蛋白表达升高（P<0.05或0.01）。转染miR-195-5p类似物组的KESE510细胞在接受X射线照射后的存活率较阴性对照组降低,EdU阳性率和克隆存活率降低,凋亡率显著升高,细胞中survivin mRNA和蛋白表达较阴性对照组均降低（P<0.05或0.01）;转染miR-195-5p拮抗物组KESE510细胞的EdU阳性率和克隆存活率较阴性对照组升高,细胞中survivin mRNA和蛋白表达均升高（P<0.01）。在KYSE150细胞中过表达miR-195-5p能够抑制BIRC5 3' UTR报告基因的荧光素酶活性（P<0.05）。结论：MiR-195-5p可能通过靶向抑制survivin基因的表达增强食管鳞癌细胞的放射敏感性。     

miR-138-5p 在结直肠癌中的表达及其生物学功能

目的：本研究旨在检测 miR-138-5p 在结直肠癌中的表达水平,研究 miR-138-5p 对结直肠癌细胞恶性生长的影响及分子机制。方法：利用公共数据库分析 miR-138-5p 在结直肠癌中的表达水平。以结直肠癌细胞为研究对象,通过转染 miRNA mimics 或 inhibitors 升高或降低 miR-138-5p 水平。CCK-8 和 EdU 检测细胞活力和增殖活性, 流式细胞术检测细胞凋亡情况,实时荧光定量 PCR 结合生物信息学筛选 miR-138-5p 靶基因。结果：miR-138-5p 在结直肠癌中呈显著低表达（P ＜ 0.05）。升高 miR-138-5p 水平后,细胞活力减弱,细胞增殖活性降低,细胞凋亡率升高;降低 miR-138-5p 水平后,细胞活力增强,细胞增殖活性升高,对 5-FU 诱导的细胞凋亡产生抵抗（均 P ＜ 0.05）。研究发现 NEBL 和 EZH2 可能是 miR-138-5p 下游靶基因,且 miR-138-5p 表达水平与 NEBL 和 EZH2 表达水平均呈显著负相关（均 P ＜ 0.05）。结论：miR-138-5p 下调可能通过靶向调控 NEBL 和 EZH2 促进结直肠癌细胞恶性生长。     

miR-106b靶向调控PTEN促进结直肠癌细胞放射抵抗

目的 阐明miR-106b在结直肠癌细胞放射敏感性中的作用及机制。方法 构建稳定过表达和干扰miR-106b结直肠癌细胞系,通过免疫荧光和平板克隆实验探讨miR-106b对结直肠癌细胞放射敏感性的影响;Western blot检测Caspase-3和γ-H2AX的表达;生物信息学预测miR-106b下游调控的靶基因,双荧光素酶报告系统、荧光定量PCR及Western blot进一步验证。在稳定过表达miR-106b的结直肠癌细胞系中转染pCDNA3.0-PTEN,探讨细胞放射敏感性变化,明确miR-106b是否通过靶向调控PTEN增加结直肠癌细胞放射抵抗。结果 在结直肠癌细胞系过表达miR-106b,经过同等条件的照射处理后,与对照组相比,过表达miR-106b组细胞存活分数升高,放射抵抗增强(P<0.05),Caspase-3及γ-H2AX表达降低。在稳定过表达miR-106b的细胞中上调PTEN后能逆转miR-106b引起的放射抵抗。结论 miR-106b通过靶向抑制PTEN从而诱导结直肠癌细胞放射抵抗,预示着miR-106b-PTEN位点可能为临床提高结直肠癌放疗效果寻找相关靶点提供理论依据。     

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。     

lncRNASNHG10靶向调控miR-532-3p促进结直肠癌SW620细胞的侵袭和迁移

目的： 探讨lncRNA SNHG10在结直肠癌组织和细胞中的表达情况及其对结直肠癌细胞侵袭和迁移的影响与可能的机制。 方法： 收集2018年1月至2019年12月在河南省人民医院行根治性结直肠癌切除术的78例患者的癌组织及对应癌旁组织标本,采用qPCR法检测结直肠癌组织、结直肠癌细胞（SW480、SW620、HT-29和LoVo）及人正常结直肠黏膜细胞FHC中lncRNA SNHG10和miR-532-3p的表达水平,并分析其与结直肠癌患者临床病理特征的关系及在组织中表达的相关性。采用双荧光素酶报告基因实验验证lncRNA SNHG10和miR-532-3p间的靶向关系。向SW620细胞中转染si-SNHG10或miR-532-3p mimic或共转染si-SNHG10+miR-532-3p inhibitor,采用Transwell实验检测其侵袭和迁移能力的改变,采用WB法检测E-cadherin,N-cadherin和vimentin蛋白表达水平变化。 结果： SNHG10 在结直肠癌组织和细胞中呈高表达（P<0.05 或 P<0.01）,其表达水平与TNM分期和远处转移有关（均P<0.05）;miR-532-3p在结直肠癌组织和细胞中呈低表达,其表达水平与TNM分期、淋巴结转移和远处转移有关（P<0.05或P<0.01）,SNHG10和miR-532-3p在结直肠癌组织中的表达呈负相关（r=-0.225, P=0.048）。双荧光素酶报告基因实验证实SNHG10靶向调节miR-532-3p的表达。下调 SNHG10 或上调 miR-532-3p 的表达后,SW620 细胞的侵袭和迁移能力显著降低（P<0.01）,E-cadherin蛋白表达水平升高（P<0.05）、N-cadherin和vimentin蛋白表达水平降低（均P<0.05）。抑制miR-532-3p表达后,敲低lncRNA SNHG10表达对结直肠癌细胞侵袭和迁移的抑制作用被逆转（均P<0.05）。 结论： lncRNA SNHG10在结直肠癌中高表达并与TNM分期和远处转移相关,lncRNASNHG10靶向调控miR-532-3p表达并通过EMT途径影响结直肠癌细胞的侵袭和转移。     

微小RNA-34家族抑制结直肠癌细胞同源重组修复的机制

目的 探讨微小RNA-34家族(miR-34s)对结直肠癌细胞同源重组修复的作用及调控机制。方法 分别在2种结直肠癌细胞系(HCT116和HT29细胞)和2种非结直肠癌细胞系(HeLa和U2OS细胞)中过表达miR-34c-5p,应用蛋白质印迹法检测polo样激酶1(PLK1)蛋白表达水平。过表达miR-34a-5p、miR-34b-5p和miR-34c-5p以及回转PLK1,应用蛋白质印迹法检测PLK1和第139位丝氨酸磷酸化的组蛋白H2AX(γH2AX)蛋白表达水平,分析miR-34s与PLK1以及DNA损伤的关系。使用DR-GFP/Ⅰ-SceI报告质粒检测miR-34a-5p、miR-34b-5p和miR-34c-5p对同源重组修复效率的影响。qRT-PCR法检测miR-34a-5p、miR-34b-5p和miR-34c-5p对PLK1 mRNA表达水平的影响。生物信息学预测和双荧光素酶报告基因实验验证miR-34a-5p、miR-34b-5p和miR-34c-5p与PLK1 mRNA的结合位点。分别在p53^wt和p53^-/-HCT11...     

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。     

Down-regulation of miR-221 inhibits proliferation of pancreatic cancer cells through up-regulation of PTEN,p27kip1, p57kip2, and PUMA

Pancreatic cancer is the fourth leading cause of cancer related death in the US and exhibits aggressive features with short survival rate and high mortality. Therefore, it is important to understand the molecular mechanism(s) involved in the aggressive growth of pancreatic cancers, and further design novel targeted therapies for its treatment with better treatment outcome. In the present study, we found that the expression of miR-221 was significantly up-regulated in pancreatic cancer cell lines and tumor tissues compared to normal pancreatic duct epithelial cells and normal pancreas tissues. Moreover, we found that the pancreatic cancer patients with high miR-221 expression had a relatively shorter survival compared to those with lower expression, suggesting that miR-221 could be an oncogenic miRNA and a prognostic factor for poor survival of patients. Interestingly, transfection of miR-221 inhibitor suppressed the proliferative capacity of pancreatic cancer cells with concomitant up-regulation of PTEN, p27^kip1, p57^kip2, and PUMA, which are the tumor suppressors and the predicted targets of miR-221. Most importantly, we found that the treatment of pancreatic cancer cells with isoflavone mixture (G2535), formulated 3,3’-diindolylmethane (BR-DIM), or synthetic curcumin analogue (CDF) could down-regulate the expression of miR-221 and consequently up-regulate the expression of PTEN, p27^kip1, p57^kip2, and PUMA, leading to the inhibition of cell proliferation and migration of MiaPaCa-2 and Panc-1 cells. These results provide experimental evidence in support of the oncogenic role of miR-221 and also demonstrate the role of isoflavone, BR-DIM, and CDF as potential non-toxic agents that are capable of down-regulation of miR-221. Therefore, these agents combined with conventional chemotherapeutics could be useful in designing novel targeted therapeutic strategy for the treatment of pancreatic cancer for which there is no curative therapy.     

In Vitro and in Vivo Anti-tumor Activity of miR-221/222 Inhibitors in Multiple Myeloma

A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.     

放射线对鼻咽癌细胞周期阻滞、凋亡和抑癌基因p57Kip2表达的影响

Objective： To study cell cycle retardation, apoptosis and the expression of antioncogene p57^kip2 by radioactive rays in nasopharyngeal carcinoma cells. Methods： Cell cycle retardation, apoptosis and cell survival rate induced by radioactive rays were tested by the methods of flow cytometry and MTT method. The expression of antioncogene p57^kip2 was detected by immunohistochemistry and Western blot. Results： After irradiation, G1 phase had no obvious retardation, S phase showed transient delay. There was a positive correlation between irradiation dosage and retardation strength in G2/M phase （P 〈 0.01）. Peak value appeared at 24 h after 12 Gy irradiation, then decreased. There was a positive correlation between apoptosis incidence and irradiation dosage or after-irradiation time extention （P 〈 0.01）. There was a negative correlation between cell survival rate and irradiation dosage or apoptosis incidence （P〈 0.01）. The expression of p57^kip2 protein was up-regulated along with the prolongation of time and dosage after irradiation （P 〈 0.01）. Conclusion： G2/M phase arrest, apoptosis and the up-regulation of the expression of p57^kip2 protein all can reflect predict the radiosensitivity of nasopharyngeal carcinoma cells.     

胆囊癌组织中p57KIP2、cyclin D1和cyclin E的表达及临床意义

背景与目的：原发性胆囊癌（primary carcinoma of the gallbladder,PCG）是死亡率极高的恶性肿瘤,其恶变的机制目前尚未明确。前期研究发现,p57^KIP2在人类多种恶性肿瘤中异常表达。本研究拟进一步探讨p57^KIP2在PCG组织中的表达及临床意义。方法：运用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）、免疫组织化学EliVision法分别检测60例PCG、20例胆囊腺瘤（adenoma of the gallbladder,AG）和20例慢性胆囊炎（chronic cholecystitis,CC）组织中p57^KIP2、cyclin D1、cyclin E mRNA表达及蛋白水平。结果：p57^KIP2 mRNA及蛋白在PCG、AG和CC中的表达逐渐升高,两两比较差异有统计学意义（P<0.05）。Cyclin D1、cyclin E mRNA及蛋白在PCG、AG和CC中的表达逐渐降低,PCG与AG比较、PCG与CC比较,差异有统计学意义（P<0.05）。在PCG组织中,p57^KIP2蛋白的表达与临床分期、组织学分级及淋巴结转移有关（P<0.05）。Cyclin D1蛋白的表达与临床分期有关(P<0.05)。p57^KIP2与cyclin D1的表达呈负相关（P<0.05）,p57^KIP2与cyclin E的表达呈负相关（P<0.05）,cyclin D1与cyclin E的表达呈正相关（P<0.05）。结论：p57^KIP2表达的降低与cyclin D1、cyclin E表达的增加可能是PCG的发生机制之一;检测p57^KIP2、cyclin D1及cyclin E对PCG的预后判断有重要意义。     

miR-221 confers lapatinib resistance by negatively regulating p27kip1 in HER2-positive breast cancer

Development of acquired resistance to lapatinib, a dual epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor, severely limits the duration of clinical response in advanced HER2-driven breast cancer patients. Although the compensatory activation of the PI3K/Akt survival signal has been proposed to cause acquired lapatinib resistance, comprehensive molecular mechanisms remain required to develop more efficient strategies to circumvent this therapeutic difficulty. In this study, we found that suppression of HER2 by lapatinib still led to Akt inactivation and elevation of FOX3a protein levels, but failed to induce the expression of their downstream pro-apoptotic effector p27^kip1, a cyclin-dependent kinase inhibitor. Elevation of miR-221 was found to contribute to the development of acquired lapatinib resistance by targeting p27^kip1 expression. Furthermore, upregulation of miR-221 was mediated by the lapatinib-induced Src family tyrosine kinase and subsequent NF-κB activation. The reversal of miR-221 upregulation and p27^kip1 downregulation by a Src inhibitor, dasatinib, can overcome lapatinib resistance. Our study not only identified miRNA-221 as a pivotal factor conferring the acquired resistance of HER2-positive breast cancer cells to lapatinib through negatively regulating p27^kip1 expression, but also suggested Src inhibition as a potential strategy to overcome lapatinib resistance.     

Ginsenoside-Rh2 blocks the cell cycle of SK-HEP-1 cells at the G1/S boundary by selectively inducing the protein expression of p27

The mechanism of action by which ginsenoside-Rh2 (G-Rh2) suppresses the proliferation of SK-HEP-1 cells is reported. The results from flow cytometric analyses show that G-Rh2 arrested the cell cycle at the G1/S transition phase. The cyclin E-dependent kinase activity which had been immunoprecipitated with cyclin E-specific antibody was down-regulated in the cells in response to G-Rh2. The IC₅₀ value required to down-regulate the kinase activity by 50% was approximately 0.75 μM. Immunoblotting analyses show that G-Rh2 selectively induced the expression of p27^kip1 in a dose-dependent manner whereas it had no effect on the levels of cyclin E, cdk2, and p21^WAF1. In addition, our data show that G-Rh2 reduced the protein levels of cdc25A at doses higher than 10 μM. Collectively, these data suggest that ginsenoside-Rh2 arrests the cell cycle at the G1/S transition phase by selectively inducing protein expression of p27^Kip1 and, as a consequence, down-regulating cyclin E-dependent kinase activity.     

放射线对鼻咽癌细胞周期阻滞、凋亡和抑癌基因p57Kip2表达的影响

Objective:To study cell cycle retardation.apoptosis and the expression of antioncogene p57kip2 by radioactive rays in nasopharngeal carcinoma cells.Methods:Cell cycle retardation.apoptosis and cell suwival rate induced by radioactive rays were tested by the methods of flow cytometry and MTT method.The expression of antioncogene p57kip2 was detected bv immunohislochemistry and Western blot.Results:After irradiation,G1 phase had no obvious retardation,S phase showed transient delay.There was a positive correlation between irradiation dosage and retardation strength in G2/M phase(P＜0.01).Peak value appeared at 24 h after 12 Gy irradiation,then decreased.There was a positive correlation between apoptosis incidence and irradiation dosage or after-irradiation time extention(P＜0.01).There was a negative correlation between cell survival rate and irradiation dosage or apoptosis incidence(P＜0.01).The expression of p57 kjP2 protein was up-regulated along with the prolongation of time and dosage after irradiation(P＜0.01).Conclusion:G2/M phase arrest,apoptosis and the up-regulation of the expression of p57kiP2 protein all can reflect predic the radiosensitivity of nasopharyngeal carcinoma cells.     

MiR-221在肝癌中的表达及其作用机制研究

  目的   分析miR-221在肝癌患者癌组织和正常组织中以及血浆中的表达水平及其表达水平与临床病理特征、诊断和预后之间的相关性。并进一步分析探讨其在肝癌发生发展中的作用机制。   方法   利用基因表达数据库（Gene Expression Omnibus database,GEO）中的芯片数据集分析miR-221在肝患者癌组织及癌旁组织中的表达水平。利用实时荧光定量PCR法验证miR-221在74例临床肝癌患者肿瘤组织和对应癌旁组织中的表达水平,进一步检测miR-221在25例肝癌患者血浆和正常人血浆中的表达水平。进一步分析miR-221在肝癌诊断及预后方面的价值。进一步进行miR-221的靶基因进行功能富集分析,及其对肝癌细胞增殖、侵袭以及EMT进程的影响。   结果  GEO数据库分析结果显示miR-221在肝癌组织中的表达水平显著高于癌旁组织（P<0.05）,同样经实时荧光定量PCR验证结果证明miR-221在肝癌患者的肿瘤组织和血浆中显著高表达,且其表达水平与肝癌患者的TNM分期和肿瘤大小显著相关。MiR-221的表达水平降低与肝癌患者的总生存时间和无病生存时间延长显著相关。功能富集分析发现miR-221所参与的信号通路主要为p53信号通路、ErbB信号通路、RNA转运和mTOR信号通路等信号通路,且体外实验表明低表达miR-221显著抑制肝癌细胞的增殖和侵袭能力,同时肝癌HepG2和MHCC97H细胞的E-cadherin mRNA和蛋白表达水平显著上调（P<0.01）,而N-cadherin和vimentin mRNA和蛋白的表达水平明显降低。   结论  MiR-221在肝癌组织和血浆中显著高表达,且与肝癌患者的预后不良显著相关,主要在肝癌中通过EMT进程而发挥作用。