端粒酶RNA和端粒酶活性在人生殖道癌细胞株中的表达

本文采用反转录套式PCR(nested RT-PCR)和PCR端粒重复扩增(TRAP)方法分别检测了5种人生殖道癌细胞株端粒酶RNA(hTR)基因及端粒酶活性的表达。结果表明,宫颈癌HeLa和SiHa细胞株,绒毛膜癌JAR和BeWo细胞株及卵巢癌OCCI细胞株hTR及端粒酶活性均呈强阳性。但正常卵巢、宫颈和胎盘组织hTR和端粒酶呈阴性或弱阳性。提示反转录套式PCR和端粒重复扩增法可以简便而有效地检测端粒酶RNA及端粒酶活性。端粒酶的激活与肿瘤细胞增殖密切相关,并可能成为一项具有临床价值的肿瘤标志物。     

miRNA-145-5p调控FMNL2基因对口腔鳞癌干细胞增殖、迁移的影响

摘要 目的：分析miRNA-145-5p调控形成素2（formin-like 2,FMNL2）基因对口腔鳞癌干细胞增殖、迁移的影响。方法：按照脂质体2000说明书对细胞进行转染miRNA-145-5p inhibitor及miRNA-145-5p mimics,按照实验设计,将其分为空白组、沉默组（miRNA-145-5p inhibitor）及过表达组（miRNA-145-5p mimics）。荧光定量PCR法检测miRNA-145-5p、FMNL2表达量,MTT检测细胞增殖能力,流式细胞仪检测细胞凋亡能力,细胞划痕实验检测细胞迁移能力,采用Western blot法检测各组细胞中Wnt/β-catenin信号通路蛋白表达量。结果：过表达组miRNA-145-5p、Bax蛋白表达量,凋亡率,细胞G0/G1比例高于沉默组,具有统计学差异（P＜0.05）;过表达组口腔鳞癌干细胞增殖率,FMNL2表达量、MMP-13、β-catenin、Bcl-2、APC蛋白表达量低于沉默组,具有统计学差异（P＜0.05）;过表达组口腔鳞癌干细胞迁移能力弱于沉默组,具有统计学差异（P＜0.05）。结论：miRNA-145-5p通过靶向调控FMNL2,作用于Wnt/β-catenin信号通路调控口腔鳞癌干细胞,进而抑制细胞增殖、迁移。     

上皮性卵巢癌中TFAP2A对hTERT表达的调节及其机制研究

摘要 目的：探讨在上皮性卵巢癌中TFA2A对hTERT表达的调节和作用机制。方法：采用免疫组织化学方法检测TFAP2A和hTERT蛋白在卵巢正常、交界及上皮性卵巢癌组织中的表达,采用Western Blot和qRT-PCR技术检测hTERT在敲减TFAP2A基因的SKOV3、CAOV3细胞中的表达水平、检测hTERT在过表达TFAP2A基因的HO8910细胞中的表达水平。在干扰TFAP2A的CAOV3细胞中或过表达TFAP2A的HO8910细胞中分别加入PI3K/AKT信号通路激动剂740-YP或抑制剂LY294002,检测相关蛋白表达变化,探讨TFAP2A、hTERT与PI3K/AKT信号通路的关系。结果：TFAP2A在71.88%的上皮性卵巢癌组织中呈高表达,hTERT在78.12%的上皮性卵巢癌组织中呈高表达; 将hTERT 和TFAP2A的免疫组化评分行Pearson相关性分析,两者间相关系数r=0.78,P＜0.001。Western Blot和qRT-PCR的结果均显示,在SKOV3和CAOV3卵巢癌细胞中,敲减TFAP2A后,hTERT的表达均明显下降,而在HO8910卵巢癌细胞中,增强TFAP2A基因表达后,hTERT的表达均明显上升。在CAOV3和HO8910处理细胞中,分别使用PI3K/AKT信号通路激动剂740-YP 或阻滞剂LY294002处理后,Western Blot 检验hTERT和PI3K/AKT通路蛋白的表达,发现激动剂740-YP 或阻滞剂LY294002可以逆转敲减或过表达TFAP2A引发的PI3K/AKT通路蛋白表达下调或上调,但不能逆转hTERT蛋白表达下调或上调。结论：在卵巢肿瘤组织中,TFAP2A和hTERT在上皮性卵巢癌组织中均呈高表达,且hTERT的表达和TFAP2A成正相关,在上皮性卵巢癌细胞中TFAP2A可调节hTERT的表达,且TFAP2A对hTERT的表达的调节不经由PI3K/AKT通路。     

端粒植入胃癌7901细胞对细胞生长、端粒和端粒酶的影响

研究外源端粒片段植入胃癌7901细胞后对细胞生长、端粒长度和端粒酶活性的影响.采用lipofectTM2000介导的转染方式,将含有端粒片段质粒pSXneo-1.6-T2AG3转染胃癌细胞SGC7901,PCR在基因水平上鉴定外源性端粒片段的植入后,采用TRAP法检测转染细胞端粒酶活性变化,TRF法检测转染细胞端粒长度变化,MTT法检测细胞生长曲线,RT-PCR测定转染细胞hTERT表达变化.染色体核型分析细胞染色体变化.结果显示端粒片段成功导入SGC7901细胞后获得稳定的细胞株,端粒片段植入后细胞生长变慢,端粒长度延长不明显,端粒酶活性明显降低,hTERT mRNA表达水平下降,核型分析显示转染前后细胞染色体数目无明显变化.实验成功将携带了1600 bp端粒TTAGGG重复序列的真核表达载体pSX-T2AG3-neo稳定转染至人胃癌7901细胞中,端粒植入降低细胞端粒酶的活性和下调端粒酶活性亚单位hTERT的表达,但对端粒长度无明显影响.     

端粒酶抑制剂

端粒酶被激活被认为是发生恶性肿瘤的主要因素,其激活及表达程度与肿瘤的发生和转移密切相关,因此端粒酶是肿瘤靶向治疗的理想靶点。研究和开发端粒酶抑制剂将为肿瘤治疗揭开新的篇章。端粒酶全酶复合物有很多可以做抑制剂的靶点,包括hTR(端粒酶RNA成分)、hTERT(端粒酶逆转录催化亚单位)、引物锚定位点、全酶的组装和那些招募端粒酶到端粒的因子。     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。     

研究报告: EIF2B5表达下调的人星形胶质细胞与人神经元在内质网应激后细胞存活及miRNA表达的差异

目的 探讨白质消融性白质脑病中胶质细胞选择性受累而神经元受累轻微的原因。方法 将EIF2B5-RNAi表达载体转染至人星形胶质细胞和人神经元,检测基础状态下及内质网应激（endoplasmic reticulum stress,ERS）后细胞凋亡和活力,检测参与ERS调控的已知和未知miRNA,筛选EIF2B5-RNAi人星形胶质细胞在ERS后miRNA变化。结果 与EIF2B5-RNAi人神经元相比,星形胶质细胞自发凋亡及细胞活力下降。较之神经元,更多miRNA参与星形胶质细胞ERS刺激后的调控,EIF2B5-RNAi组参与调控的miRNA数目显著减少。聚类分析发现,5条已知miRNA是通路连接的关键组分。结论 人星形胶质细胞在ERS后可能更加依赖众多促细胞增殖分化的miRNA修复,而EIF2B5-RNAi人星形胶质细胞存在自发凋亡,ERS后严重减少的miRNA可能导致细胞无法存活。     

Reconstitution of Telomerase Activity Extends the Proliferative Life-span and Maintains the Neurogenetic Potential of Mesenchymal Stem Cells Derived from Human Fetal Muscle

目的 :通过重建端粒酶活性延长胎儿肌肉源间充质干细胞寿命 ,并对其成神经潜能进行研究 ,为组织工程神经修复提供种子细胞。方法 :将人端粒酶催化亚基 (hTERT)基因通过脂质体转染法导入胎儿肌肉源间充质干细胞 ,RT PCR检测hTERTmRNA的表达 ,TRAP PCR检测细胞端粒酶活性。用bFGF诱导已重建端粒酶活性的肌肉源间充质干细胞向神经细胞分化 ,免疫荧光及免疫印迹法检测分化情况。结果 :转染hTERT的胎儿肌肉源间充质干细胞能稳定表达端粒酶活性。转染后传 75代的细胞经bFGF诱导仍维持着自我更新及向神经细胞分化的潜能 ,且无恶性转化倾向。结论 :重建端粒酶活性可延长胎儿肌肉源间充质干细胞寿命并维持自我更新及成神经潜能 ,为建立组织工程标准细胞系提供了新的实验手段     

特异性人端粒酶催化亚单位基因小干扰RNA对HeLa细胞生长的抑制作用

通过设计并化学合成人端粒酶催化亚单位(hTERT)特异性siRNA,观察其对hTERT表达水平及肿瘤细胞生长的影响。将hTERT-siRNA以脂质体法转染入HeLa细胞,应用RT-PCR、实时定量TRAP、Western印迹、软琼脂克隆形成实验、荷瘤裸鼠肿瘤内注射等方法检测细胞内hTERTmRNA、蛋白质表达水平及对肿瘤细胞生长的影响。RT-PCR、实时定量TRAP和Western印迹的结果显示hTERT-siRNA明显降低了HeLa细胞内hTERT的mRNA及蛋白质表达水平并伴随有端粒酶活性的下降。克隆形成实验表明hTERT-siRNA组的体外肿瘤形成能力受到抑制。荷瘤裸鼠肿瘤内注射hTERT-siRNA使肿瘤平均体积显著小于对照组。TUNEL凋亡检测表明hTERT-siRNA转染组的凋亡率明显高于对照组。研究表明hTERT特异性siRNA可以明显抑制HeLa细胞内hTERT的表达水平,对其生长有明显抑制作用,是一种有前途的肿瘤治疗新方法。     

A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Noninvasive diagnostics of bladder cancer based on analysis of telomerase activity and expression hTERT and hTR coding its subunits

In order to develop noninvasive diagnostics of bladder cancer (BC), telomerase activity has been examined by means the TRAP method (telomerase repeat amplification protocol) in tumor tissue and urine pellet samples taken from patients with bladder cancer. The levels of relative expression of genes encoding telomerase catalytic subunit (hTERT) and its RNA subunit (hTR) were evaluated by RT-PCR. Telomerase activity and expression of genes encoding its subunits were detected in both tumor tissues and in the urine cell pellet from each BC patient. Results of our study demonstrate possibility of noninvasive BC diagnostics using combination of these methods with sensitivity of 96% and specificity of 100% in the case of telomerase detection and with sensitivity of 80% and specificity of 100% in the case of hTERT detection in urine pellet samples.