Long Non-coding RNA PCED1B Antisense RNA 1 Promotes Cell Proliferation and Invasion in Hepatocellular Carcinoma by Regulating the MicroRNA-34a/CD44 Axis

Objective This study aimed to examine the role of long non-coding RNA PCED1B antisense RNA 1(PCED1B-AS1)in the development of hepatocellular carcinoma(HCC).Methods A total of 62 pairs of HCC tissues and adjacent non-tumor tissues were obtained from 62 HCC patients.The interactions of PCED1B-AS1 and microRNA-34a(miR-34a)were detected by dual luciferase activity assay and RNA pull-down assay.The RNA expression levels of PCED1B-AS1,miR-34a and CD44 were detected by RT-qPCR,and the protein expression level of CD44 was determined by Western blotting.The cell proliferation was detected by cell proliferation assay,and the cell invasion and migration by transwell invasion assay.The HCC tumor growth after PCED1B-AS1 was downregulated was determined by in vivo animal study.Results PCED1B-AS1 was highly expressed in HCC tissues,which was associated with poor survival of HCC patients.Furthermore,PCED1B-AS1 interacted with miR-34a in HCC cells,but they did not regulate the expression of each other.Additionally,PCED1B-AS1 increased the expression level of CD44,which was targeted by miR-34a.The cell proliferation and invasion assay revealed that miR-34a inhibited the proliferation and invasion of HCC in vitro,while CD44 exhibited the opposite effects.Furthermore,PCED1B-AS1 suppressed the role of miR-34a.Moreover,the knockdown of PCED1B-AS1 repressed the HCC tumor growth in nude mice in vivo.Conclusion PCED1B-AS1 may play an oncogenic role by regulating the miR-34a/CD44 axis in HCC.

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense

AIM:To determine whether an antisense RNA corresponding to the human Alu transposable element(Aluas RNA)can protect human lens epithelial cells(HLECs)from methylglyoxal-induced apoptosis.METHODS:Cell counting kit-8(CCK-8)and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays were used to assess HLEC viability.HLEC viability/death was detected using a Calcein-AM/PI double staining kit;the annexin V-FITC method was used to detect HLEC apoptosis.The cytosolic reactive oxygen species(ROS)levels in HLECs were determined using a reactive species assay kit.The levels of malondialdehyde(MDA)and the antioxidant activities of total-superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)were assessed in HLECs using their respective kits.RT-q PCR and Western blotting were used to measure m RNA and protein expression levels of the genes.RESULTS:Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxalinduced decrease in Bcl-2 m RNA and the methylglyoxalinduced increase in Bax m RNA.In addition,Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression.Aluas RNA inhibited the production of ROS induced by methylglyoxal,restored T-SOD and GSHPx activity,and moderated the increase in MDA content after treatment with methylglyoxal.Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes,including glutathione peroxidase,heme oxygenase 1,γ-glutamylcysteine synthetase and quinone oxidoreductase 1.Aluas RNA ameliorated methylglyoxal-induced increases of the m RNA and protein expression of Keap1 that is the negative regulator of Nrf2.CONCLUSION:Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Anti-aging Effects of Alu Antisense RNA on Human Fibroblast Senescence Through the MEK-ERK Pathway Mediated by KIF15

Objective:To investigate whether human short interspersed nuclear element antisense RNA(Alu antisense RNA;Alu asRNA)could delay human fibroblast senescence and explore the underlying mechanisms.Methods:We transfected Alu asRNA into senescent human fibroblasts and used cell counting kit-8(CCK-8),reactive oxygen species(ROS),and senescence-associated beta-galactosidase(SA-β-gal)staining methods to analyze the anti-aging effects of Alu asRNA on the fibroblasts.We also used an RNA-sequencing(RNA-seq)method to investigate the Alu asRNA-specific mechanisms of anti-aging.We examined the effects of KIF15 on the anti-aging role induced by Alu asRNA.We also investigated the mechanisms underlying a KIF15-induced proliferation of senescent human fibroblasts.Results:The CCK-8,ROS and SA-β-gal results showed that Alu asRNA could delay fibroblast aging.RNA-seq showed 183 differentially expressed genes(DEGs)in Alu asRNA transfected fibroblasts compared with fibroblasts transfected with the calcium phosphate transfection(CPT)reagent.The KEGG analysis showed that the cell cycle pathway was significantly enriched in the DEGs in fibroblasts transfected with Alu asRNA compared with fibroblasts transfected with the CPT reagent.Notably,Alu asRNA promoted the KIF15 expression and activated the MEK-ERK signaling pathway.Conclusion:Our results suggest that Alu asRNA could promote senescent fibroblast proliferation via activation of the KIF15-mediated MEK-ERK signaling pathway.

Targeting LncRNA LLNLR-299G3.1 with antisense oligonucleotide inhibits malignancy of esophageal squamous cell carcinoma cells in vitro and in vivo

Accumulating evidence has indicated that long non-coding RNAs(lncRNAs)play critical roles in the development and progression of cancers,including esophageal squamous cell carcinoma(ESCC).However,the mechanisms of lncRNAs in ESCC are still incompletely understood and therapeutic attempts for in vivo targeting cancer-associated lncRNA remain a challenge.By RNA-sequencing analysis,we identified that LLNLR-299G3.1 was a novel ESCC-associated lncRNA.LLNLR-299G3.1 was up-regulated in ESCC tissues and cells and promoted ESCC cell proliferation and invasion.Silencing of LLNLR-299G3.1 with ASO(antisense oligonucleotide)resulted in opposite effects.Mechanistically,LLNLR-299G3.1 bound to cancerassociated RNA binding proteins and regulated the expression of cancer-related genes,including OSM,TNFRSF4,HRH3,and SSTR3.ChIRP-seq(chromatin isolation by RNA purification and sequencing)revealed that these genes contained enriched chromatin binding sites for LLNLR-299G3.1.Rescue experiments confirmed that the effects of LLNLR-299G3.1 on ESCC cell proliferation were dependent on interaction with HRH3 and TNFRSF4.Therapeutically,intravenous delivery of placental chondroitin sulfate A binding peptide-coated nanoparticles containing antisense oligonucleotide(pICSA-BP-ANPs)strongly inhibited ESCC tumor growth and significantly improved animal survival in vivo.Overall,our results suggest that LLNLR-299G3.1 promotes ESCC malignancy through regulating gene-chromatin interactions and targeting ESCC by pICSA-BP-ANPs may be an effective strategy for the treatment of lncRNA-associated ESCC.

HOX transcript antisense intergenic RNA represses E-cadherin expression by binding to EZH2 in gastric cancer

AIM To clarify the mechanisms of HOX transcript antisense intergenic RNA(HOTAIR) in gastric cancer(GC) migration and invasion.METHODS Quantitative real-time polymerase chain reaction(qp CR) was used to detect the expression level of HOTAIR in GC tissues. The correlation of its expression with clinicopathological features was analyzed. Area under receiver operating characteristic curve(AUCROC) was constructed to evaluate the diagnostic value of HOTAIR. Wound-healing assay and Transwell assay were performed to detect the biological effects of HOTAIR in GC cells. qp CR,western blot and immunohistochemistry were used to evaluate the m RNA and protein expression of E-cadherin. RNAbinding protein immunoprecipitation was used for the analysis of EZH2 interactions with HOTAIR. Chromatin immunoprecipitation assay was performed to investigate direct interactions between EZH2 and E-cadherin.RESULTS The expression of HOTAIR was up-regulated in GC tumorous tissues compared with the para-tumorous tissues(p < 0.001). Its over-expression was correlated with tumor-node-metastasis(TNM) stage(p = 0.024),tumor invasion(p = 0.018),lymph node metastasis(p = 0.023),and poor prognosis(p < 0.001). Multivariate Cox regression analysis confirmed expression of HOTAIR as an independent predictor of overall survival(p = 0.033),together with TNM stage(p = 0.002) and lymph node metastasis(p = 0.002). The AUCROC was up to 0.709(95%CI: 0.623-0.785,p < 0.001). Knockdown of HOTAIR by si RNA in GC cells suppressed the migration and invasion of GC cells. Significantly negative correlation between HOTAIR and E-cadherin was found in GC tissues and cell lines,and HOTAIR contributed to the regulation of E-cadherin through binding to EZH2 with the E-cadherin promoter. CONCLUSION HOTAIR may play a pivotal role in tumor cell migration and invasion. It can be used as a potential diagnostic and prognostic biomarker for GC.

长链非编码RNA在急性肺损伤中的作用及分子调控机制研究进展

急性肺损伤(ALI)是临床上常见的危重症之一。长链非编码RNA(lncRNA)是一类长度超过200个核苷酸的非蛋白质编码RNA,在染色质形成、转录和转录后翻译等基因表达水平直接或间接参与ALI的发生发展。lncRNA核富集丰富转录物1、lncRNA HOX转录反义基因间RNA及lncRNA牛磺酸上调1等lncRNA可作为竞争性内源RNA,与miR-26a-5p、miR-17-5p及miR-34b-5p等微小RNA结合,调节Wnt1诱导的信号通路蛋白1、Rho关联含卷曲螺旋结合蛋白激酶1蛋白及自噬相关蛋白等下游相关蛋白的表达,参与ALI的发生发展。LncRNA可能通过调控DNA甲基化、组蛋白修饰,在表观遗传水平参与ALI的发生发展;L通过组装转录激活因子和抑制因子,调节白细胞介素6(IL-6)、IL-17、含半胱氨酸的天冬氨酸蛋白水解酶3/9等蛋白质的转录,在转录水平参与ALI的发生发展;与多种RNA结合蛋白相互作用,在转录后水平调控ALI的发生发展。

Construction and Characterization of an Antisense RNA Eukaryotic Expression Vector for Survivin

Objective: To inhibit specifically survivin expression and block its function in leukemia cells, an antisense RNA expression plasmid for survivin was constructed and transfected into a leukemia cell line.Methods: A cDNA fragment of survivin obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in the reverse direction. Antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into the cell line HL-60 by electroporation. The effect of survivin antisense RNA on survivin mRNA expression in transfected cells was examined by RT-PCR.Results: The correct construction of the recombinant plasmid has been shown by restriction enzyme digestion and DNA sequencing. As compared to controls, the level of survivin mRNA expression in transfected cells decreased significantly.Conclusion: An antisense RNA vector for survivin has been successfully constructed and may be useful as a specific inhibitor in leukemia cells. Thus, antisense therapy on the basis of survivin can be further explored in leukemia. Key words leukemia - survivin - antisense RNA This project was supported by a grant from National Key Basis Research Program of China (No. CB 513109) and the National Natural Sciences Foundation of China (No. 39970693).

Effects of long non-coding RNA Opa-interacting protein 5 antisense RNA 1 on colon cancer cell resistance to oxaliplatin and its regulation of micro RNA-137

BACKGROUND The incidence of colon cancer(CC)is currently high,and is mainly treated with chemotherapy.Oxaliplatin(L-OHP)is a commonly used drug in chemotherapy;however,long-term use can induce drug resistance and seriously affect the prognosis of patients.Therefore,this study investigated the mechanism of Opainteracting protein 5 antisense RNA 1(OIP5-AS1)on L-OHP resistance by determining the expression of OIP5-AS1 and micro RNA-137(miR-137)in CC cells and the effects on L-OHP resistance,with the goal of identifying new targets for the treatment of CC.AIM To study the effects of long non-coding RNA OIP5-AS1 on L-OHP resistance in CC cell lines and its regulation of miR-137.METHODS A total of 114 CC patients admitted to China-Japan Union Hospital of Jilin University were enrolled,and the expression of miR-137 and OIP5-AS1 in tumor tissues and corresponding normal tumor-adjacent tissues was determined.The influence of OIP5-AS1 and miR-137 on the biological behavior of CC cells was evaluated.Resistance to L-OHP was induced in CC cells,and their activity was determined and evaluated using cell counting kit-8.Flow cytometry was used to analyze the apoptosis rate,Western blot to determine the levels of apoptosisrelated proteins,and dual luciferase reporter assay combined with RNA-binding protein immunoprecipitation to analyze the relationship between OIP5-AS1 and miR-137.RESULTS OIP5-AS1 was up-regulated in CC tissues and cells,while miR-137 was downregulated in CC tissues and cells.OIP5-AS1 was inversely correlated with miR-137(P<0.001).Silencing OIP5-AS1 expression significantly hindered the proliferation,invasion and migration abilities of CC cells and markedly increased the apoptosis rate.Up-regulation of miR-137 expression also suppressed these abilities in CC cells and increased the apoptosis rate.Moreover,silencing OIP5-AS1 and up-regulating miR-137 expression significantly intensified growth inhibition of drug-resistant CC cells and improved the sensitivity of CC cells to LOHP.OIP5-AS1 targetedly inhibited miR-137 expression,and silencing OIP5-AS1 reversed the resistance of CC cells to L-OHP by promoting the expression of miR-137.CONCLUSION Highly expressed in CC,OIP5-AS1 can affect the biological behavior of CC cells,and can also regulate the resistance of CC cells to L-OHP by mediating miR-137 expression.

RNA结构对RNA沉默效率的影响

小RNA(small RNA,sRNA)介导的RNA沉默或RNA干扰(RNA silencing or RNA interference,RNAi)是真核生物基因表达调控的保守机制。内源或外源双链RNA(double-stranded RNA,dsRNA)被加工成sRNA,sRNA通过碱基互补配对识别靶标基因mRNA或DNA,通过降解mRNA、抑制翻译或者DNA甲基化在转录后或者转录水平调控基因表达。由于sRNA作用靶标特异性,RNAi技术被广泛地应用于基因功能研究、生物医药、作物分子设计育种以及开发新型农药等领域。自然界中sRNA能够在不同物种间传递并发挥作用,这一现象为RNAi应用技术的开发提供了理论基础。研究发现,dsRNA诱导RNAi的效率受多种因素影响,例如长度、剂量以及施用方式等。在细胞中,RNA结构的复杂性决定了其功能的多样性。本文概述了基于RNAi的作物病害防控技术的原理,包括宿主诱导的基因沉默(host-induced gene silencing,HIGS)技术、喷施诱导的基因沉默(spray-induced gene silencing,SIGS)技术和微生物诱导的基因沉默(microbe-induced gene silencing,MIGS)技术;总结了靶标RNA和sRNA结构影响RNAi效率的实验证据,以期加深对RNA结构影响RNAi效率地理解,为靶标筛选以及dsRNA设计提供经验,为开发高效RNAi技术提供参考;最后归纳了RNA结构检测和预测的代表性方法,为辅助设计高效诱导RNAi的dsRNA提供方法。

小细胞外囊泡及其携带的非编码RNA在非酒精性脂肪性肝病中的作用

小细胞外囊泡(small extracellular vesicles,sEVs)是由细胞分泌的一种细胞外囊泡,产生于多泡体,多泡体与质膜融合并释放到细胞外基质。由于小细胞外囊泡可以携带分子质量相对较小的核酸、蛋白质、脂质,能够执行细胞间物质传递、细胞间通讯等功能。因此,小细胞外囊泡及其携带的非编码RNA不仅参与细胞正常生理过程,也可以在多种疾病的发生发展过程中起重要作用。本文综述了小细胞外囊泡在非酒精性脂肪性肝病(NAFLD)中的作用,小细胞外囊泡及其携带的非编码RNA不仅有望成为NAFLD诊断的标志物,同时也具有治疗NAFLD的潜在作用,或能为治疗NAFLD提供新思路。

非编码RNA在颞下颌关节炎中的研究进展

颞下颌关节炎(temporomandibular joint osteoarthritis,TMJOA)是一种临床常见疾病,但发病机制尚不明确且缺乏有效的治疗手段,给患者带来很大困扰。因此,探究TMJOA的发病机制,寻找有效的治疗方法具有重要意义。近年来的研究表明,非编码RNA(noncoding RNA,ncRNA)参与调控TMJOA的发生、发展,在其诊断和治疗方面具有巨大潜力,因此有望成为新的诊断标志物和治疗靶标。本文将对ncRNA在TMJOA中的研究进展作一综述。

细胞外囊泡携带非编码RNA调控破骨细胞的活化

背景:目前已证明破骨细胞活化在骨骼系统相关疾病中发挥着重要的作用,胞外囊泡携带非编码RNA调控破骨细胞活化的机制仍未完全阐明。目的:阅读并总结国内外相关文献,按细胞外囊泡中不同的非编码RNA在不同疾病中调控破骨细胞活化进行综述,为后续研究提供一定的方向。方法:以“非编码RNA,miRNA,lncRNA,circRNA,snoRNA,破骨细胞,细胞外囊泡,外泌体,膜微粒,凋亡小体”为检索词检索中国知网、万方、维普等数据库,以“extracellular vesicles,exosome,microparticle,apoptotic bodies,non-coding RNA,miRNA,lncRNA,circRNA,snoRNA,osteoclast”为检索词检索Pub Med等数据库,最终纳入71篇文献进行综述。结果与结论:(1)破骨细胞的活化受多种因素影响,其中非编码RNA调控破骨细胞活化的具体机制尚未明确。(2)细胞外囊泡可以由成骨细胞、骨髓间充质干细胞、肿瘤细胞等多种细胞分泌,作为细胞间通讯的一种载体,能够携带非编码RNA对破骨细胞活化进行调控。(3)目前关于细胞外囊泡携带非编码RNA调控破骨细胞活化的疾病研究中,多数为骨质疏松,其次为肿瘤骨转移,而非编码RNA的种类又以mi RNA最多。(4)细胞外囊泡携带长链非编码RNA、环状RNA及核仁小RNA调控破骨细胞活化的相关研究相对较少,其调控机制主要为竞争性内源RNA机制。(5)综上,深入研究细胞外囊泡携带非编码RNA对破骨细胞活化的调控机制,有助于找到治疗骨骼系统相关疾病的关键靶点。

非编码小RNA在口腔黏膜下纤维性变中的研究进展

口腔黏膜下纤维性变(oral submucous fibrosis,OSF)是口腔黏膜最常见的癌前病变之一,其发病机制至今尚未完全阐明。非编码小RNA(small non-coding RNAs,SncRNAs)是一类不编码蛋白质的RNA分子,已被广泛报道参与多种人类疾病的调控。越来越多的研究表明多种SncRNAs在OSF发病过程中发挥重要作用。现有研究表明,微RNA(microRNAs,miRNAs)通过调控相关转录因子和基因表达或上皮间充质转化来调控成纤维细胞(fbroblasts,FB)活化而参与OSF疾病进展;长非编码RNA(long noncoding RNAs,lncRNAs)通过调控转化生长因子-β/母体抗瘫抑制因子(transforming growth factor-β/suppressor of mothers against decapentaplegic,TGF-β/Smad)信号通路或与miRNA相互作用参与OSF的发生发展;环状RNA(circular RNAs,circRNAs)通过与miRNA相互作用在OSF中发挥作用;tRNA衍生的小RNA(tRNA-derived small RNAs,tsRNAs)参与多种纤维化疾病的进展,但其在OSF中的具体作用机制仍需进一步探索。未来仍需重点关注SncRNAs介导OSF进展的作用靶点,探究其对OSF的作用功能及分子机制,以期为诊断和治疗OSF提供新思路。

Characteristics of Antisense Non-coding RNA in the INK4 Locus and Its Roles in Disease

Abstract With the development of genome-wide sequencing technology, 195 types of functional long non-coding RNAs （lncRNAs） have so far been found, and their cellular roles are gradually being revealed. Now lncRNAs have become a hotspot in the life science. These small molecules exist in almost all higher eukaryotes, and have very important regulatory roles in these organisms. This review briefly summarizes recent progress in researches on antisense non-coding RNA in the INK4 locus.

糖尿病神经病变过程中非编码RNA的作用及机制

背景:持续性高血糖症被确认为促进神经血管功能障碍,导致不可逆的内皮细胞功能障碍、神经细胞凋亡增加、氧化应激和炎症。这些协同和单独作用导致微血管和大血管病变,以及造成进行性神经病变。非编码RNAs可能为了解该疾病的病因、发病机制、治疗方法提供新的策略。目的:查阅国内外相关文献,综述非编码RNAs在糖尿病神经病变发生发展中的作用和机制,为非编码RNA在糖尿病神经病变预防和诊疗中提供新的思路和途径。方法:检索中国知网、PubMed数据库建库至2022年所发表的文献,中文关键词“非编码RNA;lncRNA;miRNA;糖尿病周围神经病变;表达谱”;英文关键词“Noncoding RNA;lncRNA;miRNA;Diabetes peripheral neuropathy;Expression profile”,将检索到的文献进行归纳、总结、分析,选取61篇文章进行综述。结果与结论:(1)非编码RNA在调节糖尿病周围神经病变的病理生理过程中起着核心作用。在研究最广泛的调节性非编码RNA物种中,有长链非编码RNAs(lncRNAs)、环状RNAs(circRNAs)和微小RNAs(miRNAs)。(2)通过非编码RNA的调节作用,引起相关细胞通路、炎症基因及下游相关细胞因子的激活或抑制,将抑制细胞凋亡,改善炎症水平,从而改变靶基因表达来参与糖尿病神经痛的进程。(3)尽管已发现多种微小RNA、长链非编码RNAs参与糖尿病神经病变疾病,但许多非编码RNAs的作用机制仍不清楚,相同的非编码RNAs可能在不同的模型中发挥不同的作用。因此,有必要进一步研究它们在疾病病因学和病理学中的作用模式,以阐明它们在糖尿病神经病变发病机制中的作用。然而,尚未建立评估非编码RNA活性的标准,需要进一步研究哪些特定非编码RNA在调节中起主导作用。(4)微小RNAs、长链非编码RNAs及其靶基因能够调节进行性神经病变,有望成为临床防治糖尿病周围神经病变的新靶点和预警糖尿病周围神经病变发生及其预后的新型生物标志物。

Inhibitory effect of IGF-Ⅱ antisense RNA on malignant phenotype of hepatocellular carcinoma

INIRODUCTIONAccording to the therapeutic effect and strategy ofantisense RNA for hepatoccllular carcinoma(HCC),we have specifically synthesized partialcDNA of human insulin-like growth factor Ⅱ(IGF-Ⅱ)and constructed IGF-Ⅱ cDNA antisenseeukaryotic expression vector.The constructedvector was introduced into hepatoma cell lineSMMC-7721 to block the intrinsic IGF-Ⅱexpression.The biological behavior changes ofhepatoma cells were observed.All these

Antisense telomerase RNA induced human gastric cancer cell apoptosis

INTRODUCTION Human tissue homeostasis is precisely regulated bycellular division,differentiation and death.Normalhuman somatic cells progressively lose telomererestriction fragment(TRF)length with eachsuccessive cell division,eventually leading tocellular quiescence,chromosomal end-degradationand apoptosis.On the contrary,stabilization oftelomere lengths by expressing telomerase,an RNA-dependent DNA polymerase,may be involved incellular immortality and carcinogenesis.

Effect of Antisense RNA Targeting Polo-like Kinase 1 on Cell Growth in A549 Lung Cancer Cells

In order to investigate the effect of Polo-like kinase-1 （Plk1） depletion on cell cycle progression and cell growth in lung cancer cells, a recombinant plasmid containing antisense RNA targeting Plk1 （pcDNA3-Plk1） was transfected into A549 cells by lipofectine. RT-PCR and Western-blot were used to detect the Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine （BrdU） labeling. Cell cycle distribution and apoptosis were examined by flow cytometry, and the inhibition rate （IR） by vinorebline （NVB） was determined by MTF assay. The results showed that after transfection of pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. In pcDNA3-Plk1 transfected groups, abnormal morphological changes of cells and growth inhibition were observed, and the BrdU labeling index was significantly lower than in the control groups （P〈0.05）. Cells in pcDNA3-Plk1 transfected groups were arresed in G2/M phase and apoptosis was detectable 72 h post transfection. IR induced by vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than in other groups. These data suggested that antisense RNA targeting Plk1 could suppress the Plk1 expression, and therefore, significantly inhibit cell proliferation and induce cell cycle arrest and apoptosis. Moreover, it sensitized lung cancer cells to chemotherapy.

Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma

AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2antisense RNA, and to explore its effects on liver cancer cell proliferation.METHODS: We studied the expression of COX-2 in 34cases of hepatocellular carcinoma (HCC) and SMMC7402and SMMC7721 by immunohistochemical technique.Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry.Cell proliferation was determined by colony-forming efficiency.RESULTS: We observed COX-2 expression in 82.4% of HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06× 1012 PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G1/G0 phase in SMMC7402 cell line.The difference of apoptotic index between the Ad-AShcox2 group and control group was statistically significant(tcontrol group= 32.62 and tAd-LacZ= 10.93, P＜0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7±0.94)% and(33.6±4.24)%, respectively.CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.

Antisense RNA of Survivin Gene Inhibits the Proliferation of Leukemia Cells and Sensitizes Leukemia Cell Line to Taxol-induced Apoptosis

The effects of survivin antisense RNA on proliferation of leukemia cell line HL-60 and taxol-induced chemotherapy was explored. A cDNA fragment of survivin obtained by RT-PCR was inserted into a plamid vector named pcDNA3 in the reverse direction. The vector encoding antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was delivered into HL-60 cells by electroporation. Growth curves were plotted based on cell counting. Trypan blue dye exclusion assay and MTT assay were carried out after the cells were incubated with taxol. DNA gel electrophoresis and nuclear staining were performed for cell apoptosis assay. The correct construction of the recombinant plasmid has been identified by restriction enzyme digestion and DNA sequencing. A stable down-regulation has been achieved in HL-60 SVVas cells after G418 selection. Compared to HL-60 cells, the proliferation of HL-60 SVVas cells was significantly inhibited （P〈0.05）. Cytotoxicity assays indicated that IC50 of HL-60 SVVas for taxol was relatively lower than controls （P〈0.01）. Apoptosis assays revealed that taxol-induced apoptosis was detected in HL-60 SVVas cells incubated with 50 ng/ml taxol for 12 h, while in HL-60 cells incubated with 100 ng/ml taxol for 72 h. It was suggested that Survivin antisense RNA could inhibit the proliferation of HL-60 cells and enhance taxol-induced apoptosis in HL-60 cells, which may lay an experimental foundation for further research on gene therapy in leukemia.