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Gene silencing efficiency of shRNA expression vectors targeting Cx43

Cuihong ZHENG, Yunxia WU, Guangying HUANG, Wei WANG

《医学前沿（英文）》 2009年 第3卷 第2期   页码 130-135 doi: 10.1007/s11684-009-0030-9

摘要： Our previous studies showed that there were close relationships between connexin 43 (Cx43) and acupoints and meridians. In order to further investigate the effect of Cx43 in acupuncture treatment, RNA interference technique was used to construct small hairpin RNA (shRNA) expression vectors targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines for further use . Aiming directly at the two targets of Cx43 mRNA sequence of the rat and mouse homology region, we synthesized two pairs of complementary oligonucleotide strands . Double strands were formed after annealing, and then inserted into Pgenesil-1 plasmid expression vector. After identification by enzyme cutting and sequencing, the recombinant plasmids named P-Cx43-shRNA (1), P-Cx43-shRNA (2) and P-con-shRNA were transfected into the NIH/3T3 cells. Immunofluorescence and Western blot assays were used to detect the protein level of Cx43 after being screened by G418.The results of enzyme cutting and sequencing showed that we successfully constructed two shRNA expression vectors targeting Cx43, and a control expression vector for rat and mouse. Also, the Cx43 protein level was decreased by 73.5% ( < 0.01) and 10.8%, accordingly. The Cx43 protein level was not influenced by the transfection of P-con-shRNA. The outcomes demonstrate that the plasmid P-Cx43-shRNA (1) can specifically silence better the expression of Cx43 in NIH/3T3 cells, which offers an experimental evidence for further investigation.

关键词： RNA interference     connexin 43     small hairpin RNA (shRNA)     acupuncture    

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Paternal environmental exposure-induced spermatozoal small noncoding RNA alteration meditates the intergenerational

《医学前沿（英文）》 2022年 第16卷 第2期   页码 176-184 doi: 10.1007/s11684-021-0885-y

摘要： Studies of human and mammalian have revealed that environmental exposure can affect paternal health conditions as well as those of the offspring. However, studies that explore the mechanisms that meditate this transmission are rare. Recently, small noncoding RNAs (sncRNAs) in sperm have seemed crucial to this transmission due to their alteration in sperm in response to environmental exposure, and the methodology of microinjection of isolated total RNA or sncRNAs or synthetically identified sncRNAs gradually lifted the veil of sncRNA regulation during intergenerational inheritance along the male line. Hence, by reviewing relevant literature, this study intends to answer the following research concepts: (1) paternal environmental factors that can be passed on to offspring and are attributed to spermatozoal sncRNAs, (2) potential role of paternal spermatozoal sncRNAs during the intergenerational inheritance process, and (3) the potential mechanism by which spermatozoal sncRNAs meditate intergenerational inheritance. In summary, increased attention highlights the hidden wonder of spermatozoal sncRNAs during intergenerational inheritance. Therefore, in the future, more studies should focus on the origin of RNA alteration, the target of RNA regulation, and how sncRNA regulation during embryonic development can be sustained even in adult offspring.

关键词： small noncoding RNAs     epigenetic inheritance     paternal intergenerational inherence     extracellular vesicles    

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Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

LIU Hongchun, CAO Zhongwei, JIN Jianjun, WANG Jiyao

《医学前沿（英文）》 2008年 第2卷 第2期   页码 166-170 doi: 10.1007/s11684-008-0030-1

摘要： This study aims to demonstrate that blocking the receptor-interacting protein2 (Rip2) expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality. Murine Rip2 small interfering RNA (siRNA) plasmids were constructed and transfected into macrophage and Rip2 expression was detected with reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cell proliferation was assayed with MTT. TNF-? concentration was assayed with ELISA and high-mobility group box 1 protein (HMGB1) level with semi-quantitative western blot after lipopolysaccharide (LPS) stimulation. LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated. Rip2 siRNA plasmid could block the mRNA and protein expression of Rip2 and promote cell proliferation. Blocking Rip2 could attenuate LPS-induced TNF-? and HMGB1 production. The HMGB1 expression in the liver decreased to (40.21 ± 11.03) pg/g, and serum TNF-? level decreased to (300.43 ± 59.26) ng/L ( < 0.05). The survival rate of mice from endotoxemia was also improved ( < 0.05). The results demonstrate that Rip2 siRNA plasmid can block the expression of Rip2, decrease the production of TNF-? and HMGB1 and protect mice from fatal endotoxemia.
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Ribozyme and the mechanisms that underlie RNA catalysis

Timothy J. Wilson,Yijin Liu,David M. J. Lilley

《化学科学与工程前沿（英文）》 2016年 第10卷 第2期   页码 178-185 doi: 10.1007/s11705-016-1558-2

摘要： Ribozymes are widespread, and catalyze some extremely important reactions in the cell. Mechanistically most fall into one of two classes, using either metal ions or general acid-base catalysis. The nucleolytic ribozymes fall into the latter class, mostly using nucleobases. A sub-set of these use a combination of guanine base plus adenine acid to catalyze the cleavage reaction. New ribozymes are still being discovered at regular intervals and we can speculate on the potential existence of ribozymes that catalyze chemistry beyond phosphoryl transfer reactions, perhaps using small-molecule coenzymes.

关键词： RNA catalysis     RNA structure     catalytic mechanism    

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5′-tiRNA-Gln inhibits hepatocellular carcinoma progression by repressing translation through the interaction with eukaryotic initiation factor 4A-I

《医学前沿（英文）》 2023年 第17卷 第3期   页码 476-492 doi: 10.1007/s11684-022-0966-6

摘要： tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases. However, their exact presence and function in hepatocellular carcinoma (HCC) remain unclear. Here, differentially expressed tsRNAs in HCC were profiled. A novel tsRNA, tRNAGln-TTG derived 5′-tiRNA-Gln, is significantly downregulated, and its expression level is correlated with progression in patients. In HCC cells, 5′-tiRNA-Gln overexpression impaired the proliferation, migration, and invasion in vitro and in vivo, while 5′-tiRNA-Gln knockdown yielded opposite results. 5′-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. The suppressed downregulated proteins include ARAF, MEK1/2 and STAT3, causing the impaired signaling pathway related to HCC progression. Furthermore, based on the construction of a mutant 5′-tiRNA-Gln, the sequence of forming intramolecular G-quadruplex structure is crucial for 5′-tiRNA-Gln to strongly bind EIF4A1 and repress translation. Clinically, 5′-tiRNA-Gln expression level is negatively correlated with ARAF, MEK1/2, and STAT3 in HCC tissues. Collectively, these findings reveal that 5′-tiRNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular G-quadruplex structure, and this process partially inhibits translation and HCC progression.

关键词： EIF4A1     G-quadruplex     hepatocellular carcinoma     tRNA-derived small RNA     translation initiation    

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Construction of multiple shRNA vectors targeting PEDV and TGEV and production of transgenic SCNT porcine

Jianwen CHEN, Kaiyuan PAN, Zhen CHEN, Biao DING, Dandan SONG, Wenbin BAO, Yunhai ZHANG

《农业科学与工程前沿（英文）》 2019年 第6卷 第1期   页码 66-72 doi: 10.15302/J-FASE-2018229

摘要：

Porcine viral diarrhea is an acute and highly contagious enteric disease of pigs that causes huge economic losses worldwide. Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are the main pathogens responsible for piglet viral diarrhea. However, currently there is no specific drug available for the effective treatment of viral diarrhea. Therefore, it is necessary to seek an effective method to diminish PEDV and TGEV infection rates. RNA interference has been applied successfully to inhibit the virus replication. It provides a potential strategy for breeding resistant pigs. In this study, four promoters and four short hairpin RNA (shRNA) vectors with LoxP sites at each end of the selectable marker genes were constructed to target PEDV and TGEV. These vectors were then transfected into porcine fetal fibroblasts, G418 resistant transfectants were confirmed by PCR and transgenic SCNT porcine blastocysts were obtained. These results have paved the way for future production of marker-free transgenic resistant to PEDV and TEGV pigs by SCNT.

关键词： piglet diarrhea     RNAi     PEDV     TGEV     transgenic resistance breeding    

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抗RNA病毒相关生物材料

姚康德,尹玉姬,张宝连,赵立国

《中国工程科学》 2003年 第5卷 第7期   页码 17-23

摘要：

在介绍RNA病毒结构、生殖、复制和转录的基础上，综述了抗病毒策略，其中包括抗SARS药物设计，RNA干扰，DNA疫苗释放系统，调控蛋白与糖胺聚糖衍生物或类似物相互作用，天然药物及肺泡组织工程等。这些实例涵盖了RNA病毒与蛋白质、DNA及多糖等生物材料的相互作用。生物材料作为基质或载体正在向细胞或/和基因活化的第三代生物材料发展，可望在抗病毒中发挥作用。

关键词： 抗RNA病毒     蛋白质     DNA     多糖     生物材料    

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基于RNA的生物防治——一种作物保护新模式 Review

Matthew Bramlett, Geert Plaetinck, Peter Maienfisch

《工程（英文）》 2020年 第6卷 第5期   页码 522-527 doi: 10.1016/j.eng.2019.09.008

摘要： 在过去的几年中，RNA干扰（RNAi）过程被认为是一种非常有前景的新方法，可作为化学和生物害虫防治剂、植物保护剂等叶面喷施、土壤或种子处理的补充。基于RNA的活性成分（AI）具有独特的作用方式，可以通过基因修饰（GM）和生物防治两种途径来实现。由于基于RNA的AI可利用自然过程来发挥控制作用，同时它们具有高度选择性，降低了非目标生物（NTO）的风险，因此基于RNA的AI有望提供未来作物保护剂所需要的选择性和可持续性。本文讨论了基于RNA的生物防治的替代方案在作物保护中的优势和局限性，以及RNA生物防治科罗拉多马铃薯甲虫（CPB）、玉米根虫（CRW）和大豆臭虫（SSB）的最新研究进展。在实现各种基于RNA的产品及其广泛使用和应用的道路上，仍然存在许多挑战。尽管如此，我们仍可预期到，基于RNA的AI将成为有价值的新工具，以补充当前的农作物保护解决方案。

关键词： 基于RNA的生物防治     RNA干扰（RNAi）     科罗拉多马铃薯甲虫（CPB）     玉米根虫（CRW）     大豆臭虫（SSB）    

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敲低特异性环状非编码RNA显著抑制骨肉瘤的进展 Article

王世东, 张红亮, 李博, 陈成龙, 任婷婷, 黄怡, 刘凯, 李敬敬, 郭卫

《工程（英文）》 2023年 第21卷 第2期   页码 187-193 doi: 10.1016/j.eng.2021.12.007

摘要： 异常表达的非编码环状RNA（circRNA）对骨肉瘤的发生和发展至关重要。本研究的目的是探索一种新的circRNA circ_000203 在骨肉瘤中的表达和作用，阐明其潜在机制。

关键词： 非编码RNA     骨肉瘤     circRNA     分子机制     敲低    

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Overexpressed long noncoding RNA CRNDE with distinct alternatively spliced isoforms in multiple cancers

Xuefei Ma, Wei Zhang, Rong Zhang, Jingming Li, Shufen Li, Yunlin Ma, Wen Jin, Kankan Wang

《医学前沿（英文）》 2019年 第13卷 第3期   页码 330-343 doi: 10.1007/s11684-017-0557-0

摘要： Alternative splicing is a tightly regulated process that contributes to cancer development. CRNDE is a long noncoding RNA with alternative splicing and is implicated in the pathogenesis of several cancers. However, whether deregulated expression of CRNDE is common and which isoforms are mainly involved in cancers remain unclear. In this study, we report that CRNDE is aberrantly expressed in the majority of solid and hematopoietic malignancies. The investigation of CRNDE expression in normal samples revealed that CRNDE was expressed in a tissue- and cell-specific manner. Further comparison of CRNDE expression in 2938 patient samples from 15 solid and hematopoietic tumors showed that CRNDE was significantly overexpressed in 11 malignancies, including 3 reported and 8 unreported, and also implicated that the overexpressed isoforms differed in various cancer types. Furthermore, anti-cancer drugs could efficiently repress CRNDE overexpression in cancer cell lines and primary samples, and even had different impacts on the expression of CRNDE isoforms. Finally, experimental profiles of 12 alternatively spliced isoforms demonstrated that the spliced variant CRNDE-g was the most highly expressed isoform in multiple cancer types. Collectively, our results emphasize the cancer-associated feature of CRNDE and its spliced isoforms, and may provide promising targets for cancer diagnosis and therapy.

关键词： long noncoding RNA     CRNDE     alternative splicing    

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Potential use of serum HBV RNA in antiviral therapy for chronic hepatitis B in the era of nucleos(t)ide

null

《医学前沿（英文）》 2017年 第11卷 第4期   页码 502-508 doi: 10.1007/s11684-017-0590-z

摘要：

Although the efficacy of nucleos(t)ide analogue (NA) has been confirmed for treatment of chronic hepatitis B, long-term therapy has been recommended due to the high frequency of off-therapy viral DNA rebound and disease relapse. In this review, the RNA virion-like particles of hepatitis B virus (HBV) are integrated into the life cycle of HBV replication, and the potential significance of serum HBV RNA is systematically described. The production of HBV RNA virion-like particles should not be blocked by NA; in this regard, serum HBV RNA is found to be a suitable surrogate marker for the activity of intrahepatic covalently closed circular DNA (cccDNA), particularly among patients receiving NA therapy. Therefore, the concept of virological response is redefined as persistent loss of serum HBV DNA and HBV RNA. In contrast to hepatitis B surface antigen (HBsAg) that can originate from either the cccDNA or the integrated HBV DNA fragment, serum HBV RNA, with pregenomic RNA origination, can only be transcribed from cccDNA. Therefore, the loss of serum HBV RNA would likely be a promising predicator for safe drug discontinuation. The clinical status of consistent loss of serum HBV RNA accompanied with low serum HBsAg levels might be implicated as a “para-functional cure,” a status nearly close to the functional cure of chronic hepatitis B, to distinguish the “functional cure” characterized as serum HBsAg loss with or without anti-HBs seroconversion.

关键词： chronic hepatitis B     serum HBV RNA     nucleos(t)ide analogs     virological response     para-functional cure    

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immune escape and microenvironment between RG-like and pri-OPC-like glioma revealed by single-cell RNA-seq

《医学前沿（英文）》 doi: 10.1007/s11684-023-1017-7

摘要： The association of neurogenesis and gliogenesis with glioma remains unclear. By conducting single-cell RNA-seq analyses on 26 gliomas, we reported their classification into primitive oligodendrocyte precursor cell (pri-OPC)-like and radial glia (RG)-like tumors and validated it in a public cohort and TCGA glioma. The RG-like tumors exhibited wild-type isocitrate dehydrogenase and tended to carry EGFR mutations, and the pri-OPC-like ones were prone to carrying TP53 mutations. Tumor subclones only in pri-OPC-like tumors showed substantially down-regulated MHC-I genes, suggesting their distinct immune evasion programs. Furthermore, the two subgroups appeared to extensively modulate glioma-infiltrating lymphocytes in distinct manners. Some specific genes not expressed in normal immune cells were found in glioma-infiltrating lymphocytes. For example, glial/glioma stem cell markers OLIG1/PTPRZ1 and B cell-specific receptors IGLC2/IGKC were expressed in pri-OPC-like and RG-like glioma-infiltrating lymphocytes, respectively. Their expression was positively correlated with those of immune checkpoint genes (e.g., LGALS3) and poor survivals as validated by the increased expression of LGALS3 upon IGKC overexpression in Jurkat cells. This finding indicated a potential inhibitory role in tumor-infiltrating lymphocytes and could provide a new way of cancer immune evasion.

关键词： single-cell RNA-seq     glioma     radial glia     primitive oligodendrocyte precursor cell     immune escape    

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Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

Zhengjuan LIU, Jie BIAN, Yuchuan WANG, Yongli ZHAO, Dong YAN, Xiaoxia WANG

《医学前沿（英文）》 2009年 第3卷 第1期   页码 57-60 doi: 10.1007/s11684-009-0003-z

摘要： Leptin resistance is a main mechanism of acquired childhood obesity, and the suppression of long form of leptin receptor (OBRb) gene expression in diet-induced obese rats indicates that the down-regulation of OBRb gene expression plays a pivotal role in the mechanism of leptin resistance. The aim of the present study was to construct the lentiviral RNA interference (RNAi) vector of rat OBRb gene and evaluate the effects of siRNA on silencing OBRb gene expression. The target sequence of siRNA-OBRb was designed, and the complementary DNA containing both sense and antisense oligonucleotides was synthesized. After phosphorylation and annealing, these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter, target sequence and Poly III terminator. Then, the products were confirmed by electrophoresis and sequencing analysis, and the effects of RNAi on reducing gene expression were further confirmed by real-time polymerase chain reaction in transfected rat glioma cells expressing OBRb. The target sequence of siRNA-OBRb was successfully cloned to pRNA-lentivector-VGFP, and the RNAi protocol specifically reduced the expression of OBRb mRNA by approximately 80% compared with controls in transfected rat glioma cells. The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation .

关键词： receptors     leptin     RNA interference     lentivirus vector    

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A ruptured recurrent small bowel gastrointestinal stromal tumour causing hemoperitoneum

null

《医学前沿（英文）》 2015年 第9卷 第1期   页码 108-111 doi: 10.1007/s11684-014-0344-0

摘要：

Hemoperitoneum is a rare and potentially life-threatening complication of GIST. We reported a 54-year-old man who developed disseminated intra-abdominal recurrence from a previously resected gastrointestinal stromal tumour (GIST) of the small bowel, and the patient presented with hemoperitoneum. Emergent debulking surgery was performed. A high dose imatinib was prescribed. Despite the presence of residual disease, the patient was well clinically 8 months after the operation. Even though, there is no evidence to support the routine use of debulking surgery in the management of GIST. In our patient, disease progression after second line targeted therapy and the absence of alternative treatment options for spontaneous rupture and hemoperitoneum prompted us to treat the patient aggressively. Resection of the ruptured GIST was carried out for control of bleeding and to prevent recurrent bleeding in this patient with good surgical risks. During the treatment decision-making, the patient’s general condition, the risk of surgery and the extent of dissemination were taken into consideration. In this patient who presented with spontaneous rupture of a small intestinal GIST, the novel use of targeted therapy and aggressive surgical treatment produced reasonably good survival outcome.

关键词： gastrointestinal stromal tumour     hemoperitoneum     small bowel GIST     small bowel neoplasm     imatinib    

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RNA m6A modification and its function in diseases

null

《医学前沿（英文）》 2018年 第12卷 第4期   页码 481-489 doi: 10.1007/s11684-018-0654-8

摘要：

N6-methyladenosine (m6A) is the most common post-transcriptional RNA modification throughout the transcriptome, affecting fundamental aspects of RNA metabolism. m6A modification could be installed by m6A “writers” composed of core catalytic components (METTL3/METTL14/WTAP) and newly defined regulators and removed by m6A “erasers” (FTO and ALKBH5). The function of m6A is executed by m6A “readers” that bind to m6A directly (YTH domain-containing proteins, eIF3 and IGF2BPs) or indirectly (HNRNPA2B1). In the past few years, advances in m6A modulators (“writers,” “erasers,” and “readers”) have remarkably renewed our understanding of the function and regulation of m6A in different cells under normal or disease conditions. However, the mechanism and the regulatory network of m6A are still largely unknown. Moreover, investigations of the m6A physiological roles in human diseases are limited. In this review, we summarize the recent advances in m6A research and highlight the functional relevance and importance of m6A modification in in vitro cell lines, in physiological contexts, and in cancers.

关键词： RNA modification     m6A     immunity     cancer     epigenetics    

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标题 作者 时间 类型 操作

Gene silencing efficiency of shRNA expression vectors targeting Cx43

Cuihong ZHENG, Yunxia WU, Guangying HUANG, Wei WANG

期刊论文

Paternal environmental exposure-induced spermatozoal small noncoding RNA alteration meditates the intergenerational

期刊论文

Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

LIU Hongchun, CAO Zhongwei, JIN Jianjun, WANG Jiyao

期刊论文

Ribozyme and the mechanisms that underlie RNA catalysis

Timothy J. Wilson,Yijin Liu,David M. J. Lilley

期刊论文

5′-tiRNA-Gln inhibits hepatocellular carcinoma progression by repressing translation through the interaction with eukaryotic initiation factor 4A-I

期刊论文

Construction of multiple shRNA vectors targeting PEDV and TGEV and production of transgenic SCNT porcine

Jianwen CHEN, Kaiyuan PAN, Zhen CHEN, Biao DING, Dandan SONG, Wenbin BAO, Yunhai ZHANG

期刊论文

抗RNA病毒相关生物材料

姚康德,尹玉姬,张宝连,赵立国

期刊论文

基于RNA的生物防治——一种作物保护新模式

Matthew Bramlett, Geert Plaetinck, Peter Maienfisch

期刊论文

敲低特异性环状非编码RNA显著抑制骨肉瘤的进展

王世东, 张红亮, 李博, 陈成龙, 任婷婷, 黄怡, 刘凯, 李敬敬, 郭卫

期刊论文

Overexpressed long noncoding RNA CRNDE with distinct alternatively spliced isoforms in multiple cancers

Xuefei Ma, Wei Zhang, Rong Zhang, Jingming Li, Shufen Li, Yunlin Ma, Wen Jin, Kankan Wang

期刊论文

Potential use of serum HBV RNA in antiviral therapy for chronic hepatitis B in the era of nucleos(t)ide

null

期刊论文

immune escape and microenvironment between RG-like and pri-OPC-like glioma revealed by single-cell RNA-seq

期刊论文

Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

Zhengjuan LIU, Jie BIAN, Yuchuan WANG, Yongli ZHAO, Dong YAN, Xiaoxia WANG

期刊论文

A ruptured recurrent small bowel gastrointestinal stromal tumour causing hemoperitoneum

null

期刊论文

RNA m6A modification and its function in diseases

null

期刊论文