Enhancement of gene knockdown efficiency by CNNC motifs in the intronic shRNA precursor

박성균,기윤,황병준 한국유전학회 2019 Genes & Genomics Vol.41 No.4

Background Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Of several methods to express shRNA, a method of transcribing shRNAs embedded in microRNA precursors has been more widely used than the one that directly expresses shRNA from RNA polymerase III promoters because the microRNA precursor form of shRNA is known to cause lower levels of cytotoxicity and off-target effects than the overexpressed shRNAs from the RNA polymerase III promoters. Objective We study the primary sequence features of microRNA precursors, which enhance their processing into mature form, helps design more potent shRNA precursors embedded in microRNA precursors. Methods We measure the enhancement of gene knockdown efficiency by adding CNNC motifs in the 3′ flanking region of shRNA precursor embedded in the human miR-30a microRNA precursor. Results By systemically adding three CNNC motifs in the 3′ flanking region of shRNA precursor, we found that addition of two CNNC motifs saturates their enhanced knockdown ability of shRNA and that the CNNC motif in the + 17 to + 20 from the drosha cleavage site is most important for the shRNA-mediated target gene knock down. We also did see little knockdown of target gene expression by the shRNA precursor lacking CNNC motif. Conclusion Since genetic studies generally require techniques that could reduce gene expression at different degrees, the findings in this study will allow us to use RNAi for genetic studies of reducing gene expression at different degrees.

이종이식 PEOH 세포주에서 clusterin에 대한 shRNA의 paclitaxel 내성 효과

신은영 ( Eun Young Shin ),정인철 ( In Cheal Jeung ),최주혁 ( Joo Hyuk Choi ),박동춘 ( Dong Choon Park ) 대한산부인과학회 2007 Obstetrics & Gynecology Science Vol.50 No.11

Objective: To evaluate the inhibition of Clusterin gene expression via shRNA decreases proliferation and metastasis and increases chemo-sensitivity to paclitaxel in xenografted PEOH cells. Methods: 1×106 paclitaxel resistant cell lines transduced with Clusterin shRNA in lentiviral inoculated subcutaneously into the flank region of 6 to 8 week-old female nude mice. Parental cells transduced with LacZ was used as a control. Tumor growth was measured twice every week and calculated by using the formula: length×width×depth×0.5236. The mice were sacrificed and examined for Clusterin expression on tumor cells and counted the metastasis sites. Results: shRNA for Cluaterin works in vivo and it is the in accord with the in vitro results. Although shRNA for Clusterin group showed decreased tumor growth and proliferation it has not statistical significance. But transfection of Clusterin shRNA on PEOH significantly increased paclitaxel-sensitivity (P<0.05). Conclusion: shRNA targeting of the Clusterin gene decreased the ovarian cancer cell`s paclitaxel resistance.

Short-Hairpin RNA-Mediated Gene Expression Interference in Trichoplusia ni Cells

( Na Young Kim ),( Jin Young Baek ),( Hong Seok Choi ),( In Sik Chung ),( Sung Ho Shin ),( Jung Ihn Lee ),( Jung Yun Choi ),( Jai Myung Yang ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.2

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The shortinterfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and β-Nacetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and β-Nacetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

A Simple and Economical Short-oligonucleotide-based Approach to shRNA Generation

Kim, Jin-Su,Kim, Hyuk-Min,Lee, Yoon-Soo,Yang, Kyung-Bae,Byun, Sang-Won,Han, Kyu-Hyung Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.3

RNAi (RNA interference) has become a popular means of knocking down a specific gene in vivo. The most common approach involves the use of chemically synthesized short interfering RNAs (siRNAs), which are relatively easy and fast to use, but which are costly and have only transient effects. These limitations can be overcome by using short hairpin RNA (shRNA) expression vectors. However, current methods of generating shRNA expression vectors require either the synthesis of long (50-70 nt) costly oligonucleotides or multi-step processes. To overcome this drawback, we have developed a one-step short-oligonucleotides-based method with preparation costs of only 15% of those of the conventional methods used to obtain essentially the same DNA fragment encoding shRNA. Sequences containing 19 bases homologous to target genes were synthesized as 17- and 31-nt DNA oligonucleotides and used to construct shRNA expression vectors. Using these plasmids, we were able to effectively silence target genes. Because our method relies on the onestep ligation of short oligonucleotides, it is simple, less error-prone, and economical.

Hiwi Knockdown Inhibits the Growth of Lung Cancer in Nude Mice

Liang, Dong,Dong, Min,Hu, Lin-Jie,Fang, Ze-Hui,Xu, Xia,Shi, En-Hui,Yang, Yi-Ju Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.2

Hiwi, a human homologue of the Piwi family, plays an important role in stem cell self-renewal and is overexpressed in various human tumors. This study aimed to determine whether an RNA interference-based strategy to suppress Hiwi expression could inhibit tumor growth in a xenograft mouse model. A rare population of $SSC^{lo}\;Alde^{br}$ cells was isolated and identified as lung cancer stem cells in our previous study. Plasmids containing U6 promoter-driven shRNAs against Hiwi or control plasmids were successfully established. The xenograft tumor model was generated by subcutaneously inoculating with lung cancer stem cell $SSC^{lo}\;Alde^{br}$ cells. After the tumor size reached about 8 mm in diameter, shRNA plasmids were injected into the mice via the tail vein three times a week for two weeks, then xenograft tumor growth was assessed. In nude mice, intravenously delivery of Hiwi shRNA plasmids significantly inhibited tumor growth compared to treatment with control scrambled shRNA plasmids or the vehicle PBS. No mice died during the experiment and no adverse events were observed in mice administered the plasmids. Moreover, delivery of Hiwi shRNA plasmids resulted in a significant suppressed expression of Hiwi and ALDH-1 in xenograft tumor samples, based on immunohistochemical analysis. Thus, shRNA-mediated Hiwi gene silencing in lung cancer stem cells by an effective in vivo gene delivery strategy appeared to be an effective therapeutic approach for lung cancer, and may provide some useful clues for RNAi gene therapy in solid cancers.

RNAi-based Knockdown of Multidrug Resistance-associated Protein 1 is Sufficient to Reverse Multidrug Resistance of Human Lung Cells

Shao, Shu-Li,Cui, Ting-Ting,Zhao, Wei,Zhang, Wei-Wei,Xie, Zhen-Li,Wang, Chang-He,Jia, Hong-Shuang,Liu, Qian Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.24

Up-regulation of multidrug resistance-associated protein 1 (MRP1) is regarded as one of the main causes for multidrug resistance (MDR) of tumor cells, leading to failure of chemotherapy-based treatment for a multitude of cancers. However, whether silencing the overexpressed MRP1 is sufficient to reverse MDR has yet to be validated. This study demonstrated that RNAi-based knockdown of MRP1 reversed the increased efflux ability and MDR efficiently. Two different short haipin RNAs (shRNAs) targeting MRP1 were designed and inserted into pSilence-2.1-neo. The shRNA recombinant plasmids were transfected into cis-dichlorodiamineplatinum-resistant A549 lung (A549/DDP) cells, and then shRNA expressing cell clones were collected and maintained. Real time PCR and immunofluorescence staining for MRP1 revealed a high silent efficiency of these two shRNAs. Functionally, shRNA-expressing cells showed increased rhodamine 123 retention in A549/DDP cells, indicating reduced efflux ability of tumor cells in the absence of MRP1. Consistently, MRP1-silent cells exhibited decreased resistance to 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and DDP, suggesting reversal of MDR in these tumor cells. Specifically, MRP1 knockdown increased the DDP-induced apoptosis of A549/DDP cells by increased trapping of their cell cycling in the G2 stage. Taken together, this study demonstrated that RNAi-based silencing of MRP1 is sufficient to reverse MDR in tumor cells, shedding light on possible novel clinical treatment of cancers.

A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with Mycobacterium tuberculosis

Fei-yu Wang,Yu-qing Zhang,Xin-min Wang,Chan Wang,Xiao-fang Wang,Jiang-dong Wu,Fang Wu,Wan-jiang Zhang,Le Zhang 한국미생물학회 2016 The journal of microbiology Vol.54 No.4

Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.

shRNA-based Gene Therapy System Using Functional Peptides for the Treatment of Psoriasis

Yoo Eun LEE,Yeong Chae RYU,Byeong Hee HWANG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Recently, the Interleukin-23 p19 subunit (IL-23p19) activating interleukin 23 only has been found to play an important role in the inflammatory response of psoriasis. This study aimed to deliver the shRNA silencing IL-23p19 with high specificity via self-assembled nanocomplexes for psoriasis treatment delivery. Nanocomplexes were formed by electrostatic attraction between fusion peptides and shRNAs and were confirmed by gel retardation assay. Moreover, the size and positive charge of nanocomplex were measured by DLS analysis. In addition, the stability of shRNAs in nanocomplex was tested under serum presence. Fluorescence imaging and flow cytometry analysis confirmed the cell permeability of the nanocomplex containing Cy3-labeled siRNAs into RAW 264.7 macrophage cells. Also, shRNAs via nanocomplexes effectively inhibited 34% of IL23-p19 mRNA expression in the cells. The fusion peptide did not reduce cell viability even at high concentrations in the cells. Thus, gene therapy based on nanocomplexes with IL-23p19 shRNAs could be effectively used to treat various related diseases, including psoriasis.

Lentivirus Mediated GOLPH3 shRNA Inhibits Growth and Metastasis of Esophageal Squamous Cancer

Wang, Qiang,Wang, Xian,Zhang, Can-Bin Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.9

Aim: To investigate the role of Golgi phosphoprotein 3 (GOLPH3) in tumour growth and metastasis of esophageal squamous cancer. Methods: A lentiviral shRNA-vector was utilized to stably knockdown GOLPH3 in Eca-109 esophageal squamous cancer cells. mRNA transcription and protein expression of GOLPH3 were examined by real-time quantitative PCR and Western blotting, respectively. Cell proliferation activity was assessed by MTT assay and invasion and migration potentials by matrigel invasion and transwell motility assays. Results: Stable knockdown in the GOLPH3 cell line was established. PD-A gene expression was significantly suppressed by lentivirus-mediated RNAi, which resulted in reducing the capacity for cell proliferation, migration, invasion and adhesion in vitro. In vivo, GOLPH3 depletion resulted in inhibition of tumour growth, with stable decrease in the expression of GOLPH3 in tumor xenografts. Conclusions: Our findings suggest that lentivirus mediated silencing of the GOLPH3 gene has a significant anti-tumour effect on esophageal squamous cancer in vitro and in vivo. In addition, the results indicate that GOLPH3 might be an effective molecular target for gene therapy in esophageal squamous cancer.

Down-regulation of the autophagy gene, ATG7, protects bone marrow-derived mesenchymal stem cells from stressful conditions

Sedigheh Molaei,Mehryar Habibi Roudkenar,Fatemeh Amiri,Mozhgan Dehghan Harati,Marzie Bahadori,Fatemeh Jaleh,Mohammad Ali Jalili,Amaneh Mohammadi Roushandeh 대한혈액학회 2015 Blood Research Vol.50 No.2

Background Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their applicationis limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defectiveorganelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or triggerthe apoptotic machinery. This study aimed to determine whether regulation of autophagywould affect the survival of MSCs under stress conditions. Methods Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, andwas inhibited via shRNA-mediated knockdown of the autophagy specific gene, ATG7. ATG7 expression in BM-MSCs was evaluated by reverse transcription polymerase chainreaction (RT-PCR), western blot, and quantitative PCR (qPCR). Cells were then exposedto harsh microenvironments, and a water-soluble tetrazolium salt (WST)-1 assay was performedto determine the cytotoxic effects of the stressful conditions on cells. Results Of 4 specific ATG7-inhibitor clones analyzed, only shRNA clone 3 decreased ATG7expression. Under normal conditions, the induction of autophagy slightly increased theviability of MSCs while autophagy inhibition decreased their viability. However, understressful conditions such as hypoxia, serum deprivation, and oxidative stress, the inductionof autophagy resulted in cell death, while its inhibition potentiated MSCs to withstandthe stress conditions. The viability of autophagy-suppressed MSCs was significantlyhigher than that of relevant controls (P<0.05, P<0.01 and P<0.001). Conclusion Autophagy modulation in MSCs can be proposed as a new strategy to improve their survivalrate in stressful microenvironments.