Hairpin RNA Expression Research Articles

Solute Carrier Family 39 Member 6 Gene Promotes Aggressiveness of Esophageal Carcinoma Cells by Increasing Intracellular Levels of Zinc, Activating Phosphatidylinositol 3-Kinase Signaling, and Up-regulating Genes That Regulate Metastasis

A common variant in the solute carrier family 39 member 6 gene (SLC39A6) has been associated with survival times of patients with esophageal squamous cell carcinoma (ESCC). We investigated the function of SLC39A6 and ways in which this variant affects tumor progression by studying ESCC samples and cell lines. SLC39A6 was expressed or knocked down by expression of short hairpin RNAs in ESCC cells (KYSE30 and KYSE450) and HeLa cells using lentiviral vectors; we analyzed effects on proliferation, colony formation, migration, and invasion invitro. Cells were grown as xenograft tumors in nude mice and tumor volume and metastases were quantified; tumors were collected and analyzed histologically. Cells were also analyzed for levels of intracellular zinc and messenger RNA (mRNA) expression patterns. We obtained ESCC and adjacent normal esophageal tissues from 94 patients who underwent esophagectomy in China from 2010 through 2014. Survival times of patients were measured from the date of diagnosis to the date of last follow-up or death. We sequenced mRNAs and compared levels between tumor and non-tumor tissues using the Wilcox rank-sum test. Total proteins in cell lines or tissue samples were measured by immunoblotting. We searched publicly available databases for variants of SLC39A6 in human tumor and non-tumor tissues. Knockdown of SLC39A6 reduced proliferation of ESCC cells in culture and metastasis of xenograft tumors in mice. Cells that overexpressed SLC39A6 had significant increases in intracellular levels of zinc and were more invasive in assays, activating phosphatidylinositol 3-kinase signaling to AKT serine/threonine kinase 1 and mitogen-activated protein kinase 1. Cells that overexpressed SLC39A6 had increased expression of mRNAs and proteins associated with metastasis, such as matrix metalloproteinase (MMP) 1, MMP3, MYC, and snail family transcriptional repressor 2 (SNAI2 or SLUG). Levels of MMP1, MMP3, MYC, and SLUG mRNAs correlated with levels of SLC39A6 mRNA in ESCC samples from patients. ESCC tissues had increased levels of SLC39A6 mRNA compared with non-tumor tissues; the increase correlated with tumor metastasis to lymph node and reduced patient survival time. In an analysis of ESCC samples and cell lines, we associated increased expression of SLC39A6 with tumor invasiveness, intracellular level of zinc, and patient survival time. ESCC cell lines that overexpress SLC39A6 up-regulate expression MMP1, MMP3, MYC, and SLUG and form metastatic xenograft tumors in mice. Up-regulation of SLC39A6 might be used to determine prognoses of patients with ESCC or as a therapeutic target.

Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice

Pancreatic stellate cells (PSCs) change from a quiescent to activated state in the tumor environment and secrete extracellular matrix (ECM) molecules and cytokines to increase the aggressiveness of tumors. However, it is not clear how PSCs are activated to produce these factors, or whether this process can be inhibited. PSCs have morphologic and functional similarities to hepatic stellate cells, which undergo autophagy to promote fibrosis and tumor growth. We investigated whether autophagy activates PSCs, which promotes development of the tumor stroma and growth of pancreatic tumors in mice. We used immunofluorescence microscopy and immunohistochemistry to analyze pancreatic tumor specimens from 133 patients who underwent pancreatectomy in Japan from 2000 to 2009. PSCs were cultured from pancreatic tumor tissues or tissues of patients with chronic pancreatitis; these were analyzed by immunofluorescence microscopy, immunoblots, quantitative reverse transcription polymerase chain reaction, and in assays for invasiveness, proliferation, and lipid droplets. Autophagy was inhibited in PSCs by administration of chloroquine or transfection with small interfering RNAs. Proteins were knocked down in immortalized PSCs by expression of small hairpin RNAs. Cells were transplanted into pancreatic tails of nude mice, and tumor growth and metastasis were quantified. Based on immunohistochemical analyses, autophagy was significantly associated with tumor T category (P= .018), histologic grade (P= .001), lymph node metastases (P < .001), stage (P= .009), perilymphatic invasion (P= .001), and perivascular invasion (P= .003). Autophagy of PSCs was associated with shorter survival times of patients with pancreatic cancer. PSC expression of microtubule-associated protein 1 light chain3, a marker of autophagosomes, was associated with poor outcomes (shorter survival time, disease recurrence) for patients with pancreatic cancer (relative risk of shorter survival time, 1.56). Immunoblots showed that PSCs from pancreatic tumor samples expressed higher levels of markers of autophagy than PSCs from chronic pancreatitis samples. Inhibitors of autophagy increased the number of lipid droplets of PSCs, indicating a quiescent state of PSCs, and reduced their production of ECM molecules and interleukin 6, as well as their proliferation and invasiveness in culture. PSCs exposed toautophagy inhibitors formed smaller tumors in nude mice (P= .001) and fewer liver metastases (P= .018) with less peritoneal dissemination (P= .018) compared to PSCs not exposed to autophagy inhibitors. Autophagic PSCs produce ECM molecules and interleukin 6 and are associated with shorter survival times and disease recurrence in patients with pancreatic cancer. Inhibitors of PSC autophagy might reduce pancreatic tumor invasiveness by altering the tumor stroma.

Agroinfiltration-based expression of hairpin RNA in soybean plants for RNA interference against Tetranychus urticae

The coatomer subunit alpha (COPA) and aquaporin 9 (AQ9) genes from the two-spotted spider mite, Tetranychus urticae, were previously determined to exhibit RNA interference (RNAi)-based lethality when their double-stranded RNAs were systemically delivered via multi-unit chambers (Kwon et al., 2016 [8]). In current study, the hairpin RNAs of the COPA and AQ9 were transiently expressed in soybean plants by agroinfiltration. When T. urticae was fed with the soybean plants agroinfiltrated with the COPA and AQ9 hairpin RNA cassettes, the cumulative mortality increased significantly at 6days post-infestation. Quantitative PCR analysis revealed that the transcript level of both COPA and AQ9 was significantly reduced in T. urticae after 2days post-infestation, thereby confirming that the significant increases in mortality resulted from the knockdown of COPA and AQ9 transcripts. Our findings demonstrate the utility of COPA and AQ9 as potential genes for plant host-mediated RNAi control of T. urticae. In addition, we proved the usefulness of agroinfiltration as a rapid validation tool for confirming the RNAi-based lethality of target genes against arthropod pests before producing transgenic plants as agroinfiltration requires less time and skill to validate transgene function. Furthermore, these findings prove the concept that hairpin RNA expressed in plant hosts can also induce RNAi and eventually kill T. urticae, a sap-sucking pest.

Genetic Manipulation of the Avian Urogenital System Using In Ovo Electroporation.

One of the advantages of the avian embryo as an experimental model is its in ovo development and hence accessibility for genetic manipulation. Electroporation has been used extensively in the past to study gene function in chicken and quail embryos . Readily accessible tissues such as the neural tube, somites, and limb bud, in particular, have been targeted. However, more inaccessible tissues, such as the embryonic urogenital system , have proven more challenging to study. Here, we describe the use of in ovo electroporation of TOL2 vectors or RCASBP avian viral vectors for the rapid functional analysis of genes involved in avian sex determination and urogenital development . In the context of the developing urogenital system , these vectors have inherent advantages and disadvantages, which will be considered here. Either vector can both be used for mis-expressing a gene and for targeting endogenous gene knockdown via expression of short hairpin RNAs (shRNAs). Both of these vectors integrate into the genome and are hence spread throughout developing tissues. Going forward, electroporation could be combined with CRISPR/Cas9 technology for targeted genome editing in the avian urogenital system .

An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.

Hydrodynamic tail vein injection (HTVI) of transposon-based integration vectors is an established system for stably transfecting mouse hepatocytes in vivo that has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon-mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon-based system for manipulating gene expression in hepatocytes in vivo. Transposon-based vector constructs were generated to enable the constitutive expression of inducible Cre recombinase (CreER) together with tetracycline-inducible transgene or miR-small hairpin RNA (shRNA) expression (Tet-ON system). Transposon and transposase expression vectors were co-injected into R26R-mTmG reporter mice by HTVI. Cre-mediated gene recombination was induced by tamoxifen, followed by the administration of doxycycline to drive tetracycline-inducible gene or shRNA expression. Expression was visualized by immunofluorescence staining in livers of injected mice. After HTVI, Cre recombination by tamoxifen led to the expression of membrane-bound green fluorescent protein in transfected hepatocytes. Activation of inducible gene or shRNA expression was detected by immunostaining in up to one-third of transfected hepatocytes, with an efficiency dependent on the promoter driving the Tet-ON system. Our vector system combines Cre-lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models.

Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant.

Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis.

Purpose: Our previous study indicated that mitochondrial DNA (mtDNA) damage and mutations are crucial to the progressive loss of retinal ganglion cells (RGCs) in a glaucomatous rat model. In this study, we examined whether high pressure could directly cause mtDNA alterations and whether the latter could lead to mitochondrial dysfunction and RGC death.Methods: Primary cultured rat RGCs were exposed to 30 mm Hg of hydrostatic pressure (HP) for 12, 24, 48, 72, 96 and 120 h. mtDNA alterations and mtDNA repair/replication enzymes OGG1, MYH and polymerase gamma (POLG) expressions were also analyzed. The RGCs were then infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting POLG (POLG-shRNA), and mtDNA alterations as well as mitochondrial function, including complex I/III activities and ATP production were subsequently studied at appropriate times. Finally, RGC apoptosis and the mitochondrial-apoptosis pathway-related protein cleaved caspase-3 were detected using a Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and western blotting, respectively.Results: mtDNA damage was observed as early as 48 h after the exposure of RGCs to HP. At 120 h after HP, mtDNA damage and mutations significantly increased, reaching >40% and 4.8 ± 0.3-fold, respectively, compared with the control values. Twelve hours after HP, the expressions of OGG1, MYH and POLG mRNA in the RGCs were obviously increased 5.02 ± 0.6-fold (p < 0.01), 4.3 ± 0.2-fold (p < 0.05), and 0.8 ± 0.09-fold (p < 0.05). Western blot analysis showed that the protein levels of the three enzymes decreased at 72 and 120 h after HP (p < 0.05). After interference with POLG-shRNA, the mtDNA damage and mutations were significantly increased (p < 0.01), while complex I/III activities gradually decreased (p < 0.05). Corresponding decreases in membrane potential and ATP production appeared at 5 and 6 days after POLG-shRNA transfection respectively (p < 0.05). Increases in the apoptosis of RGCs and cleaved caspase-3 protein expression were observed after mtDNA damage and mutations.Conclusions: High pressures could directly cause mtDNA alterations, leading to mitochondrial dysfunction and RGC death.

Effect of chicken leptin recptor short hairpin RNA on expression of JAK2, STAT3, SOCS3 and CPT1 genes in chicken preadipocytes.

In this study, we detect depressive effect on leptin receptor (LEPR) by LEPR-specific short hairpin RNA (shRNA) expression plasmids in chicken preadipocytes, and effect on messenger RNA (mRNA) expression levels of genes related to signal transduction, including JAK2, STAT3, SOCS3 as well as CPT1, which is associated with fatty acid metabolism. shRNA expression vectors targeting LEPR were constructed and transfected into chicken preadipocytes. The transfection efficiency was evaluated by fluorescence microscopy. Real-time PCR was used to detect its effect on mRNA expression levels of JAK2, STAT3, SOCS3 and CPT1. Results showed that LEPR mRNA was knocked down by 99% (P<0.01) after transfection for 72h. In the knockdown preadipocytes, the mRNA levels of JAK2 and CPT1 were down-regulated by 47.56% (P<0.01) and 42.26% (P<0.05), respectively; while expression of STAT3 and SOCS3 increased 7.72-fold (P<0.01), 1.71-fold (P<0.01), respectively. It is concluded that knockdown of LEPR influences mRNA expression of its down-stream genes, suggesting that chicken LEPR play a certain role in regulating genes in the complicated gene network of preadipocytes.

Effect of silencing key proteins in telomerase mechanism and alternative lengthening of telomeres mechanism in laryngeal cancer cells

PurposeTo explore the influences of telomerase and alternative lengthening of telomeres mechanism on telomere length and laryngeal squamous cell carcinoma in vitro and in vivo. Materials and methodsShort hairpin RNA expression vectors targeting the messenger TERT, TRF2, RAD51 and NBS1 were constructed. The mRNA and protein expression of targeted genes in human laryngeal squamous carcinoma cell line HEp-2 was evaluated by reverse transcription polymerase chain reaction and Western blotting separately. The length of telomere was analyzed by fluorescent in-situ hybridization. Cell viability was examined by cell counting Kit-8. Effects on tumor growth were also investigated in vivo. ResultsThe transfection of multiple short hairpin RNAs expression plasmid significantly inhibited the mRNA and protein expression of related genes. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes resulted in the shortening of telomere length in HEp-2 cell. However, silence of alternative lengthening of telomeres mechanism related genes could shorten the telomere length but had no significant difference. Both simultaneously and separately blocking telomerase mechanism and alternative lengthening of telomeres mechanism resulted in reduction of tumor cell viability. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes inhibited the tumor growth in vivo. ConclusionsThe inhibition of telomere related gene may be a promising strategy for the treatment of laryngeal squamous cell carcinoma.

Dickkopf-1 expression is associated with tumorigenity and lymphatic metastasis in human hilar cholangiocarcinoma.

Dickkopf-1 (DKK1) is involved in tumorigenesis and the invasion of several tumors. However, its biological function in human hilar cholangiocarcinoma (HCCA) has not yet been documented. This study was designed to investigate the clinical significance and biological function of DKK1 in HCCA. The expression of DKK1 was investigated in thirty-seven human HCCA biopsy samples by immunohistochemistry. To further explore the biological effects of DKK1 in HCCA, transient and stable knockdown of DKK1 in two human HCCA cells (QBC939 and FRH0201) were established using small interfering or short hairpin RNA expression vector. In the present study, immunohistochemistry revealed that DKK1 was up-regulated in human HCCA tissues (24/37, 64.9%). High levels of DKK1 in human HCCA correlated with metastasis to the hilar lymph nodes (P=0.038). Genetic depletion of DKK1 in HCCA cells resulted in significantly inhibited proliferation, colony formation and migration compared with controls. Most importantly, DKK1 down-regulation impaired tumor formation capacity of HCCA cells in vivo. Subsequent investigations revealed that β-catenin is an important target of DKK1 and DKK1 exerts its pro-invasion function at least in part through the β-catenin/ matrix metalloproteinase-7 (MMP-7) signaling pathway. Consistently, in human HCCA tissues, DKK1 level was positively correlated with β-catenin and MMP-7 expression, as well as tumor hilar lymphatic metastasis. Taken together, our findings indicate that DKK1 may be a crucial regulator in the tumorigenicity and invasion of human HCCA, DKK1 exerts its pro-invasion function at least in part through the β-catenin/ MMP-7 signaling pathway, suggesting DKK1 as a potential therapeutic target for HCCA.

Synthesis of Poly Linear shRNA Expression Cassettes Through Branch-PCR.

A facile and universal strategy to construct the poly linear small hairpin RNA (shRNA) expression cassettes with multiple shRNA transcription templates through polymerase chain reaction with flexible branched primers (branch-PCR) is described in this protocol. Double-stranded RNA (dsRNA) is not stable enough for the study of RNA interference (RNAi) delivery in mammalian cells. Therefore, the more stable shRNA transcription template is employed to produce the endogenous transcribed dsRNA. Then, the covalent crosslinked linear shRNA expression cassettes are constructed through the branch-PCR for the long-lasting RNAi effect in this protocol. The branched primer pair is efficiently synthesized through classic click chemistry. In one step of PCR, the much more stable poly linear shRNA expression cassettes can be produced in large scale. This strategy of efficient synthesis of the poly linear gene expression cassettes can also be applied in the field for other target gene delivery. © 2016 by John Wiley & Sons, Inc.

Abstract 5173: Role of Gli2 in mediating the progression of prostate cancer to CRPC

Abstract Background: Prostate cancer is a leading cause of cancer-related death in the US men, largely due to the development of castration resistant prostate cancer (CRPC) in the patients treated with androgen deprivation therapy. There is increasing evidence indicating that Hedgehog pathway is involved in the malignancy of prostate cancer, which includes the finding of two prostate tumors with loss-of-function mutations in the SuFu gene, correlation of tumor grade with high expression of Sonic hedgehog, Gli2 and/or hedgehog target genes including Ptch1 and HIP, and increased Gli2 expression in therapy-resistant tumor cells from patients. In addition, treatment of mice with hedgehog inhibitors significantly attenuated the growth of prostate tumor xenografts. Some recent studies also implicated a crosstalk between Hedgehog/Gli signaling and androgen signaling pathways in prostate cancer. However, the role of Gli2 in CRPC progression has not been investigated. Methods: A human Gli2 specific small hairpin RNA (shRNA) expression vector was constructed using Tet-PLKO-puro lentivector, which was delivered into the androgen-dependent human prostate cancer LNCaP cells. RT-PCR, Western Blot and Luciferase Reporter Assay were performed to confirm doxycycline-inducible Gli2 knockdown. The effect of Gli2 knockdown on anchorage-dependent or -independent growth was assessed with MTT and Soft Agar Assays respectively. Cells were cultured in androgen depleted medium or treated with the anti-androgen, Casodex, to mimic androgen deprivation condition. Mouse xenograft models were used to assess the role of Gli2 in the development of CRPC in vivo. Results: Androgen deprivation promoted the expression of Hedgehog signaling components including Gli2 in the LNCaP cells. Gli2 knockdown inhibited anchorage-dependent and -independent growth of LNCaP cells. Casodex complemented the inhibition of Gli2 knockdown on the growth of LNCaP cells. Gli2 knockdown prevented the outgrowth of androgen-independent cells from LNCaP cells cultured in androgen depleted medium. Induced-knockdown of Gli2 by doxycycline inhibited the growth of castration-resistant tumors in vivo in mice. Conclusions: Our study demonstrated the important role of Gli2 in the development of CRPC. Therefore, it may have the potential to serve as a novel therapeutic target for the prevention of CRPC. Citation Format: Lu Xia, Tai Qin, Luzhe Sun. Role of Gli2 in mediating the progression of prostate cancer to CRPC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5173.

Abstract 4577: Fibroblast growth factor receptor-4 (FGFR-4) as a novel therapeutic target for pancreatic cancer

Abstract The fibroblast growth factor receptor (FGFR) family plays crucial roles in development, tissue repair, and malignant tumors. FGFR-1, -2, and -3 each exist in two isoforms, IIIb and IIIc, due to alternative splicing of the extracellular domain, whereas FGFR-4 does not have these isoforms. FGFR-4 is reportedly over-expressed in various cancers such as breast, prostate, hepatocellular, ovarian, gastric, colorectal, and pancreatic cancers, wherein it contributes to tumor progression. Recent studies have shown that a decrease in FGFR-4 levels suppresses the aggressiveness of gastric, colorectal, and ovarian cancers. We found that pancreatic cancer cell lines expressed similar levels of the IIIb and IIIc isoforms of FGFR-1 to -3, but showed different FGFR-4 levels. These findings suggest that the cancer cells surviving after treatment with recently developed anti-FGFR drugs, which target FGFR-1 to -3, tend to express high levels of FGFR-4. Moreover, a single-nucleotide polymorphism (SNP) in exon 9 of the gene encoding FGFR-4 that results in the substitution of glycine with arginine at codon 388 (388 Gly/Arg) in the transmembrane domain is associated with poor outcomes of high-grade soft tissue sarcoma, prostate, lung, head and neck carcinoma, and advanced and treatment-resistant breast cancer. It has been reported that 40-50% of Caucasians carry at least one copy of the 388 Gly/Arg SNP of FGFR-4. In the present study, we examined the expression and roles of the 388 Gly/Arg SNP in pancreatic cancer and tried to clarify whether FGFR-4 may be a novel therapeutic target for cancer treatment. In human pancreatic tissues, FGFR-4 was weakly localized in the normal exocrine and endocrine pancreas, and was strongly expressed in 67 of 136 pancreatic ductal adenocarcinoma cases (49%). FGFR-4 expression positively correlated with larger primary tumors and more advanced stages of pancreatic cancer. FGFR-4 mRNA was expressed in 5 pancreatic cancer cell lines at various levels, and the mutation in codon 388 was detected in 3 of the cell lines, including PK-1 cells. A short hairpin RNA expression vector targeting FGFR-4 was stably transfected to these mutant-expressing PK-1 cells (388 Gly/Arg), which then showed lower growth rates and cell migration and invasion abilities compared to sham vector-transfected cells. DNA microarray analysis showed that decreased expression of FGFR-4 in PK-1 cells altered the expression levels of molecules related to cellular movement, cellular development, and cell-to-cell signaling and interactions. FGF-19—one of the major ligands for FGFR-4—was expressed in all 5 of the pancreatic cancer cell lines tested. These findings suggest that inhibition of the expression of FGFR-4 harboring the 388 Gly/Arg SNP may be a novel therapeutic strategy for pancreatic cancer, especially after treatment with the newly developed anti-FGFR-1 to -3-targeted drugs. Citation Format: Toshiyuki Ishiwata, Hisashi Yoshimura, Yoko Matsuda, Shunji Ishiwata. Fibroblast growth factor receptor-4 (FGFR-4) as a novel therapeutic target for pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4577.

Transcription elongation factor Brd4-P-TEFb accelerates intestinal differentiation-associated SLC2A5 gene expression

BackgroundExpression of the fructose transporter gene SLC2A5 and histone acetylation in the transcribed region are induced by differentiation associated-signals such as glucocorticoids and p44/42 mitogen-activated protein kinase (MAPK) inhibition in small intestinal Caco-2 cells. MethodsWe co-treated with glucocorticoid receptor agonist dexamethasone (Dex) and p44/42 MAPK inhibitor PD98059 (PD) in Caco-2 cells with or without Brd4 small hairpin (sh) RNA expression vector, and the cells were analyzed by qRT-PCR and chromatin immunoprecipitation assays. The small intestine of wild-type mice and Brd4+/− mice during weaning period were analyzed by qRT-PCR. ResultsCo-treatment with Dex and PD increased binding of the bromodomain-containing protein-4 (Brd4)–positive transcriptional elongation factor-b (P-TEFb)–RNA polymerase II complex to acetylated histones in the transcribed region of SLC2A5. Brd4-protein depletion by shRNA revealed that the association of these proteins on the transcribed region of SLC2A5 promoted gene expression in a Brd4-dependent manner. Expression of small-intestine Slc2a5, but not another intestinal gene sucrase-isomaltase, during weaning period, was significantly lower in Brd4+/− mice compared with wild-type mice. ConclusionsBrd4-P-TEFb plays a crucial role in differentiation-associated transcription of SLC2A5 gene in intestinal Caco-2 cells and in the small intestine of mice during weaning period. General significanceHistone acetylation and the transcription elongation factor Brd4 are important for SLC2A5 expression in the small intestine.

RNA interference-based technology: what role in animal agriculture?

Animal agriculture faces a broad array of challenges, ranging from disease threats to adverse environmental conditions, while attempting to increase productivity using fewer resources. RNA interference (RNAi) is a biological phenomenon with the potential to provide novel solutions to some of these challenges. Discovered just 20 years ago, the mechanisms underlying RNAi are now well described in plants and animals. Intracellular double-stranded RNA triggers a conserved response that leads to cleavage and degradation of complementary mRNA strands, thereby preventing production of the corresponding protein product. RNAi can be naturally induced by expression of endogenous microRNA, which are critical in the regulation of protein synthesis, providing a mechanism for rapid adaptation of physiological function. This endogenous pathway can be co-opted for targeted RNAi either through delivery of exogenous small interfering RNA (siRNA) into target cells or by transgenic expression of short hairpin RNA (shRNA). Potentially valuable RNAi targets for livestock include endogenous genes such as developmental regulators, transcripts involved in adaptations to new physiological states, immune response mediators, and also exogenous genes such as those encoded by viruses. RNAi approaches have shown promise in cell culture and rodent models as well as some livestock studies, but technical and market barriers still need to be addressed before commercial applications of RNAi in animal agriculture can be realised. Key challenges for exogenous delivery of siRNA include appropriate formulation for physical delivery, internal transport and eventual cellular uptake of the siRNA; additionally, rigorous safety and residue studies in target species will be necessary for siRNA delivery nanoparticles currently under evaluation. However, genomic incorporation of shRNA can overcome these issues, but optimal promoters to drive shRNA expression are needed, and genetic engineering may attract more resistance from consumers than the use of exogenous siRNA. Despite these hurdles, the convergence of greater understanding of RNAi mechanisms, detailed descriptions of regulatory processes in animal development and disease, and breakthroughs in synthetic chemistry and genome engineering has created exciting possibilities for using RNAi to enhance the sustainability of animal agriculture.

A novel multi-target RNAi adenovirus inhibits hepatoma cell proliferation, migration, and induction of angiogenesis.

The pathogenesis of hepatocellular carcinoma (HCC) is a multi-step process involving many genes. Consequently, single gene targeting therapy has limited efficacy, making combination therapy targeting multiple genes a necessity. Based on our previous findings, we constructed a single vector mediating simultaneous expression of multiple short hairpin RNAs (shRNAs) against human vascular endothelial growth factor receptor 2 (VEGFR2), chemokine C-C motif receptor 1 (CCR1), and epithelial cell adhesion molecule (EpCAM), three genes closely related to HCC progression that act through separate pathways. The shRNA vector efficiently downregulated the mRNA and protein of all three molecules in Huh7 hepatoma cells. The vector also inhibited cell proliferation and migration and reduced angiogenesis. Furthermore, this shRNA vector can be recombined into adenovirus, a gene therapy vector, for better in vivo application. It thus offers a potentially effective future gene therapy approach to treating human liver cancer.

Use of microalgae Chlamydomonas reinhardtii for production of double-stranded RNA against shrimp virus

RNA interference has been proposed to be a promising tool for combating shrimp viruses. Antiviral double-stranded (ds)RNA has been mostly produced in Escherichia coli-expression system because of its high efficiency and inexpensive operations. However, overusing the bacteria may raise concerns regarding public health and environmental contamination, and seeking for a new dsRNA production platform would be alternative for future molecular farming. In this study, we exploited the green microalgae Chlamydomonas reinhardtii to produce dsRNA targeting the lethal shrimp yellow head virus (YHV). The expression plasmid pSL18 for C. reinhardtii was constructed to contain YHV-specific hairpin RNA expression cassette, and the successful assembly of pSL18-YHV was confirmed by PCR and enzymatic digestions. Glass bead method was employed for transformation of C. reinhardtii nuclear genome with pSL18-YHV. Microalgal expression of dsRNA-YHV, approximately 45ng from 100-mL culture, was detected by qRT-PCR. Oral feeding experiment on postlarval shrimp revealed that the formulated feed with C. reinhardtii expressing dsRNA-YHV, at the ratio of 1×108 transformants per gram feed, improved 22% survival rate after YHV challenge. The present study suggests that C. reinhardtii can be bioengineered to produce viral-specific dsRNA for shrimp viral disease control, and the developed qRT-PCR could detect microalgal dsRNA with detection limit of subpicogram.

Efficient inhibition of human immunodeficiency virus replication using novel modified microRNA-30a targeting 3'-untranslated region transcripts.

RNA interference (RNAi)-based gene therapy is currently considered to be a combinatorial anti-human immunodeficiency virus-1 (HIV-1) therapy. Although artificial polycistronic microRNAs (miRs) can reduce HIV-1 escape mutant variants, this approach may increase the risk of side effects. The present study aimed to optimize the efficiency of anti-HIV RNAi gene therapy in order to reduce the cell toxicity induced by multi-short hairpin RNA expression. An artificial miR-30a-3'-untranslated region (miR-3-UTR) obtained from a single RNA polymerase II was used to simultaneously target all viral transcripts. The results of the present study demonstrated that HIV-1 replication was significantly inhibited in the cells with the miR-3-UTR construct, suggesting that miR-3'-UTR may serve as a promising tool for RNAi-based gene therapy in the treatment of HIV-1.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

Matrix Metallopeptidase 14 Plays an Important Role in Regulating Tumorigenic Gene Expression and Invasion Ability of HeLa Cells.

The objectives of this study were to investigate the functional effect of matrix metallopeptidase 14 (MMP14) on cell invasion in cervical cancer cells (HeLa line) and to study the underlying molecular mechanisms. Expression vector of short hairpin RNA targeting MMP14 was treated in HeLa cells, and then, transfection efficiency was verified by a florescence microscope. Transwell assay was used to investigate cell invasion ability in HeLa cells. Quantitative polymerase chain reaction and Western blotting analysis were used to detect the expression of MMP14 and relative factors in messenger RNA and protein levels, respectively. Matrix metallopeptidase 14 short hairpin RNA expression vector transfection obviously decreased MMP14 expression in messenger RNA and protein levels. Down-regulation of MMP14 suppressed invasion ability of HeLa cells and reduced transforming growth factor β1 and vascular endothelial growth factor B expressions. Furthermore, MMP14 knockdown decreased bone sialoprotein and enhanced forkhead box protein L2 expression in both RNA and protein levels. Matrix metallopeptidase 14 plays an important role in regulating invasion of HeLa cells. Matrix metallopeptidase 14 knockdown contributes to attenuating the malignant phenotype of cervical cancer cell.