Vector-mediated RNA Interference Research Articles

Low frequency‑pulsed electromagnetic fields promote osteogenic differentiation of bone marrow‑derived mesenchymal stem cells by regulating connexin 43 expression.

The present study investigated the effect of connexin 43 (Cx43) on the regulation of osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) using low-frequency-pulsed electromagnetic fields (LPEMF). The BMSCs were isolated and cultured in vitro using adherent whole-bone marrow cultures. CCK-8 assay was used to detect the effects of LPEMF on the proliferation ability of BMSCs and alkaline phosphatase (ALP) activity and the levels of osteogenic marker genes were detected to evaluate the osteogenic ability change following LPEMF treatment. Lentiviral vector-mediated RNA interference was transfected into BMSCs to inhibit the expression of Cx43 and western blotting was used to detect Cx43 expression. The BMSCs showed the highest proliferation following LPEMF treatment at 80 Hz for 1 h. The results of ALP activity, osteogenic marker genes and Alizarin Red S staining showed that the osteogenic ability was notably increased following LPEMF treatment at 80 Hz for 1 h. Cx43 expression increased during the osteogenic differentiation of BMSCs following LPEMF treatment at 80 Hz. The enhanced osteogenic differentiation of the LPEMF-treated BMSCs were partially reversed when Cx43 expression was inhibited. LPEMF may promote the osteogenic differentiation of BMSCs by regulating Cx43 expression and enhancing osteogenic ability.

Development of a Ligation-Independent Cloning-Based Dual Vector System for RNA Interference in Plants.

RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing mechanism that responds to double-stranded RNA (dsRNA) by sequence-specific downregulation of target genes. The dsRNA-mediated RNAi technology has become one of the most widely used and powerful tools for functional genomic studies in diverse organisms. However, its application has been limited due to the technical difficulty of making RNAi constructs caused by the inverted repeat structure that is required for the formation of hairpin RNA. Here, we present a ligation-independent cloning-based dual vector-mediated RNAi system for silencing specific genes in plants. This approach is simple, efficient, and cost-effective and can be readily adapted to other binary vectors for functional analysis of target genes and the development of sustainable disease and pest control strategies in a broad range of plant species.

Adenovirus type 5 vectors encoding short hairpin RNAs targeting dengue virus 5’ non-translated region and capsid gene suppress pre-established dengue infection in cultured epithelial and myeloid cells

Dengue, a mosquito-borne viral disease, caused by any of four serotypes of dengue viruses (DENV-1, -2, -3 and -4), is estimated to affect >1 million of the world's population daily. We showed earlier that a recombinant human adenovirus type 5 (HuAd5) vector, encoding a short hairpin RNA (shRNA), targeting a conserved sequence in the DENV genome, could effectively suppress pre-established DENV-2 infection in Vero cells. In this study, we identified an additional conserved shRNA target in the DENV genome, developed a HuAd5 vector to target this site, and evaluated if HuAd5-delivered shRNAs suppress pre-established infection by the remaining three DENV serotypes, not only in Vero cells, but also in macrophages, the in vivo sites of DENV replication in infected individuals. We also assessed the effect of anti-HuAd5 antibodies on shRNA delivery.We show that recombinant HuAd5 vectors, encoding shRNAs targeting conserved DENV genomic sequences, in the 5’ non-translated region and capsid gene, can suppress ongoing replication of all four prototypic DENV serotypes in Vero cells and in a HuAd5-refractory human macrophage cell line expressing a DENV attachment factor. DENV suppression was assessed on the basis of inhibition of viral antigen secretion, viral RNA replication and progeny virus generation. Interestingly, HuAd5 vector-mediated DENV suppression in the macrophage cell line was dependent on the presence of anti-HuAd5 antibody. This suggests that HuAd5 vector complexed to its antibody enters these cells through the Fc receptor pathway. This may have implications for specific targeting of HuAd5 vector-mediated antiviral RNA interference therapy to macrophages.

Inhibiting PHD2 in human periodontal ligament cells via lentiviral vector-mediated RNA interference facilitates cell osteogenic differentiation and periodontal repair.

Periodontal defect regeneration in severe periodontitis remains a challenging task in clinic owing to poor survival of seed cells caused by the remaining oxidative stress microenvironment. Recently, the reduction of prolyl hydroxylase domain-containing protein 2 (PHD2), a primary cellular oxygen sensor, has shown an incredible extensive effect on skeletal muscle tissue regeneration by improving cell resistance to reactive oxygen species, whereas its role in periodontal defect repair is unclear. Here, through lentivirus vector-mediated RNA interference, the PHD2 gene in human periodontal ligament cells (hPDLCs) is silenced, leading to hypoxia-inducible factor-1α stabilization in normoxia. In vitro, PHD2 silencing not only exhibited a satisfactory effect on cell proliferation, but also induced distinguished osteogenic differentiation of hPDLCs. Real-time polymerase chain reaction and Western blotting revealed significant up-regulation of osteocalcin, alkaline phosphatase (ALP), runt-related transcription factor 2, and collagen type I (COL I). Under oxidative stress conditions, COL I and ALP expression levels, suppressed by 100 μM H2 O2 , were elevated by PHD2-gene-silencing in hPDLCs. In vivo, periodontal fenestration defects were established in 18 female Sprague-Dawley rats aged 6wk old, followed by implantation of PHD2 silencing hPDLCs in situ for 21 d. Persistent and stable silencing of PHD2 in hPDLCs promoted better new bone formation according to microcomputed tomography 3D reconstruction and related bone parameter analysis. This work demonstrates the therapeutic efficiency of PHD2 gene interference in osteogenic differentiation and periodontal defect repair for highly efficient periodontal regeneration.

Topographic transcriptomics of the nucleus accumbens shell: Identification and validation of fatty acid binding protein 5 as target for cocaine addiction

Substance use disorders for cocaine are major public health concerns with few effective treatment options. Therefore, identification of novel pharmacotherapeutic targets is critical for future therapeutic development. Evolution has ensured that genes are expressed largely only where they are needed. Therefore, examining the gene expression landscape of the nucleus accumbens shell (NAcSh), a brain region important for reward related behaviors, may lead to the identification of novel targets for cocaine use disorder. In this study, we conducted a novel two-step topographic transcriptomic analysis using five seed transcripts with enhanced expression in the NAcSh to identify transcripts with similarly enhanced expression utilizing the correlation feature to search the more than 20,000 in situ hybridization experiments of the Allen Mouse Brain Atlas. Transcripts that correlated with at least three seed transcripts were analyzed with Ingenuity Pathway Analysis (IPA). We identified 7-fold more NAcSh-enhanced transcripts than our previous analysis using single voxels in the NAcSh as the seed. Analysis of the resulting transcripts with IPA identified many previously identified signaling pathways such as retinoic acid signaling as well as novel pathways. Manipulation of the retinoic acid pathway specifically in the NAcSh of male rats via viral vector-mediated RNA interference targeting fatty acid binding protein 5 (FABP5) decreased cocaine self-administration and modulates excitability of medium spiny neurons in the NAcSh. These results not only validate the prospective strategy of conducting a topographic transcriptomic analysis, but also further validate retinoic acid signaling as a promising pathway for pharmacotherapeutic development against cocaine use disorder.

Studying the Role and Molecular Mechanisms of MAP4K3 in Sorafenib Resistance of Hepatocellular Carcinoma.

Sorafenib is the first FDA-approved therapeutic drug for molecular target medication on advanced-stage hepatocellular carcinoma. It is reported that sorafenib could improve the survival of progression-free patients for 4 to 6 months; however, most of the patients developed drug resistance. Thus, it is critical to reveal the biological mechanisms behind sorafenib resistance. In this study, a sorafenib-resistant model was developed by exposing HepG2 cells to sorafenib with gradient increasing concentration, and the resistance-related genes were screened by microarray. Real-time qPCR was used to validate selected gene expression of the resistance model, and lentivirus vector-mediated RNA interference was applied for specific gene knockdown. In addition, high-throughput High Celigo Select (HCS) and flow cytometry were used to measure the effect on cellular proliferation and apoptosis. As a result, our study established a sorafenib-resistant model with IC50 of 9.988 μM. The Affymetrix expression profile of the sorafenib-resistant model showed 35 resistant-related genes, and 91.4% of the resistant genes showed upregulation in HepG2 resistance cells. In addition, 20 genes were knocked down to measure cell proliferation, and MAP4K3 with high proliferation inhibiting phenotype was chosen for further study. Meanwhile, the HCS results revealed that shMAP4K3 transfection could downregulate resistant cell proliferation, and the flow cytometry results showed that cell apoptosis was significantly increased in the MAP4K3 knockdown group. In summary, MAP4K3 is a novel molecular marker for improving the drug sensitivity of sorafenib treatment in hepatocellular carcinoma.

CGRP Modulates Orofacial Pain through Mediating Neuron-Glia Crosstalk

Calcitonin gene-related peptide (CGRP) plays a crucial role in the modulation of orofacial pain, and we hypothesized that CGRP mediated a neuron-glia crosstalk in orofacial pain. The objective of this study was to elucidate the mechanisms whereby CGRP mediated trigeminal neuron-glia crosstalk in modulating orofacial pain. Orofacial pain was elicited by ligating closed-coil springs between incisors and molars. Trigeminal neurons and satellite glial cells (SGCs) were cultured for mechanistic exploration. Gene and protein expression were determined through immunostaining, polymerase chain reaction, and Western blot. Orofacial pain was evaluated through the rat grimace scale. Our results revealed that the expressions of CGRP were elevated in both trigeminal neurons and SGCs following the induction of orofacial pain. Intraganglionic administration of CGRP and olcegepant exacerbated and alleviated orofacial pain, respectively. The knockdown of CGRP through viral vector-mediated RNA interference was able to downregulate CGRP expressions in both neurons and SGCs and to alleviate orofacial pain. CGRP upregulated the expression of inducible nitric oxide synthase through the p38 signaling pathway in cultured SGCs. In turn, L-arginine (nitric oxide donor) was able to enhance orofacial pain by upregulating CGRP expressions in vivo. In cultured trigeminal neurons, L-arginine upregulated the expression of CGRP, and this effect was diminished by cilnidipine (N-type calcium channel blocker) while not by mibefradil (L-type calcium channel blocker). In conclusion, CGRP modulated orofacial pain through upregulating the expression of nitric oxide through the p38 signaling pathway in SGCs, and the resulting nitric oxide in turn stimulated CGRP expression through N-type calcium channel in neurons, building a CGRP-mediated positive-feedback neuron-glia crosstalk.

Abstract 6336: Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma

Abstract BACKGROUND: It has been demonstrated in our previous study that poorly-differentiated hepatoma cells with low α-feto-protein (AFP) expression and a CD133-/EpCAM- surface profile exhibited aberrant hedgehog signaling (Hh) activity, epithelial-mesenchymal transition and chemoresistance. However, expression of canonical Hh molecules (Sonic ligand and SMO) was not increased in poorly-differentiated hepatoma cells in contrast to well-differentiated hepatoma cells, indicating that Hh transcription factors Gli-1/2 may be activated through a non-classic pathway. The present study aims to investigate whether the TGF-β/Smad3 pathway is responsible for Gli-2 activation through a non-canonical pathway. METHODS: Expression of Sonic Hh (Shh) and Smad3 was assessed in both hepatoma cells and HCC specimens. Gli-2 and Smad3 expression was suppressed by lentiviral vector-mediated RNA interference in poorly-differentiated hepatoma Huh-7-DN and HLE cells. Chemosensitivity was evaluated in hepatoma cells with a Gli-2 or Smad3 inhibitor. RESULTS: Significantly increased Gli-1 and Gli-2 protein levels were observed in 78% (11 out of 14) and 50% (7 out of 14) HCC specimens. In 7 HCC patients with a normal serum AFP value (<20 μg/L), Gli-1/2 protein levels were obviously higher than their pericancerous tissue. Moreover, increased Gli-2 and ABCC1 protein levels were accompanied with increased TGF-β1 and p-Smad3 protein levels in 5 out 7 HCC specimens; whereas Shh protein levels were similar to paired pericancerous tissue. Consistent with the findings from HCC specimens, significantly higher Gli-2 and p-Smad3 nuclear protein levels were associated with increased ABCC1 protein levels in poorly-differentiated hepatoma cells (Huh-7-trans, Huh-7-DN, HLE and HLF); whereas Shh and SMO protein levels were not significantly different from well-differentiated hepatoma Huh-7 and Hep3B cells. Suppressing Gli-2 expression by specific shRNA caused marked decreased expression of Gli-2 and ABCC1 in Huh-7-DN, Huh-7-trans and HLE cells. In addition, expression of Smad3, Gli-2 and ABCC1 was significantly down-regulated in HLE cells after infected with a lentiviral vector harboring shSmad3, indicating that Gli-2 is a downstream target of Smad3. Sorafenib IC50 was significantly reduced from 35.76 to 17.45 μM or 14.04 μM in HLE cells when treated with a GLI inhibitor (GANT-61) or a Smad3 inhibitor (SIS3) at 5 μM. A potential GLI-binding site in the ABCC1 promoter was identified. ABCC1-Gli-luciferase reporter activity was increased up to 39-fold in HLE cells when transfected with constructs containing the potential GLI-binding site, compared to the construct that lacked a GLI-binding site. CONCLUSION: TGF-β/Smad3 activated Gli-2 and its direct transcriptional target ABCC1 in poorly-differentiated and chemoresistant hepatoma cells. Suppressing TGF-β/Smad3/Gli-2 signaling activity confers a potential molecular target in partially alleviating Sorafenib resistance for the treatment of refractory hepatoma. Citation Format: Jia Ding, Hui-yan Li, Li Zhang, Xiao-tian Zhou, Jian Wu. Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6336.

Trib1 Contributes to Recovery From Ischemia/Reperfusion-Induced Acute Kidney Injury by Regulating the Polarization of Renal Macrophages.

Increasing evidence suggests that macrophage polarization is involved in the recovery from ischemia-reperfusion (I/R)-induced acute kidney injury (AKI), implying that the regulation of macrophage polarization homeostasis might mediate AKI recovery. Trib1 is a key regulator of macrophage differentiation, but its role in AKI remains unclear. Here, we aimed to investigate the role of Trib1 and its link with the macrophage phenotype in the process of adaptive recovery from I/R-induced renal injury. Lentiviral vector-mediated RNA interference (RNAi) was used to knock down Trib1 expression in vitro and in vivo, and a mouse model of moderate AKI was established by the induction of I/R injury. Renal function measurements and inflammatory factors were determined by the corresponding kits. Histomorphology was assessed by hematoxylin-eosin, Masson and PAS staining. Western blot and flow cytometry were employed for the analysis of signal transduction, cell apoptosis and macrophage phenotypes. Trib1 knockdown inhibited cell viability of tubular epithelial cells (TECs) by inhibiting proliferation and enhancing apoptosis in vitro. I/R-induced AKI significantly impaired renal function in mice via increasing the levels of BUN, Scr, NGAL and renal tubular damage, leading to renal fibrosis from days 1 to 3. Through the adaptive self-repair mechanism, renal dysfunction recovered over time and returned to almost normal levels on day 28 after I/R intervention. However, Trib1 depletion worsened renal damage on day 3 and blunted the adaptive repair process of the renal tissue. Mechanistically, Trib1 inhibition suppressed renal tubular cell proliferation under adaptive self-repair conditions by affecting the expression of the proliferation-related proteins cyclin D1, cyclin B, p21, and p27, the apoptosis-related proteins Bcl-2 and Bax, and the fibrosis-related proteins collagen I and III. Furthermore, the M1/M2 macrophage ratio increased in the first 3 days and decreased from day 7 to day 28, consistent with changes in the expression of inflammatory factors, including TNFα, IL-6, IL-12, IL-10, and IL-13. Trib1 inhibition blocked macrophage polarization during adaptive recovery from I/R-induced moderate AKI. Our results show that Trib1 plays a role in kidney recovery and regeneration via the regulation of renal tubular cell proliferation by affecting macrophage polarization. Thus, Trib1 might be a viable therapeutic target to improve renal adaptive repair following I/R injury.

Inhibition of steroid receptor coactivator-1 in the hippocampus impairs the consolidation and reconsolidation of contextual fear memory in mice

AimsSteroid receptor coactivator-1 (SRC-1) is a key coactivator for the efficient transcriptional activity of steroids in the regulation of hippocampal functions. However, the effect of SRC-1 on hippocampal memory processes remains unknown. Our aim was to investigate the roles of hippocampal SRC-1 in the consolidation and reconsolidation of contextual fear memory in mice. Main methodsContextual fear conditioning paradigm was constructed in adult male C57BL/6 mice to examine the fear learning and memory processes. Adeno-associated virus (AAV) vector-mediated RNA interference (RNAi) was infused into hippocampus to block hippocampal SRC-1 level. Immunofluorescent staining was used to detect the efficiency of transfection. High plus maze and open field test were used to determine anxiety and locomotor activity. Western blot analyses were used to detect the expression of SRC-1 and synaptic proteins in the hippocampus. Key findingsWe first showed that the expression of SRC-1 was regulated by fear conditioning training in a time-dependent manner, and knockdown of SRC-1 impaired contextual fear memory consolidation without affecting innate anxiety or locomotor activity. In addition, hippocampal SRC-1 was also regulated by the retrieval of contextual fear memory, and downregulation of SRC-1 disrupted fear memory reconsolidation. Moreover, knockdown of SRC-1 reversed the increased GluR1 and PSD-95 levels induced by contextual fear memory retrieval. SignificanceOur data indicate that hippocampal SRC-1 is required for the consolidation and reconsolidation of contextual fear memory, and SRC-1 may be a potential therapeutic target for mental disorders that are involved in hippocampal memory dysfunction.

Down-regulation of CD19 expression inhibits proliferation, adhesion, migration and invasion and promotes apoptosis and the efficacy of chemotherapeutic agents and imatinib in SUP-B15 cells

The survival rate of childhood acute lymphoblastic leukemia (ALL) has increased while that of Philadelphia-positive (Ph+) ALL remains low. CD19 is a B-cell specific molecule related to the survival and proliferation of normal B cells. However, there is little information available on the effects of CD19 on the biological behavior of Ph+ ALL cells. In this study, we explored a lentiviral vector-mediated short hairpin RNA (shRNA) expression vector to stably reduce CD19 expression in Ph+ ALL cell line SUP-B15 cells and investigated the effects of CD19 downregulation on cell proliferation, apoptosis, drug sensitivity, cell adhesion, cell migration and cell invasion in vitro. CD19 mRNA and protein expression levels were inhibited significantly by CD19 shRNA. Down-regulation of CD19 could inhibit cell proliferation, adhesion, migration and invasion, and increase cell apoptosis and the efficacy of chemotherapeutic agents and imatinib in SUP-B15 cells. Moreover, we found that down-regulation of CD19 expression inhibits cell proliferation and induces apoptosis in SUP-B15 cells in a p53-dependent manner. Taken together, our results suggest that lentiviral vector-mediated RNA interference of CD19 gene may be a promising strategy in the treatment of Ph+ ALL.

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.

Inhibiting PHD2 in bone marrow mesenchymal stem cells via lentiviral vector-mediated RNA interference facilitates the repair of periodontal tissue defects in SD rats.

Hypoxia-inducible factors (HIFs) play an important role in angiogenesis, and they can activate the expression of several downstream angiogenic factors. HIF-1 is a major transcriptor of HIFs, composed of α and β subunits. Prolyl hydroxylase domain-containing protein 2 (PHD2) is the main catabolic enzyme for HIF-1α, and it can accelerate its degradation under normoxic conditions. PHD2 expression in bone marrow mesenchymal stem cells (BMMSCs) of SD rats was down-regulated under normoxic conditions in this study by utilizing lentiviral vector-mediated RNA interference to promote HIF-1α accumulation, thus enhancing the expression of angiogenic factors. A tissue-engineered compound was constructed using the composite collagen membrane of BMMSCs after PHD2 gene silencing to repair periodontal fenestration defects in SD rats. The results of this study indicated that, after PHD2 gene silencing, the osteogenic differentiation of BMMSCs was enhanced in vitro, the resistance of cells to oxidative stress was also validated in vitro, thereby illustrating the promotion of the repair of artificially constructed periodontal tissue defects in rats. The results of this study provide a reference and guidance for future applications of RNA interference in periodontal tissue engineering and serve as a basis for improving the survival of seed cells in recipient tissues.

Herpes simplex virus-mediated RNA interference targeting vesicular glutamate transporter 3 attenuates tactile allodynia in mice

To construct a replication-deficient herpes simplex virus (HSV-1) for delivering a short hairpin RNA (shRNA) targeting vesicular glutamate transporter 3 (VGLUT3) and observe its effect in alleviating allodynia in mice. The recombinant HSV-1 vector carrying the shRNA targeting Vglut3 (HSV-1-shvglut3) was constructed and inoculated in the sciatic nerve in a mouse model of mechanical allodynia to test its analgesia effect. Mechanical allodynia and heat hypersensitivity of the mice were tested by von Frey filaments and Hargreaves' test, respectively. VGLUT3 expression in the dorsal root ganglion (DRG) was evaluated by immunohistochemistry and Western blotting. Following inoculation in the sciatic nerve, the HSV vector HSV-1-shvglut3 was retrogradely transported to the DRG. Mechanical withdraw thresholds of the mouse models receiving HSV-1-shvglut3 inoculation were reversed to nearly the baseline level, and VGLUT3 expression in the DRG was down-regulated 2 weeks after vector inoculation. The analgesic effect lasted for over 2 weeks in these mice without obvious systematic side effects or changes in heat hypersensitivity threshold. Vglut3 in the DRG is a promising therapeutic target for alleviating mechanical allodynia, and HSV-1 vector-mediated RNA interference is safe and efficient for inducing long-lasting analgesia after peripheral inoculation of the vector.

Enhancing chemosensitivity in oral squamous cell carcinoma by lentivirus vector-mediated RNA interference targeting EGFR and MRP2

Oral cancer is the eighth most common type of cancer among men worldwide, with an age-standardized rate of 6.3 per 100,000, and is the fourth leading cause of cancer-associated mortality among men in Taiwan. Cisplatin and 5-fluorouracil (5-FU) are two of the most frequently utilized chemotherapy drugs for the treatment of oral cancer. Although oral cancer patients initially benefit from chemotherapy with these drugs, they may develop resistance to them, which worsens their prognosis and reduces survival rates. It has been reported that increased levels of epidermal growth factor receptor (EGFR) and multidrug resistance-associated protein 2 (MRP2) induce drug resistance in numerous types of human cancer. Therefore, the present study employed lentivirus vector-mediated RNA interference (RNAi) in order to target the genes encoding EGFR and MRP2 in the oral squamous cell carcinoma cell line OC2. It was observed that RNAi-mediated downregulation of EGFR or MRP2 increased the sensitivity to 5-FU and cisplatin in OC2 cells. Downregulation of EGFR resulted in significant suppression of OC2 tumor growth following 5-FU administration. However, simultaneous downregulation of the two genes did not further suppress the tumor growth, indicating that MRP2 does not have a significant role in the chemosensitivity of EGFR-downregulated cells to 5-FU. In contrast, downregulation of MRP2 was demonstrated to significantly enhance the therapeutic effects of cisplatin in EGFR-downregulated OC2 tumors. The observation that the expression of MRP2 was positively correlated with the level of cisplatin resistance in cells suggests that RNAi-mediated downregulation of MRP2 may be applicable as a therapeutic approach toward reversing MRP2-dependent cisplatin resistance in oral cancer.

Non-viral Vector Mediated RNA Interference Technology for Central Nerve System Injury.

Neuronal axons damaged by traumatic injury are unable to spontaneously regenerate in the mammalian adult central nervous system (CNS), causing permanent motor, sensory, and cognitive deficits. Regenerative failure in the adult CNS results from a complex pathology presenting multiple barriers, both the presence of growth inhibitors in the extrinsic microenvironment and intrinsic deficiencies in neuronal biochemistry, to axonal regeneration and functional recovery. There are many strategies for axonal regeneration after CNS injury including antagonism of growth-inhibitory molecules and their receptors, manipulation of cyclic nucleotide levels, and delivery of growth-promoting stimuli through cell transplantation and neurotrophic factor delivery. While all these approaches have achieved varying degrees of improvement in plasticity, regeneration, and function, there is no clinically effective therapy for CNS injury. RNA interference technology offers strategies for improving regeneration by overcoming the aspects of the injured CNS environment that inhibit neurite growth. This occurs through the knockdown of growth-inhibitory molecules and their receptors. In this review, we discuss the current state of RNAi strategies for the treatment of CNS injury based on non-viral vector mediated delivery.

CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

Lentiviral Vector Mediated Claudin1 Silencing Inhibits Epithelial to Mesenchymal Transition in Breast Cancer Cells.

Breast cancer has a high incidence and mortality rate worldwide. Several viral vectors including lentiviral, adenoviral and adeno-associated viral vectors have been used in gene therapy for various forms of human cancer, and have shown promising effects in controlling tumor development. Claudin1 (CLDN1) is a member of the tetraspan transmembrane protein family that plays a major role in tight junctions and is associated with tumor metastasis. However, the role of CLDN1 in breast cancer is largely unexplored. In this study, we tested the therapeutic potential of silencing CLDN1 expression in two breast cancer (MDA-MB-231 and MCF7) cell lines using lentiviral vector mediated RNA interference. We found that a CLDN1 short hairpin (shRNA) construct efficiently silenced CLDN1 expression in both breast cancer cell lines, and CLDN1 knockdown resulted in reduced cell proliferation, survival, migration and invasion. Furthermore, silencing CLDN1 inhibited epithelial to mesenchymal transition (EMT) by upregulating the epithelial cell marker, E-cadherin, and downregulating mesenchymal markers, smooth muscle cell alpha-actin (SMA) and Snai2. Our data demonstrated that lentiviral vector mediated CLDN1 RNA interference has great potential in breast cancer gene therapy by inhibiting EMT and controlling tumor cell growth.

The effect of lentiviral vector-mediated RNA interference targeting hypoxia-inducible factor 1α on the uptake of fluorodeoxyglucose ((18)f) in the human pancreatic cancer cell line, patu8988.

Hypoxia can stimulate (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in cultured tumor cells. This study has investigated the effect of lentiviral vector-mediated RNA interference (RNAi) targeting hypoxia-inducible factor 1α (HIF-1α) on the changes in HIF-1 and glucose transporter 1 (Glut-1) expression, the cell growth, and the uptake of (18)F-FDG in the human pancreatic cancer cell line, Patu8988. Lentiviral RNAi vector targeting the HIF-1α gene (LV-HIF-1αRNAi) was constructed and used to treat cells at various concentrations (25-200 nM). The expression changes of HIF-1α and Glut-1 in hypoxic Patu8988 cells after RNAi treatment were determined using real time reverse transcription-polymerase chain reaction (real-time PCR). The inhibition rate of cell proliferation 48 hours after the addition of 10 μL of different concentrations of LV-HIF-1αRNAi (25-200 nM) was assayed using the MTT method. Meanwhile, the cell uptake of (18)F-FDG was also assessed. After RNAi transfection, the relative expression levels of HIF-1α mRNA and Glut-1 under hypoxia were reduced and the relative expression levels of HIF-1α protein also decreased. Compared with the control group, the inhibition rates of cell proliferation under different viral dosages were 5.98%, 15.65%, 26.42%, and 40.81%, respectively, positively correlated with the viral doses (r=0.558, p<0.05). Under hypoxia, Glut-1 mRNA expression in Patu8988 cells treated with 200 nM of LV-HIF-1αRNAi for 24, 48, and 72 hours, respectively, was positively correlated with the inhibition rate of cell proliferation (r=0.618, p<0.05) as well as the inhibition rate of (18)F-FDG uptake (r=0.664, p<0.05), while the latter two displayed a positive correlation with each other too (r=0.582, p<0.05). Under hypoxia, RNAi targeting HIF-1α significantly inhibited the expression of Glut-1 mRNA in Patu8988 pancreatic cancer cells and their uptake of (18)F-FDG. These results suggest that LV-HIF-1αRNAi may form a new treatment for pancreatic cancer, and the effectiveness of the treatment can be readily assessed with (18)F-FDG imaging.

Establishment of stable MRP1 knockdown by lentivirus-delivered shRNA in the mouse testis Sertoli TM4 cell line

Sertoli cells around germ cells are considered a barrier that protects spermatogenesis from harmful influences. The transporter multidrug-resistance-associated protein 1 (MRP1) is a xenobiotic efflux pump that can export glutathione S-conjugated metabolites and xenobiotics from cells. In this study, the Mrp1 gene was stably knocked down in a mouse Sertoli cell line (TM4) using lentivirus vector-mediated RNA interference (RNAi) technology. Four shRNA interference sequences were chosen and designed to screen for the most effective shRNA in candidate cells. The results indicate that lentivirus vectors with high titres were generated and successfully transfected into TM4 cells with high efficiency. Puromycin was added to the culture medium to maintain constant selection during the establishment of the stable cell lines. The expression levels of Mrp1 mRNA and MRP1 protein in stably transfected TM4 cells were significantly lower than those in the control group. Importantly, the transport activity of MRP1 to Calcein and 5-carboxyseminaptharhodafluor (SNARF-1) were significantly reduced because of MRP1 silencing. Moreover, the silencing of the Mrp1 gene in the transfected TM4 cell lines remained highly stable for more than 6 months. These results suggest that the lentivirus-based RNAi stably knocks down the expression of the Mrp1 gene in the established TM4 cell line. This transfected TM4 cell line will provide a new and powerful tool to study the underlying mechanism of MRP1-mediated drug resistance and detoxication in the reproductive system.