AIM: To explore the possibility of reversing multi-drug resistance (MDR) to HepG2/mdr1 in vitro and in vivo with RNA interference (RNAi).
METHODS: HepG2/mdr1 was obtained by cloning the whole gene mdr1 into HepG2 cells. shRNA targeting sequence was designed to be homologous to the P-gp encoding MDR1 mRNA consensus sequence. pSUPER-shRNA/mdr1 was constructed using the enzyme-digested technique. HepG2/mdr1 cells were transfected with vectors of pSUPER-shRNA/mdr1 to measure their efficacy by real-time PCR for mdr1 mRNA, flow cytometry (FCM) for P-gp expression, and Rhodamine efflux, MTT method for HepG2/mdr1 function, respectively. In vivo, mice tumors were treated by injecting pSUPER-shRNA/mdr1 in situ and into intra-abdominal cavity. Tumors were collected to create cell suspension and cryosections after chemothearpy with adiramycin and mytomycin. The cell suspension was incubated in RPMI-1640 supplemented with G418 to screen stable cells for appreciating the reversal of MDR. Cryosections were treated with immunohistochemistry technique to show the effectiveness of transfection and the expression of P-gp.
RESULTS: pSUPER-shRNA/mdr1 was successfully constructed, which was confirmed by sequencing. The MDR phenotype of HepG2/mdr1 was decreased significantly in vitro transfection. HepG2/mdr1 showing its MDR was reversed notably in P-gp expression (11.0% vs 98.2%, P < 0.01). Real-time PCR showed that mRNA/mdr1 was lower in test groups than in control groups (18.73 ± 1.33 vs 68.03 ± 2.21, P < 0.001). Compared with HepG2, the sensitivity of HepG2/mdr1 and HepG2/mdr1-dsRNA cells to ADM was decreased by 1.64 times and 15.6 times, respectively. The accumulation of DNR in positive groups was decreased evidently. In vivo, the p-gp expression in positive groups was significantly lower than that in control groups (65.1% vs 94.1%, P < 0.05). The tumor suppressing rate in test groups was 57.8%. After chemotherapy, the growth rate in test groups was lower than that in control groups (700.14 ± 35.61 vs 1659.70 ± 152.54, P < 0.05). Similar results were also observed under fluorescence microscope, and confirmed by Image-Pro Plus 4.5 analysis.
CONCLUSION: pSUPER-shRNA/mdr1 vector system allows simple, stable and durable nonviral knockdown of P-gp by RNAi in malignant cells and animals to restore their sensitivity to adriamycin.
Hepatocellular carcinoma; Multi-drug resistance; RNA interference
Drug resistance caused by overexpression of P-glycoprotein (P-gp), the MDR1 (ABCB1) gene product, limits the therapeutic outcome. Expression of MDR1 can be induced by divergent stimuli, and involves a number of transcriptional factors. We found that the expression of CtBP1 (C-terminal-binding protein 1), a transcriptional co-regulator, was increased (~4 – fold) in human multidrug resistant (MDR) cancer cell lines, NCI/ADR-RES and A2780/DX, as compared to their sensitive counterparts. Silencing of CtBP1 expression by RNAi decreased the MDR1 mRNA and P-gp. Knockdown of CtBP1 also enhanced the sensitivity of MDR cells to chemotherapeutic drugs that are transported by P-gp and increased intracellular drug accumulation. In a reporter gene assay, co-transfection of MDR1 promoter constructs with a CtBP1 expression vector resulted in a ~2–4-fold induction of MDR1 promoter activity. CtBP1 appeared to contribute to the activation of MDR1 transcription through directly interacting with the MDR1 promoter, as evidenced by its physical binding to the promoter region of the MDR1 gene in chromatin immunoprecipitation and electromobility shift assays. Histone modifications at the MDR1 promoter, such as mono-methylation, di-methylation, and acetylation of histone H3, were not found to be affected by silencing of CtBP1 expression. Our results reveal a novel role for CtBP1 as an activator of MDR1 gene transcription, and suggest that CtBP1 might be one of the key transcription factors involved in the induction of MDR1 gene. Therefore, CtBP1 may represent a potentially new target for inhibiting drug resistance mediated by overexpression of the MDR1 gene.
Multidrug resistance; MDR1 gene; P-glycoprotein; CtBP1; transcription; cancer
RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.
RNAi-mediated knockdown of gene expression in target cells, controlled by a modified Tet-repressor (TetR) in the presence of doxycycline (Dox) was robust. Expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or an improved second-generation Tet-responsive promoter element (TRE) placed upstream of the promoter was tight and reversible as judged using quantitative protein measurements. We also established and tested a novel hU6 promoter system in which the distal sequence element (DSE) of the hU6 promoter was replaced with a second-generation TRE. In this system, positive regulation of shRNA production is mediated by novel Tet-dependent transactivators bearing transactivation domains derived from the human Sp1 transcription factor.
Our modified lentiviral vector system resulted in tight and reversible knockdown of target gene expression in unsorted cell populations. Tightly regulated target gene knockdown was observed with vectors containing either a single TO sequence or a second-generation TRE using carefully controlled transduction conditions. We expect these vectors to ultimately find applications for tight and reversible RNAi in mammalian cells in vivo.
RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells.
The limiting factor in in vivo RNA interference (RNAi) is delivery. Drug delivery methods that are effective in cell culture may not be practical in vivo for intravenous RNAi applications. Nucleic acid drugs are highly charged and do not cross cell membranes by free diffusion. Therefore, the in vivo delivery of RNAi-therapeutics must using targeting technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. For RNAi of the brain, the nucleic acid-based drug must first cross the brain capillary endothelial wall, which forms the blood-brain barrier (BBB) in vivo, and then traverse the brain cell plasma membrane. Similar to the delivery of non-viral gene therapies, plasmid DNA encoding for short hairpin RNA (shRNA) may be delivered to brain following intravenous administration with pegylated immunoliposomes (PILs). The plasmid DNA is encapsulated in a 100 nm liposome, which is pegylated, and conjugated with receptor specific targeting monoclonal antibodies (MAb). Weekly, intravenous RNAi with PILs enables a 90% knockdown of the human epidermal growth factor receptor, which results in a 90% increase in survival time in mice with intra-cranial brain cancer. Similar to the delivery of antisense agents, short interfering RNAi (siRNA) duplexes can be delivered with the combined use of targeting MAb’s and avidin-biotin technology. The siRNA is mono-biotinylated in parallel with the production of a conjugate of the targeting MAb and streptavidin. Intravenous RNAi requires the combined use of RNAi technology and a drug targeting technology that is effective in vivo.
blood-brain barrier; RNAi; endothelium; transferrin receptor; insulin receptor; monoclonal antibody
Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication.
Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively.
Co-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group.
Lentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.
ATP-binding cassette (ABC) proteins include the best known mediators of resistance to anticancer drugs. In particular, ABCB1 (MDR1/P-gp) extrudes many types of drugs from cancer cells, thereby conferring resistance to those agents. Attempts to overcome P-gp-mediated drug resistance using specific inhibitors of P-gp has had limited success, and has faced many therapeutic challenges. As an alternative approach to using P-gp inhibitors, we characterize a thiosemicarbazone derivative (NSC73306) identified in a generic screen as a compound that exploits, rather than suppresses, P-gp function to induce cytotoxicity. Cytotoxic activity of NSC73306 was evaluated in vitro using human epidermoid, ovarian, and colon cancer cell lines expressing various levels of P-gp. Our findings suggest that cells become hypersensitive to NSC73306 in proportion to the increased P-gp function and multidrug resistance (MDR). Abrogation of both sensitivity to NSC73306 and resistance to P-gp substrate anticancer agents occurred with specific inhibition of P-gp function using either a P-gp inhibitor (PSC833, XR9576) or RNA interference (RNAi), suggesting that cytotoxicity was linked to MDR1 function, not to other, nonspecific factors arising during the generation of resistant or transfected cells. Molecular characterization of cells selected for resistance to NSC73306 revealed loss of P-gp expression and consequent loss of the MDR phenotype. Although hypersensitivity to NSC73306 required functional expression of P-gp, biochemical assays revealed no direct interaction between NSC73306 and P-gp. This work demonstrates that NSC73306 kills cells with intrinsic or acquired P-gp-induced MDR and indirectly acts to eliminate resistance to MDR1 substrates.
multidrug resistance (MDR); P-glycoprotein; inhibitors; cancer; hypersensitivity; thiosemicarbazones; NCI 60; targeted therapy
RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death.
As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.
Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.
Apatinib, a small-molecule multi-targeted tyrosine kinase inhibitor, is in phase III clinical trial for treatment of patients with non-small cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr and HEK293/ABCB1 cells overexpressing ABCB1 and S1-M1-80, MCF-7/FLV1000 and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photolabeling of both ABCB1 and ABCG2 with [125I]-iodoarylazidoprazosin in a concentration-dependent fashion. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration the produced a reversal of MDRl, did not significantly alter the expression of the ABCB1 or ABCG2 protein or mRNA levels or the phosphorylation of AKT and ERK1/2. Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1 resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs.
Apatinib; Multidrug resistance; ATP-binding cassette transporters; P-glycoprotein; ABCG2; Xenograft
Aim: To construct short hairpin RNAs (shRNAs) and miR30-based shRNAs against heparanase (HPSE) to compare their safety and their effects on HPSE down-modulation in vitro and in vivo to develop a more ideal therapeutic RNA interference (RNAi) vector targeting HPSE.
Methods: First, we constructed shRNAs and miR30-based shRNAs against HPSE (HPSE-shRNAs and HPSE-miRNAs) and packed them into lentiviral vectors. Next, we observed the effects of the shRNAs on knockdown for HPSE expression, adhesion, migration and invasion abilities in human malignant melanoma A375 cells in vitro. Furthermore, we compared the effects of the shRNAs on melanoma growth, metastasis and safety in xenograft models.
Results: Our data showed that these artificial miRNAs targeting HPSE could be effective RNAi agents mediated by Pol II promoters in vitro and in vivo, although these miRNAs were not more potent than the HPSE-shRNAs. It was noted that obvious lung injuries, rarely revealed previously, as well as hepatotoxicity could be caused by lentivirus-mediated shRNAs (LV shRNAs) rather than lentivirus-mediated miRNAs (LV miRNAs) in vivo. Furthermore, enhanced expression of pro-inflammatory cytokines IL-6 and TGF-β1 and endogenous mmu-miR-21a-5p were detected in lung tissues of shRNAs groups, whereas the expression of mmu-let-7a-5p, mmu-let-7b-5p and mmu-let-7c-5p were down-regulated.
Conclusion: These findings suggest that artificial miRNAs display an improved safety profile of lowered lung injury or hepatotoxicity relative to shRNAs in vivo. The mechanism of lung injuries caused by shRNAs may be correlated with changes of endogenous miRNAs in the lung. Our data here increase the flexibility of a miRNA-based RNAi system for functional genomic and gene therapy applications.
RNA interference; microRNA(miRNA); heparanase; metastasis; safety
Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters through efflux of antineoplastic agents from cancer cells is a major obstacle to successful cancer chemotherapy. The inhibition of these ABC transporters is thus a logical approach to circumvent MDR. There has been intensive research effort to design and develop novel inhibitors for the ABC transporters to achieve this goal. In the present study, we evaluated the ability of UMMS-4 to modulate P-glycoprotein (P-gp/ABCB1)-, breast cancer resistance protein (BCRP/ABCG2)- and multidrug resistance protein (MRP1/ABCC1)-mediated MDR in cancer cells. Our findings showed that UMMS-4, at non-cytotoxic concentrations, apparently circumvents resistance to ABCB1 substrate anticancer drugs in ABCB1-overexpressing cells. When used at a concentration of 20 μmol/L, UMMS-4 produced a 17.53-fold reversal of MDR, but showed no effect on the sensitivity of drug-sensitive parental cells. UMMS-4, however, did not significantly alter the sensitivity of non-ABCB1 substrates in all cells and was unable to reverse ABCG2- and ABCC1-mediated MDR. Additionally, UMMS-4 profoundly inhibited the transport of rhodamine 123 (Rho 123) and doxorubicin (Dox) by the ABCB1 transporter. Furthermore, UMMS-4 did not alter the expression of ABCB1 at the mRNA and protein levels. In addition, the results of ATPase assays showed that UMMS-4 stimulated the ATPase activity of ABCB1. Taken together, we conclude that UMMS-4 antagonizes ABCB1-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1. These findings may be useful for the development of safer and more effective MDR modulator.
UMMS-4; multidrug resistance; ATP binding cassette transporters; ABCB1; chemotherapeutic drugs
Gastric cancer is highly aggressive disease. Despite advances in diagnosis and therapy, the prognosis is still poor. Various genetic and molecular alterations are found in gastric cancer that underlies the malignant transformation of gastric mucosa during the multistep process of gastric cancer pathogenesis. The detailed mechanism of the gastric cancer development remains uncertain. In present study we investigated the potential role of stathmin1 gene in gastric cancer tumorigenesis and examined the usefulness of RNA interference (RNAi) targeting stathmin1 as a form of gastric cancer treatment.
A lentiviral vector encoding a short hairpin RNA (shRNA) targeted against stathmin1 was constructed and transfected into the packaging cells HEK 293 T and the viral supernatant was collected to transfect MKN-45 cells. The transwell chemotaxis assay and the CCK-8 assay were used to measure migration and proliferation of tumor cells, respectively. Quantitative real-time PCR and western blotting were used to detect the expression levels of stathmin1.
Lentivirus mediated RNAi effectively reduced stathmin1 expression in gastric cells. Significant decreases in stathmin1 mRNA and protein expression were detected in gastric cells carrying lentiviral stathmin-shRNA vector and also significantly inhibited the proliferation, migration in gastric cancer cells and tumorigenicity in Xenograft Animal Models.
Our findings suggest that stathmin1 overexpression is common in gastric cancer and may play a role in its pathogenesis. Lentivirus mediated RNAi effectively reduced stathmin1 expression in gastric cells. In summary, shRNA targeting of stathmin1 can effectively inhibits human gastric cancer cell growth in vivo and may be a potential therapeutic strategy for gastric cancer.
Stathmin1; Gastric cancer; Lentivirus; shRNA
AIM: To reverse the multidrug resistance (MDR) by RNA interference (RNAi)-mediated MDR1 suppression in hepatoma cells.
METHODS: For reversing MDR by RNAi technology, two different short hairpin RNAs (shRNAs) were designed and constructed into pGenSil-1 plasmid, respectively. They were then transfected into a highly adriamycin-resistant HepG2 hepatoma cell line (HepG2/ADM). The RNAi effect on MDR was evaluated by real-time PCR, cell cytotoxicity assay and rhodamine 123 (Rh123) efflux assy.
RESULTS: The stably-transfected clones showed various degrees of reversal of MDR phenotype. Surprisingly, the MDR phenotype was completely reversed in two transfected clones.
CONCLUSION: MDR can be reversed by the shRNA-mediated MDRI suppression in HepG2/ADM cells, which provides a valuable clue to make multidrug-resistant hepatoma cells sensitive to anti-cancer drugs.
Multidrug resistance; ShRNA; MDR1; Hepatocellular carcinoma
Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells.
Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter.
MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues.
Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors.
Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV.
To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting.
Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective.
This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials.
ABCB6 is a mitochondrial transporter that regulates porphyrin biosynthesis. ABCB6 expression is upregulated in hepatocellular carcinoma (HCC) but the significance of this upregulation to HCC is not known. In the present study, we investigated: 1) ABCB6 expression in 18 resected human hepatocellular carcinoma (HCC) tissues and 3 human hepatoma cell lines; 2) pattern of ABCB6 expression during liver disease progression; and 3) functional significance of ABCB6 expression to HCC using the hepatoma cell line Huh7. ABCB6 expression was determined by real-time quantitative reverse transcription-polymerase chain reaction and western blotting. ABCB6 expression was upregulated in all the HCC specimens and the three-hepatoma cell lines. Increased ABCB6 expression correlated with liver disease progression with the pattern of expression being HCC > cirrhosis > steatosis. Small hairpin RNA (shRNA)-mediated knockdown of ABCB6 in Huh7 cells lead to decreased cellular proliferation and colony formation. Attenuation of ABCB6 expression did not affect Huh7 apoptosis but lead to a delay in G2/M phase of the cell cycle. In contrast, ABCB6 overexpression resulted in increased growth and proliferation of Huh7 cells. Since ABCB6 expression is induced in multiple tumor types we explored the role of ABCB6 in other cancer cells. ShRNA mediated knockdown of ABCB6 in HEK293 and K562 cells reduced cellular proliferation leading to a delay in G2/M phase, while ABCB6 overexpression promoted cell growth and proliferation. Collectively, these findings, obtained by loss of function and gain of function analysis, suggest that ABCB6 plays a role in cell growth and proliferation by targeting the cell cycle.
ABC transporters; carcinoma; hepatocellular carcinoma; growth and proliferation
Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.
Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.
By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.
RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For HIV-1, both approaches resulted in profound inhibition of virus replication. However, the therapeutic use of a single siRNA/shRNA appears limited due to the rapid emergence of RNAi-resistant escape viruses. These variants contain deletions or point mutations within the target sequence that abolish the antiviral effect. To avoid escape from RNAi, the virus should be simultaneously targeted with multiple shRNAs. Alternatively, long hairpin RNAs can be used from which multiple effective siRNAs may be produced. In this study, we constructed extended shRNAs (e-shRNAs) that encode two effective siRNAs against conserved HIV-1 sequences. Activity assays and RNA processing analyses indicate that the positioning of the two siRNAs within the hairpin stem is critical for the generation of two functional siRNAs. E-shRNAs that are efficiently processed into two effective siRNAs showed better inhibition of virus production than the poorly processed e-shRNAs, without inducing the interferon response. These results provide building principles for the design of multi-siRNA hairpin constructs.
RNA interference (RNAi) is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium.
The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein). RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs) targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1) was used. Cell types, the efficiency of both transfections and RNAi were all evaluated.
Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low.
In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.
Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to
multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the
potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a
treatment for drug-resistant breast cancer and to investigate the effect of
CIAPIN1 on the drug resistance of breast cancer in
vivo. We used lentivirus-vector-based RNAi to knock down
CIAPIN1 in nude mice bearing MDR breast cancer tumors and found
that lentivirus-vector-mediated silencing of CIAPIN1 could
efficiently and significantly inhibit tumor growth when combined with chemotherapy
in vivo. Furthermore, Western blot analysis showed that both
CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was
upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi
targeting of CIAPIN1 is a potential approach to reverse MDR of
breast cancer. In addition, CIAPIN1 may participate in MDR of breast
cancer by regulating P-glycoprotein and P53 expression.
CIAPIN1 gene; Multidrug resistance; RNA interference; MDR1 gene; Breast neoplasms
RNA interference (RNAi) is a widely used gene suppression tool that holds great promise as a novel antiviral approach. However, for error-prone viruses including human immunodeficiency virus type 1(HIV-1), a combinatorial approach against multiple conserved sequences is required to prevent the emergence of RNAi-resistant escape viruses. Previously, we constructed extended short hairpin RNAs (e-shRNAs) that encode two potent small interfering RNAs (siRNAs) (e2-shRNAs). We showed that a minimal hairpin stem length of 43 base pairs (bp) is needed to obtain two functional siRNAs. In this study, we elaborated on the e2-shRNA design to make e-shRNAs encoding three or four antiviral siRNAs. We demonstrate that siRNA production and the antiviral effect is optimal for e3-shRNA of 66 bp. Further extension of the hairpin stem results in a loss of RNAi activity. The same was observed for long hairpin RNAs (lhRNAs) that target consecutive HIV-1 sequences. Importantly, we show that HIV-1 replication is durably inhibited in T cells stably transduced with a lentiviral vector containing the e3-shRNA expression cassette. These results show that e-shRNAs can be used as a combinatorial RNAi approach to target error-prone viruses.
ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [125I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.
ATP-binding-cassette family membrane proteins play an important role in multidrug resistance. In this study, we investigated BIRB796, an orally active inhibitor of p38 mitogen-activated protein kinase, reversed MDR induced by ABCB1, ABCG2 and ABCC1. Our results showed that BIRB796 could reverse ABCB1-mediated MDR in both the drug selected and transfected ABCB1-overexpressing cell models, but did not enhance the efficacy of substrate-chemotherapeutical agents in ABCC1 or ABCG2 overexpression cells and their parental sensitive cells. Furthermore, BIRB796 increased the intracellular accumulation of the ABCB1 substrates, such as rhodamine 123 and doxorubicin. Moreover, BIRB796 bidirectionally mediated the ATPase activity of ABCB1, stimulating at low concentration, inhibiting at high concentration. However, BIRB796 did not alter the expression of ABCB1 both at protein and mRNA level. The down-regulation of p38 by siRNA neither affected the expression of ABCB1 nor the cytotoxic effect of paclitaxel on KBV200. The binding model of BIRB796 within the large cavity of the transmembrane region of ABCB1 may form the basis for future lead optimization studies. Importantly, BIRB796 also enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBV200 cell xenografts in nude mice. Overall, we conclude that BIRB796 reverses ABCB1-mediated MDR by directly inhibiting its transport function. These findings may be useful for cancer combinational therapy with BIRB796 in the clinic.
RNA interference is a post transcriptional gene silencing mechanism that is triggered by double-stranded RNA (dsRNA). Various attributes of the 3′ end structure, including overhang length and sequence composition, plays a primary role in determining the position of the Dicer cleavage in both dsRNA and unimolecular, short hairpin RNA (shRNA). The specificity and robustness of RNAi have triggered an immense interest in using RNAi as a tool in various settings. RNAi is a mechanism for controlling normal gene expression which has recently began to be employed as a potential therapeutic agent for a wide range of disorders, including cancer, infectious diseases and metabolic disorders. Clinical trials with RNAi have now begin, but major obstacles, such as off-target effects, toxicity and unsafe delivery methods, have to be overcome before RNAi can be considered as a conventional drug. It is also used as a tool to improve crops by providing resistance against parasites and modified versions of siRNA that are directed against disease causing genes are being developed, some of which are already tested in clinical trials. In this paper, we first reviewed the RNAi mechanism and then focussed on some of its applications in biomedical research such as treatment for HIV, viral hepatitis and several other diseases.
SiRNAs, small interfering RNAs; RISC, RNA induced silencing complex; MiRNAs, microRNAs; RNAi; Gene silencing; Antiviral; Dicer
Therapeutic RNA interference has emerged as a promising approach for the treatment of many incurable diseases, including cancer, infectious disease or neurodegenerative disorders. Demonstration of efficacy and safety in animal models is necessary before planning human application. Our group and others have previously shown the potential of this approach for the dominantly-inherited neurological disease DYT1 dystonia by achieving potent shRNA-mediated silencing of the disease protein, torsinA, in cultured cells. To establish the feasibility of this approach in vivo, we pursued viral delivery of shRNA in two different mouse models. Surprisingly, intrastriatal injections of AAV2/1 vectors expressing different shRNAs, whether targeting torsinA expression or mismatched controls, resulted in significant toxicity with progressive weight loss, motor dysfunction and animal demise. Histological analysis showed shRNA-induced neurodegeneration. Toxicity was not observed in animals that received control AAV2/1 encoding no shRNA, and was independent of genotype, occurring in both DYT1 and wild type animals. Interestingly, the different genetic background of both mouse models influenced toxicity, being earlier and more severe in 129/SvEv than C57BL/6 mice. In conclusion, our studies demonstrate that expression of shRNA in the mammalian brain can lead to lethal toxicity. Furthermore, the genetic background of rodents modifies their sensitivity to this form of toxicity, a factor that should be taken into consideration in the design of preclinical therapeutic RNAi trials.
RNAi; AAV; DYT1 dystonia; torsinA; TOR1A