Short Hairpin RNA Expression Vector Research Articles

Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer

Background and aims:Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification...

Application of RNAi Technology to the Inhibition of Zebrafish GtHα, FSHβ, and LHβ Expression and to Functional Analyses

Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHalpha, common alpha), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHalpha RNAi expression compared with the other two subunits. A GtHalpha morpholino analysis showed that the GtHalpha morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHalpha -shRNA. Taken together, these results show that GtHalpha-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.

Long-Term Suppression of Hepatitis B Virus Replication by Short Hairpin RNA Expression Using the Scaffold/Matrix Attachment Region-Based Replicating Vector System pEPI-1

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.

TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome‐associated protein NA14

TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sjögren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14DeltaN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40DeltaC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DeltaN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.

Effect of blocking VEGF, hTERT and Bcl-xl by multiple shRNA expression vectors on the human laryngeal squamous carcinoma xenograft in nude mice

In our previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human telomerase reverse transcriptase (hTERT) messenger RNA, and we demonstrated that these vectors could inhibit the growth of cancer cells by 76.5% in vivo. In order to maximize the efficiency and versatility of the vector-based siRNA approach, here we have constructed multiple shRNA expression vectors that simultaneously targeted 3 different genes in cancer cells, and then investigated their effect on human laryngeal squamous carcinoma (Hep-2) in vivo. Materials and methods Short hairpin RNA expression vectors targeting the mRNA of VEGF, hTERT and Bcl-xl were constructed and subsequently transfected by direct injections into the tumors formed by Hep-2 cells implanted in nude mice. The expression of targeted genes as well as apoptosis of tumor cells were evaluated after treatment with multiple shRNA vectors or control vectors. Results We found that expression of multiple shRNAs led to a significant reduction in VEGF, hTERT and Bcl-xl RNA and protein expression. Tumor growth curves showed that those tumors treated with the shRNAs were obviously smaller than control, non-transfected tumors. Analysis at 14 days following the final injection showed a tumor inhibition ratio of 91.2%. However, the control shRNA vectors showed none of these effects. Conclusions Our results demonstrate that the application of vector-based RNAi technology that involves blocking multiple targets will be a promising therapeutic modality in the gene therapy of human cancers. The data strongly suggest that simultaneously blocking multiple genes in human cancers using an RNAi approach should be considered in cancer therapy.

Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.

Reduction of AHSP synthesis in hemin-induced K562 cells and EPO-induced CD34 + cells leads to α-globin precipitation, impairment of normal hemoglobin production, and increased cell death

alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression. Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains. RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

Gankyrin oncoprotein overexpression as a critical factor for tumor growth in human esophageal squamous cell carcinoma and its clinical significance

To elucidate the possible involvement of gankyrin in ESCC progression and the effect of its down-regulation in ESCC, we investigated the expression of gankyrin in ESCC tissues comparing it with the corresponding normal esophageal epithelia and tested a short-hairpin RNA (shRNA) expression vector for gankyrin in ESCC cell lines. Gankyrin protein expression in 11 ESCC cell lines (KYSE series) was examined by RT-PCR and western blot. The expression of gankyrin mRNA in 30 ESCC tissues was compared with the corresponding normal epithelia by Real-time PCR. Expression of gankyrin protein was immunohistochemically analyzed in the ESCC of 103 patients. A gankyrin-shRNA vector was stably transfected into KYSE 170 cells to assess the role of gankyrin in cell motility, invasion and proliferation in vitro and tumor formation in vivo. Gankyrin expression increased in all 11 ESCC cell lines. Real-time PCR revealed that gankyrin expression was higher in the cancerous tissue for all 30 patients. In immunohistochemistry, gankyrin overexpression was correlated with lower survival rate (p = 0.0001), extent of the primary tumor, lymph node metastasis, distant lymph node metastasis and stage (p = 0.0072, p = 0.0004, p = 0.0172 and p = 0.0002, respectively). A shRNA vector against gankyrin repressed growth, cell motility, invasiveness in vitro and tumor formation in vivo. Gankyrin overexpression is associated with poor prognosis. It may play an important role in ESCC tumor progression and could be a potentially important therapeutic gene target in ESCC.

C-Myc depletion inhibits proliferation of human tumor cells at various stages of the cell cycle

A major role for c-Myc in the proliferation of normal cells is attributed to its ability to promote progression through G(1) and into S phase of the cell cycle. The absolute requirement of c-Myc for cell cycle progression in human tumor cells has not been comprehensively addressed. In the present work, we used a lentiviral-based short hairpin RNA (shRNA) expression vector to stably reduce c-Myc expression in a large number of human tumor cell lines and in three different types of normal human cells. In all cases, cell proliferation was severely inhibited, with normal cells ultimately undergoing G(0)/G(1) growth arrest. In contrast, tumor cells demonstrated a much more variable cell cycle response with cells from several lines accumulating in S or G(2)/M phases. Moreover, in some tumor lines, the phase of cell cycle arrest caused by inhibition of c-Myc could be altered by depleting tumor suppressor protein p53 or its transcriptional target p21(CIP/WAF). Our data suggest that, as in the case of normal cells, c-Myc is essential for sustaining proliferation of human tumor cells. However its rate-limiting role in cell cycle control is variable and is reliant upon the status of other cell cycle regulators.

Selection and Inhibitory Effect Analysis of siRNAs Specific to ORF2–4 of Porcine Reproductive and Respiratory Syndrome Virus

RNA interference (RNAi) is a powerful tool for gene function research. In order to investigate the role of GP2, GP3 and GP4 of porcine reproductive and respiratory syndrome virus (PRRSV) in the viral replication, small interference RNAs (siRNAs) directed to ORF2, ORF3 and ORF4 were designed and 12 short hairpin RNA (shRNA) expression vectors were constructed (designated as 21, 22, 23, 24, 31, 32, 33, 34, 41, 42, 43 and 44). Cells treated with shRNA expression vectors were infected by PRRSV. The effective shRNA expression vectors were selected by cytopathic effect (CPE) and fluorescence quantitative PCR (FQ-PCR). The virus titer of culture supernatant of cells treated with effective shRNA expression vectors (23, 24, 31, 34 and 41) were reduced by 184 to 4.65 folds to compare with that of controls.

Malignant phenotype of PC3 cell line was inhibited by siRNA targeting PAR gene

To investigate the effects of down-regulation of prostate androgen regulated (PAR) expression on proliferation of PC3 cells by using RNA interference (RNAi), suppression of PAR expression was achieved by transfection of PC3 cells with short hairpin RNA (shRNA) expression vectors against PAR, designated as psiRNA-PAR1, psiRNA-PAR2 and psiRNA-PAR3. The inhibitory effects were confirmed by RT-PCR. The growth features of PC3 transfectants were analyzed by cell counts, colon formation in soft agar and flow cytometry. The expression of PAR was suppressed by the three shRNA expression vectors. psiRNA-PAR1 was shown to inhibit the PAR expression most efficiently, with the inhibitory rate reaching a peak at (81.18+/-1.68)% 48 h after the transfection. PC3 transfectants exhibited a decreased proliferation in cell culture and a low efficiency of colon formation in soft agar. Flow cytometry revealed a G(2)/M arrest and induced apoptosis. Down-regulated PAR expression inhibited the growth of PC3 cells by inducing G(2)/M arrest and activating apoptotic pathway. As a potential proto-oncogene that triggers and/or has persistent malignant proliferation, PAR may serves as a very target for the gene therapy.

The comparative and functional study between two construction methods of shRNA expression vector targeted LMP1 gene encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.

Crucial role of Bysl in mammalian preimplantation development as an integral factor for 40S ribosome biogenesis.

Blastocyst formation during mammalian preimplantation development is a unique developmental process that involves lineage segregation between the inner cell mass and the trophectoderm. To elucidate the molecular mechanisms underlying blastocyst formation, we have functionally screened a subset of preimplantation embryo-associated transcripts by using small interfering RNA (siRNA) and identified Bysl (bystin-like) as an essential gene for this process. The development of embryos injected with Bysl siRNA was arrested just prior to blastocyst formation, resulting in a defect in trophectoderm differentiation. Silencing of Bysl by using an episomal short hairpin RNA expression vector inhibited proliferation of embryonic stem cells. Exogenously expressed Bysl tagged with a fluorescent protein was concentrated in the nucleolus with a diffuse nucleoplasmic distribution. Furthermore, the loss of Bysl function by using RNA interference or dominant negative mutants caused defects in 40S ribosomal subunit biogenesis. These findings provide evidence for a crucial role of Bysl as an integral factor for ribosome biogenesis and suggest a critical dependence of blastocyst formation on active translation machinery.

Attenuation of Adipocyte Triacylglycerol Hydrolase Activity Decreases Basal Fatty Acid Efflux

Fatty acids released from adipose triacylglycerol stores by lipolysis provide vertebrates with an important source of energy. We investigated the role of microsomal triacylglycerol hydrolase (TGH) in the mobilization of adipocyte triacylglycerols through inactivation of the TGH activity by RNA interference or chemical inhibition. Attenuation of TGH activity resulted in decreased basal but not isoproterenol-stimulated efflux of fatty acids from 3T3-L1 adipocytes. Lack of TGH activity was accompanied by accumulation of cellular triacylglycerols and cholesteryl esters without any changes in the expression of enzymes catalyzing triacylglycerol synthesis (diacylglycerol acyltransferases 1 and 2) or degradation (adipose triglyceride lipase and hormone-sensitive lipase). Inhibition of TGH-mediated lipolysis also did not affect insulin-stimulated Glut4 translocation from intracellular compartments to the plasma membrane or glucose uptake into adipocytes. These data suggest that TGH plays a role in adipose tissue triacylglycerol metabolism and may be a suitable pharmacological target for lowering fatty acid efflux from adipose tissue without altering glucose import.

Inhibition of Telomerase Activity in Cancer Cells using Short Hairpin RNA Expression Vectors

Telomerase activity is mainly regulated by the human telomerase reverse transcriptase (hTERT) gene. Our objective was to investigate the effect of short hairpin RNA (shRNA) directed against hTERT mRNA on telomerase activity in laryngeal cancer cells (Hep-2), nasopharyngeal carcinoma cells (NEC), and human bone marrow mesenchyme stem cells (hMSCs). Short hairpin RNA expression vectors targeting the messenger RNA of hTERT were constructed. Cells were treated with shRNA expression vectors directed against hTERT mRNA and control vectors that included mismatched shRNA. We found that treatment of special shRNA expression vectors induced significantly decrease in hTERT expression, telomerase activity, and cell viability in Hep-2 and NEC cells. In contrast, the shRNA control showed none of these effects. And none of these effects appeared in hMSCs cells. Our results suggest that shRNA against hTERT mRNA inhibits telomerase activity and cell viability through suppression of the hTERT expression in cancer cells. And this treatment has no side effect on healthy cells lack of telomerase activity. RNA interfering technology may be a promising strategy for the treatment of cancers.

Uniform amplification of multiple DNAs by emulsion PCR

When several DNAs are amplified by PCR in one PCR tube, biased amplification is known to occur because amplification efficiency differs from one DNA to another. Therefore, we conducted PCR in the water in oil-emulsion (W/O emulsion) to examine whether the procedure allows the uniform amplification of several DNAs. In the amplification of a model library consisting of two clones, the emulsification of the PCR mixture successfully reduced the difference in its amplification efficiency to approximately one-seventh the value obtained without emulsification. Furthermore, we conducted repeated PCR to amplify a model library consisting of ten short hairpin RNA (shRNA) expression vectors as a model experiment for gene discovery using an shRNA expression library. Consequently, the emulsification of the PCR mixture successfully reduced PCR bias. Our results indicate that emulsion PCR is capable of uniformly amplifying libraries of shRNA, ribozyme, cDNA, and others, and is useful also for gene discovery using these libraries.

Transmembrane Gla Protein 4 as a Novel Modulator of ERK2.

Transmembrane Gla protein 4 (TMG4) is a single pass, type I transmembrane protein with a vitamin-K dependent γ-carboxy-glutamic acid-rich domain (Kulman et al PNAS,2001). Bioinformatic analysis of the primary sequence indicates a very high degree of conservation (ca. 80%) among mammals and to-date no known functional roles have been assigned to this protein. In our efforts to elucidate molecular functions of TMG4, we first analyzed sub cellular localization of this protein. Microscopic analysis of HEK293T cells expressing a TMG4-GFP fusion construct showed juxtanuclear localization of the tagged protein at 24h post transfection. Staining with organelle specific fluorescent dyes revealed that the TMG4-GFP primarily localizes to the trans Golgi network in the cell. Recently, there have been increasing number of reports of golgi-localized proteins participating in compartmentalized signal transduction pathways and affecting cell differentiation and proliferation by activating major signaling cascades like the Ras-Raf/MAPK signaling cascades. Moreover, the transmembrane localization and the current lack of evidence for a direct role for TMG4 in the coagulation pathways encouraged us to hypothesize that TMG4 may play a role in signal transduction. To test this we generated a TMG4 over-expressing cell line (high-TMG4) by stably transfecting HEK293T cells with a CMV-promoter driven TMG4 plasmid , and a TMG4 knock-down cell line (low-TMG4) by stable transfections with a TMG4 short-hairpin RNA (shRNA) expression vector. The levels of TMG4 gene expression were confirmed in both the cell lines by RNA and protein expression analysis. The low-TMG cells showed >90% knockdown of TMG4 in comparison to untransfected control cells. The cells were grown under standard tissue culture conditions for 48h after which they were harvested. Total cell lysates from the cell lines were analyzed by western blotting using primary antibodies against a panel of signal transduction proteins representing major signaling pathways including threonine kinase, tyrosine kinase and ERK2. The results showed no significant changes in activation of signal transduction proteins tested except for extracellular-signal-regulated kinase (ERK) -2. The high-TMG cells showed a down modulation of phosphorylated ERK2 while the low-TMG4 cell lysates showed a 500-fold up regulation of phosphorylated ERK2 when compared to the levels in the low-TMG cells. Our observations were confirmed visually by assaying the cells for intracellular localization of ERK2. We used immuno-fluorescent staining to show that TMG4 levels regulate ERK2 activation based on TMG4 dependent translocation of ERK2 using anti phospho-ERK2 and anti-ERK2 antibodies. More than 85% of high-TMG4 cells showed extra nuclear localization of ERK2 while phosphorylated ERK2 was localized in the nucleus in the >80% of low-TMG4 cells. Thus there is an inverse correlation between TMG4 levels and ERK2 phosphorylation. We conclude that TMG4 is a molecular modulator of ERK2. Whether TMG4 has a direct interaction or affects ERK2 indirectly is under investigation. These studies suggest novel roles for vitamin-K dependent Gla-proteins in regulating thrombopoietin dependent megakaryocytic differentiation, platelet activation and myelopoiesis and myeloproliferation.

Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages

AbstractAlthough successful attempts to inhibit HIV-1 replication in T cells using RNAi have been reported, the effect of HIV-specific RNAi on macrophages is not well known. Macrophages are key targets for anti–HIV-1 therapy because they are able to survive long after the initial infection with HIV and can spread the virus to T cells. In this study, we identified a putative RNAi target of HIV, consisting of the portion of the nef gene overlapping the U3 region (Nef366), and generated a lentivirus-based short hairpin RNA (shRNA) expression vector (Lenti shNef366). We show that Lenti shNef366 inhibits (1) HIV-1 replication in a monocytic cell line and in primary monocyte-derived macrophages (MDMs), (2) reactivation of latent HIV-1 infection, and (3) the production of secondary HIV-1 from MDMs harboring a genomic copy of Nef366. Moreover, we found that the up-regulated production of macrophage inflammatory protein 1β (MIP-1β), but not MIP-1α, in MDMs by Nef expression was considerably suppressed by Lenti shNef366, which suggests that HIV-1 dissemination to T cells through its interaction with HIV-1–infected MDMs can also be controlled by Lenti shNef366. Thus, lentivirus-mediated shRNA expression targeting the U3-overlapping region of HIV nef represents a feasible approach to genetic vaccine therapy for HIV-1.

Lentivirus-Mediated RNA Interference of HMGA1 Promotes Chemosensitivity to Gemcitabine in Pancreatic Adenocarcinoma

The high mobility group A1 (HMGA1) proteins are overexpressed in pancreatic cancers. They are architectural nuclear proteins, which regulate expression of multiple genes implicated in the malignant phenotype. In this study, we hypothesized that HMG A1 silencing will promote chemosensitivity in pancreatic adenocarcinoma. We studied highly malignant pancreatic adenocarcinoma cell lines (MiaPaCa2 and PANC1). Lentiviral short-hairpin RNA (shHMGA1) expression vectors targeting HMGA1 were used for generation of lentiviral particles. Stable transfectants were developed after lentiviral transduction. Nuclear expression of HMGA1 was assayed using Western blot analysis. Chemosensitivity to gemcitabine was determined by IC50 analysis. Caspase activity was quantitated using fluorometric caspase profiling. Apoptosis was assessed by flow cytometric analysis. Lentivirus-mediated RNA interference resulted in 90% silencing of HMGA1 expression in each of MiaPaCa2 and PANC1 cell lines. HMGA1 silencing enhanced chemosensitivity to gemcitabine with an approximately 50% reduction in IC50 in each cell line. Lentivirus-mediated HMGA1 silencing promoted the activation of caspases 3, 2, 9, and 8, on exposure to gemcitabine. HMGA1 silencing resulted in reduction in Akt kinase activity. Lentivirus-mediated RNA interference of HMGA1 promoted chemosensitivity to gemcitabine in pancreatic adenocarcinoma. HMGA1 may represent a novel therapeutic target in pancreatic cancer.

Complete Reversal of ABCG2-Depending Atypical Multidrug Resistance by RNA Interference in Human Carcinoma Cells

In the chemotherapeutic treatment of patients with disseminated neoplasms, multidrug resistance (MDR) is a major obstacle. ABCG2 (BCRP/MXR), a member of the superfamily of adenosine triphosphate-binding cassette (ABC) transporters, was demonstrated to be associated with "atypical" forms of multidrug-resistant phenotypes of cancer cells. To overcome the ABCG2-depending MDR, two specific anti-ABCG2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the human gastric carcinoma cell line EPG85-257RNOV, exhibiting an atypical MDR phenotype. Because both siRNAs showed biological activity, for stable inhibition of ABCG2 corresponding short hairpin RNA (shRNA) expression vectors were constructed. By treatment of EPG85-257RNOV cells with these constructs, expression of the targeted ABCG2-encoding mRNA and transport protein was inhibited completely. Furthermore, anti-ABCG2 shRNA-treated cells increased cellular drug accumulation to the same level measured in drug-sensitive parental cells. These effects were accompanied by complete reversal of the drug-resistant phenotype. Thus, the data indicate that siRNA- and shRNA-mediated RNAi-based gene therapy may be applicable in preventing and reversing ABCG2-depending atypical MDR.