Short Hairpin RNA Silencing Research Articles

Targeting hexokinase 1 alleviates NLRP3-mediated inflammation in apical periodontitis: A laboratory investigation.

The aim of the study was to explore whether hexokinase 1 (HK1) is involved in the inhibition of inflammation mediated by nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signalling pathway in the development of apical periodontitis (AP). Human AP tissues and normal control tissues were collected in the clinic. First, the levels of glucose, pyruvate, lactate and hexokinase activity were examined in human AP tissues. ECAR and OCR were further measured to detect the level of glycolysis in vitro model of inflammation, which established with lipopolysaccharide (LPS)-stimulated RAW264.7 cell line. Secondly, the expression of HK1, NLRP3, caspase-1 and interleukin (IL)-1β were measured by Western blot, immunohistochemistry or RT-qPCR. Finally, lentiviral short hairpin RNA (shRNA) silencing technique or the inhibitor 2-deoxy-d-glucose (2-DG) were used to further detect the relationship between HK1-mediated glycolysis and NLRP3-mediated inflammation in the development of AP in vitro. Initially, the level of glycolysis was significantly increased in human AP tissues. Subsequently, the expression of HK1, NLRP3, caspase-1 and IL-1β were upregulated significantly in human AP tissues. Furthermore, in the model of AP in vitro, a high level of glycolysis and the high expression of HK1, NLRP3, caspase-1 and IL-1β was observed. Finally, the expression of NLRP3, caspase-1 and IL-1β mediated by LPS stimulation was significantly reduced via HK1 knockdown or 2-DG treatment in vitro. Our data support that HK1-mediated glycolysis plays a crucial role in the development of AP via upregulating the NLRP3 signalling pathway. Moreover, targeting HK1 may contribute to prevent the progression of AP, which has a potential clinical translation.

DOT1L Epigenetically Regulates Autophagy and Mitochondria Fusion in Cell Lines of Renal Cancer.

DOT1L, a histone methylase, is overexpression in renal cell cancer. However, the role and detailed molecular mechanism of DOT1L involved in renal cancer development remain unknown. The inhibition of DOT1L was used by SGC0946 and short hairpin RNA silencing. Monodansylcadaverine staining and transmission electron microscope were performed to detect autophagy changes as a result of the inhibition of DOT1L. MitoTracker Red assay was used to analyze mitochondrial morphology. The autophagy markers and mitochondria-related proteins were analyzed by Western blot, qPCR, or immunofluorescence. ChIP assay was performed to demonstrate H3K79me2 is involved in the direct regulation of Farnesoid X receptor transcription. DOT1L inhibition increased autophagy activity and promoted mito chondria fusion in cell lines of renal cancer. Inhibition of DOT1L upregulated levels of LC3α/β, P62, MFN1, and MFN2, which contributed to autophagy activity or mitochondria fusion. DOT1L knockdown showed a similar the above process. DOT1L inhibition or silencing resulted in AMP-activated protein kinase activation and mammalian target of rapamycin inhibition. Mechanistically, the DOT1L inhibitor and its short hairpin RNAs decreased the expression of Farnesoid X receptor in a histone methylase-dependent manner. We revealed the essential role of Farnesoid X receptor in regulating DOT1L-induced autophagy and mitochondrial fission through the AMP-activated protein kinase/mammalian target of rapamycin pathway in cell lines of renal cancer, which may provide new insights into the pathogenesis of renal cell cancer.

Ras suppressor 1 long form (RSU1L) silencing promotes apoptosis in invasive breast cancer cells

Ras Suppressor-1 (RSU1) is a cell-extracellular matrix (ECM) adhesion protein implicated in breast cancer (BC) cell metastasis. Nevertheless, its role in apoptosis is yet unknown. In the present study, we used bioinformatics tools to evaluate the association of RSU1 expression and BC patient survival, the expression of basic pro- and anti-apoptotic genes in metastatic BC samples and their correlation with the expression of RSU1. Then, we specifically depleted RSU1 long form (RSU1L) using a short hairpin RNA (shRNA) silencing approach in two BC cell lines, the non-invasive MCF-7 and the highly invasive MDA-MB-231-LM2 cells and assessed gene expression of pro-and anti-apoptotic genes, as well as cell survival and apoptosis. Our results showed that high RSU1 expression was correlated with poor survival and significant changes were found in the expression of apoptosis-related genes (PUMA, TP53, BCL-2 and BCL-XL) in metastatic BC. Moreover, silencing of the long and most common isoform of RSU1 (RSU1L) resulted in the upregulation of PUMA and TP53 and concomitant downregulation of anti-apoptotic BCL-2 and BCL-XL, with the effect being more prominent in invasive MDA-MB-231-LM2 cells. Finally, RSU1L depletion leads to a dramatic increase in apoptosis of MDA-MB-231-LM2 cells, while no change was observed in the apoptotic rate of MCF-7 cells. This is the first study linking RSU1L with apoptosis and provides evidence for its differential role in cell lines of different invasive potential. This indicates that RSU1L represses apoptosis in aggressive BC cells helping them evade cell death and survive.

Long noncoding RNA-LINC00478 promotes the progression of clear cell renal cell carcinoma through PBX3.

Long noncoding RNAs play an important regulatory role in the development and progression of tumors. Our study found that LINC00478 was upregulated in clear cell renal cell carcinoma (ccRCC), so we made an in-depth exploration into its mechanism. In Caki-2 cells, we established the oe-LINC00478 cell line overexpressing LINC00478, and established underexpressing sh-LINC00478 cell line by short hairpinRNA silencing. The abilities of oe-LINC00478 cell invasion and metastasis were significantly enhanced, and the cell proliferative potential was also improved. The cellular expressions of PBX3, CDCA8, and CDK2 were upregulated, while in the sh-LINC00478 cells, the proliferative potential and metastatic and invasive abilities were weakened. Similarly, we established the PBX3-overexpressing oe-PBX3 cell line and the PBX3-underexpressing sh-PBX3 cell line, finding that the PBX3 overexpression enhanced the metastatic and invasive abilities of Caki-2 cells. When we overexpressed LINC00478 in PBX3-knockout Caki-2-PBX3- / - cells, no significant changes were noted in the metastatic or invasive ability. Through RNA pull-down and RNA-binding protein immunoprecipitation assays, we found that LINC00478 could facilitate the transcription-translation processes of PBX3 by binding to it, thus further promoting the expression of downstream cyclins to exert its action. In animal experimentation, the oe-LINC00478 and sh-LINC00478 Caki-2 cells were separately seeded, revealing that the tumor volume was significantly larger in the oe-LINC00478 group than in the sh-LINC00478 group. This study finds that by promoting the PBX3 transcription, LINC00478 can further regulate the expressions of downstream cyclins, thereby facilitating the metastasis and invasion of ccRCC.

Long Non-Coding RNA GABPB1-AS1 Augments Malignancy of Glioma Cells by Sequestering MicroRNA-330 and Reinforcing the ZNF367/Cell Cycle Signaling Pathway.

BackgroundDeregulation of long non-coding RNAs (lncRNAs) is frequently relevant to the malignant phenotypical changes. This study aimed to explore the role of lncRNA GABPB1-AS1 in the malignancy of glioma cells.MethodsAbnormally expressed genes in glioma were analyzed using a GEO GSE2223 dataset. Short hairpin (sh) RNA silencing of GABPB1-AS1 was introduced in glioma cells to explore its correlation with the proliferation, apoptosis, and invasiveness of cancer cells. The target transcripts of GABPB1-AS1 were predicted by bioinformatics analyses. MicroRNA (miR)-330 inhibition was additionally introduced in the glioma cells after GABPB1-AS1 knockdown for rescue experiments. Animal studies were performed by inducing xenograft tumors in nude mice.ResultsGABPB1-AS1 was highly expressed in the glioma tissues and associated with advanced WHO grades. GABPB1-AS1 knockdown reduced proliferation and invasiveness of glioma cells in vitro and in vivo. miR-330 was a target transcript of GABPB1-AS1. miR-330 inhibition restored the malignancy of glioma cells. miR-330 directly bound to ZNF367. ZNF367 was highly expressed in glioma tissues and positively correlated with GABPB1-AS1 expression, and it was relevant to the cell cycle signaling pathway. Downregulation of GABPB1-AS1 reduced the expression of ZNF367 and reduced the levels of cell cycle-related proteins PCNA, CDC20, CDC7 and CCNA1 in cells.ConclusionThis study demonstrated that GABPB1-AS1 competitively bound to miR-330 and de-repressed ZNF367 expression, leading to activation of the cell cycle signaling pathway and the growth and metastasis of glioma cells.

Cathelicidin antimicrobial peptide (CAMP) gene promoter methylation induces chondrocyte apoptosis

ObjectiveThe occurrence of osteoarthritis is related to genetic and environmental factors. Among them, the change of chondrocyte gene expression pattern regulated by epigenetic modification is an important participant. This study analyzed the effect of CAMP gene methylation on the level of oxidative stress and inflammation of chondrocytes.MethodsWe analyzed the changes of the transcriptome in the articular cartilage tissue of osteoarthritis (OA) patients from the GSE117999 dataset. The GSE48422 dataset was used to analyze the changes in the methylation level of osteoarthritis cells. Cell Counting Kit-8 (CCK-8) and flow cytometry analysis of short hairpin RNA (shRNA) silencing CAMP gene and 5-μM 5-Aza-2’-Deoxycytidine (AZA) treatment on the proliferation and apoptosis of Human chondrocytes osteoarthritis (HC-OA) cells. The Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was used to detect the level of reactive oxygen species (ROS), and the expression level of inflammatory factors was analyzed by Western Blot.ResultsThe expression of CAMP in cartilage tissue of OA patients was upregulated, and the level of methylation was downregulated. CAMP was highly expressed in osteoarthritis articular cartilage cells. Silencing CAMP inhibited the proliferation of HC-OA cells and promoted their apoptosis. CAMP gene methylation inhibited ROS levels and tumor necrosis factor-α (TNF-α) expression levels in HC-OA cells, and promoted transforming growth factor beta (TGF-β) expression. CAMP gene methylation inhibited the proliferation of HC-OA cells and promoted their apoptosis.ConclusionCAMP gene promoter methylation inhibits ROS levels and inflammation and induces chondrocyte apoptosis.

NF-κB–induced R-loop accumulation and DNA damage select for nucleotide excision repair deficiencies in adult T cell leukemia

Constitutive NF-κB activation (NF-κBCA) confers survival and proliferation advantages to cancer cells and frequently occurs in T/B cell malignancies including adult T cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1). Counterintuitively, NF-κBCA by the HTLV-1 transactivator/oncoprotein Tax induces a senescence response, and HTLV-1 infections in culture mostly result in senescence or cell-cycle arrest due to NF-κBCA How NF-κBCA induces senescence, and how ATL cells maintain NF-κBCA and avert senescence, remain unclear. Here we report that NF-κBCA by Tax increases R-loop accumulation and DNA double-strand breaks, leading to senescence. R-loop reduction via RNase H1 overexpression, and short hairpin RNA silencing of two transcription-coupled nucleotide excision repair (TC-NER) endonucleases that are critical for R-loop excision-Xeroderma pigmentosum F (XPF) and XPG-attenuate Tax senescence, enabling HTLV-1-infected cells to proliferate. Our data indicate that ATL cells are often deficient in XPF, XPG, or both and are hypersensitive to ultraviolet irradiation. This TC-NER deficiency is found in all ATL types. Finally, ATL cells accumulate R-loops in abundance. Thus, TC-NER deficits are positively selected during HTLV-1 infection because they facilitate the outgrowth of infected cells initially and aid the proliferation of ATL cells with NF-κBCA later. We suggest that TC-NER deficits and excess R-loop accumulation represent specific vulnerabilities that may be targeted for ATL treatment.

High expression of CIN85 promotes proliferation and invasion of human esophageal squamous cell carcinoma.

SH3 domain-containing kinase-binding protein 1 (CIN85), an 85 kDa protein known to be a member of the signal adaptor family, is abnormally expressed in several human malignancies and has been found to be involved in the growth, migration and invasion of these tumors. The objective of the present study was to clarify the clinical significance of CIN85 in human esophageal squamous cell carcinoma (ESCC), as well as its in vitro functions. CIN85 expression was evaluated in 129 cases of ESCC and its adjacent normal tissues using immunohistochemistry to explore its clinical relevance and prognostic value. The functions of CIN85 in the ESCC TE1 cell line were analyzed in vitro using the interfering short hairpin RNA silencing technique. MTS, wound healing, clone formation and Transwell assays were used to detect the proliferation, migration and invasion of ESCC cells. CIN85 expression was identified mainly in ESCCs and their adjacent normal tissues, and the high expression of CIN85 was significantly associated with advanced Tumor Node Metastasis stage and lymph node metastasis. CIN85 gene silencing significantly inhibited TE1 cell proliferation, migration and invasion. These results demonstrated that CIN85 was highly expressed in advanced stage ESCC and lymph node metastasis, and played a critical role in tumor proliferation and progression. Therefore, CIN85 may be a promising therapeutic target for human ESCC.

Silencing of MYH7 ameliorates disease phenotypes in human iPSC-cardiomyocytes.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy (HCM) to date have focused on mouse models of disease. We here examine allele-specific silencing in a human-cell model of HCM. We investigate two methods of silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. These findings are the first exploration of allele-specific silencing in a human HCM model and provide a foundation for further exploration of silencing as a therapeutic treatment for MYH7-mutation-associated cardiomyopathy.

Multiple myeloma: Combination therapy of BET proteolysis targeting chimeric molecule with CDK9 inhibitor.

Cyclin Dependent Kinase 9 (CDK9) associates with Bromodomain and Extra-Terminal Domain (BET) proteins to promote transcriptional elongation by phosphorylation of serine 2 of RNAP II C-terminal domain. We examined the therapeutic potential of selective CDK9 inhibitors (AZD 4573 and MC180295) against human multiple myeloma cells in vitro. Short-hairpin RNA silencing of CDK9 in Multiple Myeloma (MM) cell lines reduced cell viability compared to control cells showing the dependency of MM cells on CDK9. In order to explore synergy with the CDK9 inhibitor, proteolysis targeting chimeric molecule (PROTAC) ARV 825 was added. This latter drug causes ubiquitination of BET proteins resulting in their rapid and efficient degradation. Combination treatment of MM cells with ARV 825 and AZD 4573 markedly reduced their protein expression of BRD 2, BRD 4, MYC and phosphorylated RNA pol II as compared to each single agent alone. Combination treatment synergistically inhibited multiple myeloma cells both in vitro and in vivo with insignificant weight loss. The combination also resulted in marked increase of apoptotic cells at low dose compared to single agent alone. Taken together, our studies show for the first time that the combination of a BET PROTAC (ARV 825) plus AZD 4573 (CDK9 inhibitor) is effective against MM cells.

LncRNA AWPPH participates in the development of non-traumatic osteonecrosis of femoral head by upregulating Runx2.

AWPPH is a newly discovered long noncoding (lnc)RNA that plays an oncogenic role in development of several types of malignancies, whiles its involvement in non-traumatic osteonecrosis of femoral head (ONFH) is unknown. Therefore, the present study aimed to investigate the functionality of AWPPH in non-traumatic ONFH. Blood and mesenchymal stem cells (MSCs) were obtained from both non-traumatic ONFH patients and healthy controls, and expression of AWPPH in those tissues was detected by RT-qPCR. Receiver operating characteristic curve analysis was performed to investigate the diagnostic value of lncRNA AWPPH expression for non-traumatic ONFH. Bone morphogenic protein (BMP-2) was used to treat MSCs to induce osteogenic differentiation and the effects on lncRNA AWPPH expression was detected by RT-qPCR. LncRNA AWPPH overexpression and short hairpin (sh)RNA silencing cell lines were established and the effects on runt-related transcription factor 2 (Runx2) expression were detected by western blotting. It was demonstrated that AWPPH was significantly downregulated in non-traumatic ONFH patients compared with in healthy controls in both MSCs and serum. Expression of AWPPH in MSCs and serum is a sensitive diagnostic marker for non-traumatic ONFH. Expression of AWPPH exhibited no significant correlation with patients' age, gender and living habits, but was significantly correlated with course of disease. BMP-2 treatment significantly increased the expression level of AWPPH in human MSCs from bone marrow (hMSC-BM). AWPPH overexpression promoted, while AWPPH short hairpin RNA silencing inhibited the expression of Runx2 expression in hMSC-BM cells. Therefore, it was concluded that lncRNA AWPPH may participate in the development of ONFH by upregulating Runx2.

The orphan nuclear receptor EAR-2 (NR2F6) inhibits hematopoietic cell differentiation and induces myeloid dysplasia in vivo

BackgroundIn patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased predisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)-- suggesting that factors regulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as a gene involved in control of growth ability.MethodsBone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP. Ex vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments cells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin RNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo.ResultsHere, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant chimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR-2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating experiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization of immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were cytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed that EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic differentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid and myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential in EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone deacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain.ConclusionsThis data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the NR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS.

Formin-dependent TGF-β signaling for epithelial to mesenchymal transition.

The role of distinct actin filament architectures in epithelial plasticity remains incompletely understood. We therefore determined roles for formins and the Arp2/3 complex, which are actin nucleators generating unbranched and branched actin filaments, respectively, in the process of epithelial to mesenchymal transition (EMT). In clonal lung, mammary, and renal epithelial cells, the formin activity inhibitor SMIFH2 but not the Arp2/3 complex activity inhibitor CK666 blocked EMT induced by TGF-β. SMIFH2 prevented the proximal signal of increased Smad2 phosphorylation and hence also blocked downstream EMT markers, including actin filament remodeling, decreased expression of the adherens junction protein E-cadherin, and increased expression of the matrix protein fibronectin and the transcription factor Snail. The short hairpin RNA silencing of formins DIAPH1 and DIAPH3 but not other formins phenocopied SMIFH2 effects and inhibited Smad2 phosphorylation and changes in Snail and cadherin expression. Formin activity was not necessary for the cell surface expression or dimerization of TGF-β receptors, or for nuclear translocation of TAZ, a transcription cofactor in Hippo signaling also regulated by TGF-β. Our findings reveal a previously unrecognized role for formin-dependent actin architectures in proximal TGF-β signaling that is necessary for Smad2 phosphorylation but not for cross-talk to TAZ.

Cullin7 promotes epithelial‑mesenchymal transition of esophageal carcinoma via the ERK‑SNAI2 signaling pathway.

Cullin7 (CUL7) is a member of Cullin protein family and exhibits a tumor‑promoting role in several types of tumors, including breast, liver and ovarian. However, its roles in esophageal carcinoma (EC) have not yet been reported. In the present study, CUL7 expression in EC tissue was revealed to be significantly higher compared with nontumoral tissues, as detected by immunohistochemistry (IHC; P=0.000). χ2analysis confirmed that CUL7 expression was positively associated with invasion depth (P=0.000), lymph node involvement (P=0.033) and advanced clinical stage (P=0.000). Survival analysis demonstrated that CUL7 was positively associated with poor overall survival (P=0.001) and poor disease‑free survival (P=0.0019). An association of CUL7 with endothelial‑mesenchymal transition (EMT) was examined, and IHC results indicated that high CUL7 expression was associated with increased zinc finger protein SNAI2 (SNAI2) expression (P=0.000) and decreased E‑cadherin (P=0.000). Western blot analysis demonstrated that short hairpin RNA silencing CUL7 in EC1 cells increased epithelial (E)‑cadherin protein expression level, and decreased expression of Vimentin and SNAI2; cell migration was also reduced. Western blot analysis demonstrated that over expression of CUL7 in EC9706 cells increased Vimentin and SNAI2 protein expression, but decreased E‑cadherin expression, and the number of migratory cells. Investigation into the potential molecular mechanisms demonstrated that over expressing CUL7 in EC9706 cells stimulated the phosphorylation of ERK. Inhibiting ERK through treatment with U0126 significantly abrogated CUL7‑induced alterations in Vimentin, SNAI2 and E‑cadherin expression levels. Results from the present study demonstrated that CUL7 expression was associated with EC progression and poor prognosis. CUL7 may promote EMT via the ERK‑SNAI2 pathway in EC. These data may improve our understanding of the role of CUL7 in tumors and provide supporting evidence for the development of novel therapeutic targets for EC.

Effects of inhibition of neural precursor cell-expressed developmentally down regulated 8 gene on biological behavior, and androgen receptor of human prostate cancer cells

Objective To explore the effect of lentivirus-mediated short hairpin RNA (shRNA) silencing neural precursor cell-expressed developmentally down regulated 8 (NEDD8) gene on androgen receptor (AR), prostate specific antigen (PSA), proliferation, apoptosis and invasion of human prostate cancer LNCap and VCaP cells. Methods LNCap and VCaP cells were transfected with lentivirus-mediated NEDD8-shRNA. The mRNA and protein expression of NEDD8, AR and PSA was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting, respectively. The proliferation, apoptosis and invasion abilities were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry and Transwell TM assay, respectively. Results After transfection with NEDD8-shRNA, NEDD8 mRNA expression in LnCaP and VCaP was (0.11±0.03) fold and (0.16±0.04) fold, respectively, as compared to the blank control group; AR mRNA expression was (0.52±0.04) fold and (0.42±0.05) fold; PSA mRNA expression was (0.49±0.04) fold and (0.59±0.06) fold. NEDD8, AR and PSA protein expression in LnCaP and VCaP was (11.26±2.65)% and (6.26±2.04)%, (30.36±4.35)% and (15.36±3.37)%, and (13.37±2.70)% and (11.36±2.67)%, respectively. As compared with the control groups, the mRNA and protein expression levels of NEDD8, AR and PSA were significantly inhibited after transfection with NEDD8-shRNA (P=0.000). As compared with the control groups, the proliferation of LnCaP and VCaP was significantly inhibited after transfection with NEDD8-shRNA. The apoptosis rate of LNCap and VCaP cells was (15.08±1.23)% and (20.37±1.76)% respectively, which was significantly enhanced as compared with the control groups (P=0.001, P=0.000). After transfection, the number of LnCaP and VCaP cells invading the Matrigel was (55.5±12.5) and (60.8±11.3) respectively, which was significantly reduced as compared with the control groups (P=0.000, P=0.004). Conclusion NEDD8 may downregulate AR at the transcriptional level. The lentivirus-mediated shRNA silencing NEDD8 gene could inhibit the proliferation and invasion, and promote apoptosis of LNCap and VCaP cells. Key words: Prostate cancer; Neural precursor cell-expressed developmentally down regulated factor 8; Androgen receptor; RNA interference

RNAi-mediated silencing of TNF-α converting enzyme to down-regulate soluble TNF-α production for treatment of acute and chronic colitis

Elevated level of tumor necrosis factor-α (TNF-α), one of the inflammatory cytokines, is considered to be a potential target for the inflammatory bowel disease (IBD) therapy. Recently, TNF-α converting enzyme (TACE), a sheddase playing an important role in cleaving and releasing bioactive soluble TNF-α, has been challenged with inhibitors to treat inflammatory diseases. Here, we report a novel anti-TNF-α strategy using a short hairpin RNA silencing TACE (shTACE) to prevent and treat colitis. The shTACE formed stable complexes with nona-arginine-based bio-cleavable disulfide bond-linked poly (arginine) (PAs-s) for enhanced gene delivery. Systemically administered shTACE/PAs-s peptoplexes efficiently decreased TNF-α levels, increased survival and alleviated pathophysiological parameters representing colitis severity. Our results demonstrate effectiveness and safety of shTACE/PAs-s peptoplexes with the capacity of overcoming acute and chronic ulcerative colitis through modulation of excessive inflammatory responses in the colon, providing a strong potential as a therapeutic agent for a broad variety of inflammatory diseases.

Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Short hairpin RNA silencing of TGF-βRII and FZD-7 synergistically suppresses proliferation and metastasis of hepatocellular carcinoma cells.

Transforming growth factor-β (TGF-β) is a multifunctional regulator of cell growth, apoptosis, differentiation and migration. The Wnt/β-catenin signaling pathway has been implicated in a wide spectrum of diseases, including numerous cancers and degenerative disease. The aim of the present study was to investigate if simultaneous blocking of TGF-β and Wnt/β-catenin signaling pathways exerts synergistic anti-tumor effects on hepatocellular carcinoma (HCC) cells. Short hairpin (sh) RNA eukaryotic expression vectors, specific to TGF-β receptor II (RII) and Frizzled receptor (FZD)-7, were constructed by gene recombination. The expression vectors were transfected into human HCC HepG2 and Huh-7 cells using Lipofectamine 2000 to investigate the synergistic effects between TGF-β and Wnt/β-catenin signaling pathways on HCC cell proliferation, invasion and migration and the cell-cycle distribution. Western blot analysis was used to identify the expression of β-catenin, c-Myc and cyclin D1 in transfected cells to investigate the underlying mechanisms that cause TGF-β and Wnt/β-catenin signaling in HCC cells. shTGF-βRII-c and shFZD-7–2 were selected as the most efficient plasmids. A cell growth assay and colony-forming assay consistently demonstrated that the proliferative activity of the co-transfected group was significantly decreased compared to the single-transfected group. A wound healing invasion and migration assay demonstrated that co-transfection of shTGF-βRII-c and shFZD-7-2 decreased the invasion and migration abilities of the cells compared with either single-transfected group. In addition, the present study demonstrated that the observed reduction in cell proliferation was due to the cells arresting at the G1 phase of the cell cycle, and the downregulation of β-catenin, c-Myc and cyclin D1 impaired the proliferative and invasive abilities of the HCC cells. The present results demonstrate that simultaneous blocking of TGF-β and Wnt/β-catenin signaling by targeting TGF-βRII and FZD-7 may inhibit the proliferation and metastasis of HCC cells more effectively compared with blocking either the TGF-β or Wnt/β-catenin pathway.

Short hairpin RNA silencing of interleukin-6 in human bone marrow-derived mesenchymal stromal cells inhibits multiple myeloma cell growth

Short hairpin RNA silencing of interleukin-6 in human bone marrow-derived mesenchymal stromal cells inhibits multiple myeloma cell growth

Effects of lentiviral short hairpin RNA silencing of Toll-like receptor 4 on the lens epithelial cell line HLEC.

The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.