Scramble Control Research Articles

Expression of microRNA let-7 in cleavage embryos modulates cell fate determination and formation of mouse blastocysts†.

After fertilization, the zygote undergoes cell division. Up to the 8-cell stage, the blastomeres of mouse preimplantation embryos are morphologically identical. The first cell differentiation starts in the morula leading to the formation of trophectoderm cells and inner cell mass cells of the blastocyst. The regulation of the differentiation event and the formation of blastocysts are not fully known. Lethal-7 (let-7) is a family of evolutionarily conserved microRNAs. Here, we showed that the expression of let-7a and let-7g decreased drastically from the 1-cell stage to the 2-cell stage, remained low up to the 8-cell stage and slightly increased after the morula stage of mouse embryos. The expression of let-7 in the inner cell mass was higher than that in the trophectoderm. Forced expression of let-7a in embryos at the 1-cell and 4-cell stage inhibited blastocyst formation and downregulated the expression of CDX2 but maintained that of OCT4 in the trophectoderm. Forced expression of other let-7 isoforms exhibited similar inhibitory action on blastulation. On the other hand, inhibition of let-7a at the 4-cell stage and the 8-cell stage enhanced blastocyst formation. Co-injection of green fluorescent protein (GFP) mRNA (lineage tracer) with either precursor of let-7a (pre-let-7a) or scramble control into one blastomere of 2-cell embryos showed that ~75% of the resulting blastocysts possessed GFP+ cells in their inner cell mass only. The biased development towards the inner cell mass with forced expression of let-7 was reproduced in 2-cell chimeric embryos consisting of one wildtype blastomere and one GFP mRNA-injected blastomere from another 2-cell embryo carrying a doxycycline-inducible let-7g gene. Bioinformatics analysis indicated that Tead4 was a potential target of let-7. Let-7 bound to the 3'UTR of Tead4 and let-7 forced expression downregulated the expression of Tead4 in mouse blastocysts. Co-injection of Tead4 mRNA partially nullified the modulatory roles of let-7a in the inner cell mass cell fate. In conclusion, a high level of let-7 at the 2-cell stage favored the formation of the inner cell mass, whereas a low level of let-7 at the 4-cell to 8-cell stage enhanced blastocyst formation. Tead4 mediated the action of let-7 on the inner cell mass cell-fate determination.

Abstract A028: Growth factor signaling and kinase inhibitors regulate oncogenesis in desmoid fibromatosis by modulating activity of the beta-catenin transcription target ABL1

Abstract Introduction: While activation of beta-catenin is associated with desmoid fibromatosis (DF), mechanisms by which this oncogene initiates tumorigenesis are unclear as are factors underlying variable biologic behavior in the disease and vulnerability to targeted therapies. This study sought to define downstream pathways dysregulated by beta-catenin that may be modulated to potentially affect patient outcome. Methods: Multiple primary DF cell lines were developed from surgical specimens, validated by Sanger sequencing, and immortalized by ectopic expression of TERT. Gene expression was assessed in DF tumors (n=45) with U133A arrays and by RNA-seq in cells. Direct targets of beta-catenin were identified by CHIP-seq. Lentiviral systems were used to deliver shRNA (or scramble control) and overexpression constructs. Cell proliferation, protein levels/phosphorylation and gene expression were assessed by CyQuant DNA quantification, immunoblot, and RT-PCR, respectively. Endothelial cell (HUVEC) tube formation was quantitated using light microscopy. Results: Gene set enrichment analysis performed on RNA-seq data comparing DF cells treated with shRNA directed against CTNNB1 showed downregulation of hypoxia-regulated genes, and unsupervised analysis clustered 45 DF tumors separately from normal mesenchymal tissue based on the expression levels of these genes. CTNNB1 knockdown (KD) was associated with reduction in HIF1A and ability of DF cells to induced endothelial tube formation in HUVEC co-cultures (71%, p<0.001); ectopic expression of HIF1A in CTNNB1 KDs rescued this effect. HIF1A KD itself inhibited DF induction of HUVEC tube formation (49%, p<0.001), but did not affect DF cell proliferation. CHIP-seq nominated ABL1, a known regulator of HIF1 translation, as a direct target of beta-catenin. CTNNB1 KD caused 65% (p=0.01) decrease in ABL1 expression, and reduction in levels of c-ABL, its downstream target p-CRKL, and HIF1-alpha. Unlike HIF1A, ABL1 KD also reduced proliferation in multiple DF cell lines (up to 90%) as did direct inhibition of c-ABL with its inhibitor dasatinib (IC50 <50nM). Dasatinib and sorafenib, a PDGFR-beta inhibitor of clinical benefit in DF, both reduced cellular levels of p-ABL, p-CRKL and HIF1a expression in DF cells. Sorafenib also inhibited HUVEC tube-formation (59% at 1uM, p<0.05) induced by DF. Conversely, exogenous PDGF-BB stimulated DF proliferation (53% increase at 20ng/ml, p<0.05), increased p-ABL, p-CRKL and HIF1a in DF and promoted endothelial cell tube formation (2-fold, p<0.05) when added to DF and HUVEC co-cultures but not HUVEC cell cultures alone. Conclusion: ABL1 is a transcriptional target of beta-catenin in DF cells and is necessary for proliferation and maintenance of HIF1-alpha levels. Regulation of c-ABL activity by PDGFR-beta and targeted therapies modulates DF cell proliferation and paracrine signaling, suggesting a reason for variable biologic behavior between tumors and a mechanism for sorafenib activity in DF. This finding may point to markers predictive of outcome in patients. Citation Format: Jia Hu, Anthony Villano, Rachael O'Connor, Yuliy Rozenberg, Alankrta Venkatesh, Mrinal Gounder, Nicholas Socci, Samuel Singer, Meera Hameed, Aimee M. Crago. Growth factor signaling and kinase inhibitors regulate oncogenesis in desmoid fibromatosis by modulating activity of the beta-catenin transcription target ABL1 [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A028.

Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.

The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference

PurposeThe purpose of this study was to evaluate the pathological involvement of erythropoietin (EPO) in experimental choroidal neovascularization (CNV) and its association with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in the Chinese population.MethodsTreatment effect of recombinant EPO protein were assessed by human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, and ex vivo choroid-sprouting ability. The effect of intravitreal injection of Epo siRNA against neovascularization was evaluated in the laser-induced CNV mouse model. In addition, the association of EPO variants with neovascular AMD and PCV was determined.ResultsExogenous supplementation of EPO significantly enhanced the migration and tube formation of HUVECs and promoted ex vivo choroid sprouting in mouse retinal pigment epithelium (RPE)-choroid-sclera complex culture. In the experimental CNV mouse model, Epo expression was found to be significantly upregulated by 3.5-folds in RPE-choroid-sclera complex at day 10 after laser induction as compared to the baseline. Immunofluorescence analysis showed that Epo was mainly expressed around the vascular endothelial cells in the RPE-choroid-sclera complex. Intravitreal injection of siRNA targeting Epo reduced 40% Epo expression and 40% CNV lesion areas as compared to the scramble control. However, EPO variants were not associated with neovascular AMD nor PCV in the Chinese population.ConclusionsThis study revealed the promotion of human endothelial cell tube formation in vitro and choroid sprouting ex vivo by EPO, and the reduction of laser-induced CNV in vivo by Epo RNA interference.Translational RelevanceTargeting EPO could be a potential additional treatment for CNV-related diseases.

Intratunical injection of rat-derived bone marrow mesenchymal stem cells prevents fibrosis and is associated with increased Smad7 expression in a rat model of Peyronie’s disease

ObjectivePeyronie’s disease (PD) is a fibrotic disorder of the penis, but effective treatments are lacking. Here, we observed the effects of rat-derived bone marrow mesenchymal stem cells (BMSCs) injection in the active phase and chronic phase in a rat model of PD, and the possible mechanism was analysed with fibroblasts derived from rat penile tunica albuginea (TA).MethodsThirty-two male Sprague-Dawley rats were divided into four groups. In sham group, the rats were injected with 50 µL of vehicle. In the PD group, the rats were injected with 50 µg TGF-β1. In the PD + BMSCs early treatment group, the rats were injected with 50 µg TGF-β1 and injected with 1 × 106 BMSCs after 1 day. In the PD + BMSCs late treatment group, the rats were injected with 50 µg TGF-β1 and injected with 1 × 106 BMSCs after 28 days. Twenty-seven days after the last injection, the erectile function of the rats was measured, and then, penile fibrosis was analysed by histology and western blot. In vitro, fibroblasts derived from rat penile TA were used to identify a possible antifibrotic mechanism of BMSCs, and a Smad7 expression vector was used as a positive control. Fibroblasts were pretreated with the Smad7 expression vector or BMSCs for 48 h and then activated with 10 ng/mL TGF-β1 for 24 h. Cells viability was assessed, and Smad7, collagen 3, elastase-2B and osteopontin expression levels were analysed by immunofluorescence and western blot. Furthermore, fibroblasts were transfected with Smad7 siRNA or scramble control to observe whether the effects of BMSCs could be offset.ResultsErectile function obviously improved, and fibrosis of penile TA was prevented after BMSCs treatment compared with that in the rats with PD. Furthermore, the effects of BMSCs treatment in the active phase were better than those in the chronic phase. After cocultured with BMSCs, cell viability was not affected, Smad7 expression was upregulated, and collagen 3, elastase-2B and osteopontin levels were decreased in the TGF-β1-treated fibroblasts. After transfection with Smad7 siRNA, the antifibrotic effects of BMSCs were offset.ConclusionsThe antifibrotic effects of BMSCs treatment in the active phase of the PD rat model were better than those in the chronic phase. A possible mechanism of BMSCs treatment was related to increased Smad7 expression, suggesting a possible effective and safe procedure for the treatment of PD.

Hepatotropic Peptides Grafted onto Maleimide-Decorated Nanoparticles: Preparation, Characterization and In Vitro Uptake by Human HepaRG Hepatoma Cells.

We recently demonstrated the strong tropism of George Baker (GB) Virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB) derived synthetic peptides towards hepatoma cells. In a first approach, these peptides were covalently bound to poly(benzyl malate) (PMLABe73) and poly(ethylene glycol)-block-PMLABe73 (PEG62-b-PMLABe73) (co)polymers, and corresponding peptide-decorated nanoparticles (NPs) were prepared by nanoprecipitation. We showed that peptide enhanced NPs internalization by hepatoma cells. In the present work, we set up a second strategy to functionalize NPs prepared from PMLABe73 derivates. First, maleimide-functionalized PMLABe73 (Mal-PMLABe73) and PEG62-b-PMLABe73 (Mal-PEG62-b-PMLABe73) were synthesized and corresponding NPs were prepared by nanoprecipitation. Then, peptides (GBVA10-9, CPB and their scramble controls GBVA10-9scr and CPBscr) with a thiol group were engrafted onto the NPs’ maleimide groups using the Michael addition to obtain peptide functionalized NPs by post-formulation procedure. These peptide-modified NPs varied in diameter and dispersity depending on the considered peptides and/or (co)polymers but kept their spherical shape. The peptide-functionalized NPs were more efficiently internalized by HepaRG hepatoma cells than native and maleimide-NPs with various levels relying on the peptide’s nature and the presence of PEG. We also observed important differences in internalization of NPs functionalized by the maleimide-thiol-peptide reaction compared to that of NPs prepared from peptide-functionalized PMLABe73 derivatives.

Abstract 792: Knockdown of MK2 in bladder cancer cells increases their radiosensitivity

Abstract We have previously identified the p38 substrate MAPK-activated protein kinase-2 (MK2) as a radiation response stress pathway regulating inflammatory cytokine production that facilitates EMT pathway activation and tumor growth in head and neck cancer. Here we examine whether MK2 knockdown in bladder cancer cells alters their ability to produce immune mediators and to resist radiation.In order to study the impact of MK2 in bladder cancer (i.e., T24, HTB9), we generated both scrambled control (SCR) and MK2 shRNA using a lentiviral transduction system and selected for clones via puromycin selection. Generally, all MK2 shRNA clones demonstrated >88% reduction in MK2 protein expression by immunoblot. Increased phosphorylation of MK2 (p272) compared to control was detected 48 hours after the exposure of cells to 10 Gy radiation which was not seen in shRNA cells. Gene expression analysis of the bladder cells at 48 or 72 hours after 10 Gy radiation showed an increase in the expression of the inflammatory cytokines IL-6 and TNFα and genes that regulate epithelial to mesenchymal transition; SNAI1 and vimentin. A reduced expression of these genes was seen in the shRNA cells following radiation demonstrating MK2 as an important signaling molecule in this response. We then examined whether MK2 also impacted tumor proliferation, radiosensitivity and the cell cycle. The proliferation rates of shRNA cells compared to SCR cells showed no difference under control conditions in both cell lines. However, in response to radiation both T24 and HTB9 shRNA cells showed a greater loss of cells compared to SCR as determined by CyQUANT DNA fluorescence at 24, 48, 72 and 144 hours. Furthermore, shRNA cells showed reduced clonogenic activity after radiation compared to SCR as determined by colony-forming assays using 0-10 Gy doses in 2 Gy increments. Analysis of cell cycle changes induced by radiation at 28 hours showed a pronounced shift of HTB9 cells from 57.8 (% G1 Phase), 19.1 (% S phase) and 22.1 (% G2/M) phase to 7.6, 3.4, 86.7. This pattern was significantly different in shRNA cells that showed proportions of 46.2, 17.8, 32.0 without radiation which shifted to 20.6, 5.9 and 67.3 28 hours after irradiation. The percentage of cells in G1, and G2/M phase 28 hours after irradiation was significantly different between SCR and shRNA (p<0.001) suggesting that MK2 knockdown altered radiosensitivity by impacting cell cycle kinetics.In summary, we measured a significant increase in the sensitivity of bladder cancer cells to radiation following knockdown of MK2. The data suggests a reduction in survival of tumors cells exposed to radiation with MK2 knockdown through cell cycle changes and reduced production of survival and growth signals. Citation Format: Deri Morgan, Grace Millington, Hanna Smith, Colby Spiess, Kiersten L. Berggren, Xinglei Shen, Gregory N. Gan. Knockdown of MK2 in bladder cancer cells increases their radiosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 792.

Abstract 6168: Beta-catenin and PDGFRB modulate HIF1a in desmoid cells to promote sorafenib-sensitive paracrine signaling pathways

Abstract Introduction: Sorafenib has been shown to have clinical benefit in desmoid-type fibromatosis (DF), a mesenchymal neoplasm associated with activating CTNNB1 mutations. This study sought to better understand the mechanisms of sorafenib activity in DF by defining downstream pathways dysregulated by β-catenin. Methods: Primary DF cell lines (DES9525, 3276, 8163) were developed from surgical specimens and validated by Sanger sequencing of CTNNB1. Gene expression in DF tumors was assessed with U133A arrays and in cell lines using RNA-seq. Lentiviral systems were used to deliver shRNA (or scramble control) and overexpression constructs. Luciferase reporter assay was employed to assess transcription activity. Cell proliferation, protein levels/phosphorylation and gene expression were assessed by CyQuant DNA quantification, immunoblot, and RT-PCR, respectively. Endothelial cell (HUVEC) tube formation was quantitated manually using light microscopy. Results: Gene set enrichment analysis of DES9525 CTNNB1 knock-down (KD) showed decreased expression of genes commonly associated with hypoxia and angiogenesis. Unsupervised clustering confirmed in vivo relevance as expression levels of hypoxia-associated genes clustered 45 DF tumors separately from normal mesenchymal tissue (fat and muscle). Higher baseline levels of HIF1α were observed in DF cell lines compared to mesenchymal stem cells and abrogation of CTNNB1 reduced cellular levels of HIF1α and HIF transcriptional activity. DF co-culture with endothelial cells induced VEGFR phosphorylation on HUVECs and tube formation; HIF1A KD inhibited VEGFR phosphorylation and reduced tube formation >50% but had no effect on desmoid cell proliferation. Similar inhibition was observed after co-culture of DF CTNNB1 KD cells with endothelial cells (>50% reduction in tube formation, p<0.05), but not after overexpression of HIF1A in CTNNB1 KD cells. Sorafenib inhibited this tube-formation (60%, p<0.05) induced by DF at lower concentrations (1μM) as compared to those required to inhibit DF cell proliferation (IC50 10uM). While sorafenib directly inhibited VEGFR phosphorylation on endothelial cells, it also inhibited phosphorylation of PDGFRB and decreased HIF1α levels in DF cells. Exogenous PDGF-BB conversely increased levels of HIF1α in DF cells and endothelial tube formation (2 times, p<0.05) when added to DF but not to endothelial cells grown in the absence of DF cells. Conclusion: β-catenin and PDGFRB signaling modulate cellular levels of HIF1α in DF cells to affect endothelial cells via VEGFR. Sorafenib inhibits a PDGFR/β-catenin/HIF1α axis by modulating signaling pathways both on the DF cells themselves as well as RTK targets of sorafenib on stromal cells. Improved understanding of these interactions may allow for more specific therapeutic selection in DF patients. Citation Format: Jia Hu, Anthony Villano, Rachael Connor, Yuliy Rozenberg, Alankrta Venkatesh, Nicholas Socci, Samuel Singer, Aimee Crago. Beta-catenin and PDGFRB modulate HIF1a in desmoid cells to promote sorafenib-sensitive paracrine signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6168.

Abstract 2597: Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a grim prognosis. Though the overall 5-year survival rates have recently improved to 10%, there is still an unmet need for better therapies. PARP inhibitors (PARPi)/platinum therapies have demonstrated clinical efficacy in BRCA1/2 mutated PDAC and has recently led to the FDA approval of PARPi (olaparib) in the maintenance setting, offering the best personalized treatment approach for such patients. Although promising, the benefits of these therapeutic strategies are currently limited to only a few patients. We have recently established that BARD1 (BRCA1- Associated-Ring-Domain-1), the main binding partner of BRCA1 in regulating homologous recombination repair is upregulated in PDAC cells exposed to PARPi/platinum compounds, by a post-transcriptional RNA binding mechanism. We therefore hypothesized that inhibition of BARD1 will sensitize cells to these agents. We found that inhibition of BARD1 (both by siRNA/shRNA and CRISPR-KO) not only sensitized the cells to olaparib or oxaliplatin but enhanced DNA damage in cells, as analyzed by comet assays and γ-H2AX immunofluorescence staining. BARD1 inhibition led to a decrease in homologous repair efficiency of cells as analyzed by the well established pDRGFP assay. To further understand BARD1 dependent downstream signaling, we performed comparative transcriptomic analysis of BARD1 silenced cells compared to scramble control. RNA seq analysis revealed modulation of several cell cycle checkpoints and DNA damage response pathways. Notably, through gene set enrichment analysis (GSEA), we found that silencing BARD1 causes de-enrichment of pathway regulating Polo-Like-Kinase-1 (PLK1, a serine/threonine kinase) activity (p-val=0.05, FDR=0.07). PLK1 is frequently upregulated in PDAC cells and promotes progression of cells through mitosis. Based on these results, we found that a PLK1 specific inhibitor (onvansertib) synergized with olaparib to reduce cell survival of homologous repair proficient (HR-P) PDAC cells. Importantly, the mechanism of drug synergy involved induction of apoptosis and DNA damage, as assessed by cleaved caspase-3 and γ-H2AX protein analysis. These results are suggestive of targeting the BARD1-PLK1 axis in order to enhance sensitivity of PARPi/platinums in HR proficient cells. Our ongoing and future studies are aimed at understanding the interaction of BARD1 and PLK1 in PDAC cells and analyze the effects of onvansertib and olaparib on tumor growth in vivo. Accomplishment of these studies will establish novel therapeutic targets and synthetic lethal combinations that have not been explored yet for deadly pancreatic cancers, ultimately benefiting patient survival in the long run. Citation Format: Carolyn Coats, Anoohya Arkala, Eric Londin, Avinoam Nevler, Jonathan R. Brody, Charles J. Yeo, Aditi Jain. Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2597.

MEDB-76. Evaluating the B7-H3 checkpoint in Medulloblastoma

BACKGROUND: There is currently no curative therapy for recurrent/refractory MB. Novel approaches to MB include immunotherapy, such as targeting the immune checkpoint molecule B7-H3. B7-H3 is implicated in tumor metastasis and is highly expressed in MB. This study explores the effects of genetically knocking down B7-H3 in a murine model of recurrent/refractory medulloblastoma. METHODS: Murine MB cells were transduced with a CRISPR/Cas9 lentivirus to create a B7-H3 knockout. Knockout population was sorted twice via FACS by the AECOM flow cytometry CORE and confirmed by western blot and flow cytometry. Three healthy clones were used in subsequent studies, and compared to the wild type and the scramble control. IncuCyte live imaging technology was used to evaluate spheroid growth. Matrigel Boyden chambers were used to evaluate migration. Bulk RNA-seq was performed by the Yale University Core. RESULTS: B7-H3 knockout was successful in the murine MB model. Morphological differences were noted in the B7-H3 knockout cells. Spheroid formation assays show one of the clones with statistically slower growth kinetics compared to controls. Migration results are pending. RNA seq revealed similar clustering amongst knockouts, separate from controls with an enrichment in genes of morphologic development, WNT signaling and amoeboid migration. CONCLUSIONS: The morphologic changes in the B7-H3 knockouts suggest a potential growth differential. Although in vitro growth assays have shown mixed results regarding the effect of knocking out B7-H3 in spheroid formation, B7-H3 has been more directly implicated in migration and immune signaling. If migration is impaired, this will suggest that B7-H3 enhances malignant and metastatic potential in MB. Functional in vivo immune studies in syngeneic mice will investigate immune mediated effects of B7-H3 knockout in this tumor. If our studies support a role for B7-H3 in the development of MB, it may have important clinical implications, particularly for relapsed patients.

Toll Like Receptor 4 Inhibition Improves Antitumor Responses to MBQ‐167 in Triple Negative Breast Cancer Cells in vitro

Triple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen, progesterone, and human epithelial growth factor receptors. Due to the scarce availability of receptor‐targeted or hormonal treatments, TNBC patients are submitted to taxane‐based chemotherapies like paclitaxel. Patients initially respond to paclitaxel, but 4 out of 10 patients may develop tumor recurrence along with high metastasis and mortality rates. Several studies demonstrate that paclitaxel may induce chemoresistance and metastasis through the activation of Toll‐Like Receptor 4 (TLR4)/NFKB signaling pathway, a known cancer prognostic marker in breast cancer. Hence, new treatments are needed to improve TNBC prognosis in patients. Recently, our laboratory and collaborators developed MBQ‐167, a potent Rac and Cdc42 inhibitor that decreases tumor burden, cell proliferation, and metastasis in TNBC mouse models. In preliminary studies with TNBC mice, we observed that MBQ‐167/Paclitaxel combination treatment prevented metastasis progression compared to paclitaxel treatment alone. This led us to hypothesize that MBQ‐167 chemosensitizes aggressive breast cancer cells and reduces metastasis by blocking Rac/Cdc4, hence decreasing their downstream target NfkB activity. This research project aims to evaluate cellular and molecular events that explain how MBQ‐167 prevents paclitaxel‐induced metastasis. Here, we generated TLR4‐knockdowns (TLR4‐KD) in MDA‐MB‐231 TNBC cell lines using lentiviral particles with shRNAs against the TLR4 mRNA. After knockdown validation with western blot analysis (WB), Wildtype (MDA‐MB‐231), Scramble control knockdowns (Sc‐KD), and TLR4‐KD cells were treated with different concentrations of MBQ‐167, paclitaxel, doxorubicin, or combinations. After treatments, we evaluated the effect on cell viability from MTT assays at 96 and 120 hours, cell apoptosis through Caspase‐Glo 3/7 Assays at 48 hours, and cellular migration through scratch wound healing assays. Our results demonstrate that partial inhibition of TLR4 decreases the cell viability of cells, increases apoptosis, and decreases cell migration after treatment with MBQ‐167 (250 nM and 500 nM), Paclitaxel (5 nM and 10 nM), or Doxorubicin (250 nM and 500 nM). We also observed an additive response upon combinatorial treatments with MBQ‐167/Paclitaxel and MBQ‐167/Doxorubicin when TLR4‐KD cells were compared to wild‐type and Sc‐KD cells. Altogether, these results suggest that TLR4‐KD chemosensitizes cells to Paclitaxel and Doxorubicin chemotherapies. In addition, TLR4 inhibition improves cellular response to Rac/Cdc42 inhibitor, MBQ‐167. Therefore, TLR4‐KD may reduce Rac and NfkB signaling, improving treatment response to MBQ‐167, MBQ‐167/Paclitaxel, and MBQ‐167/Doxorubicin. Other cellular studies, including Rac and NfkB activation assays, are being evaluated in our laboratory.

Cell-directed aptamer therapeutic targeting for cancers including those within the central nervous system

ABSTRACT Osteopontin (OPN) is produced by tumor cells as well as by myeloid cells and is enriched in the tumor microenvironment (TME) of many cancers. Given the roles of OPN in tumor progression and immune suppression, we hypothesized that targeting OPN with aptamers that have high affinity and specificity could be a promising therapeutic strategy. Bi-specific aptamers targeting ligands for cellular internalization were conjugated to siRNAs to suppress OPN were created, and therapeutic leads were selected based on target engagement and in vivo activity. Aptamers as carriers for siRNA approaches were created including a cancer targeting nucleolin aptamer Ncl-OPN siRNA and a myeloid targeting CpG oligodeoxynucleotide (ODN)-OPN siRNA conjugate. These aptamers were selected as therapeutic leads based on 70–90% OPN inhibition in cancer (GL261, 344SQ, 4T1B2b) and myeloid (DC2.4) cells relative to scramble controls. In established immune competent 344SQ lung cancer and 4T1B2b breast cancer models, these aptamers, including in combination, demonstrate therapeutic activity by inhibiting tumor growth. The Ncl-OPN siRNA aptamer demonstrated efficacy in an immune competent orthotopic glioma model administered systemically secondary to the ability of the aptamer to access the glioma TME. Therapeutic activity was demonstrated using both aptamers in a breast cancer brain metastasis model. Targeted inhibition of OPN in tumor cells and myeloid cells using bifunctional aptamers that are internalized by specific cell types and suppress OPN expression once internalized may have clinical potential in cancer treatment.

Syndecan-1 and -4 influence Wnt signaling and cell migration in human breast cancers

Heparan sulfate proteoglycans (HSPGs) participate in numerous normal and pathophysiological cellular functions. HSPGs are crucial components of the extracellular matrix (ECM) binding signalling molecules such as fibroblast growth factors (FGF) and Wnts to mediate various cellular processes including cell proliferation, migration, and cancer invasion. The syndecans (SDCs1-4) are a major family of four HSPGs, implicated in the development of breast carcinomas. This study examined syndecan-1 (SDC1) and syndecan-4 (SDC4; SDC1/4) in breast cancer (BC) in vitro cell models and their role in tumorigenesis. Gene expression of HSPG core proteins, biosynthesis and modification enzymes along with Wnt/FGF morphogen pathway components were examined following inhibition of SDC1 and SDC4 via small interfering RNA (siRNA), and enhancement of HSPGs via addition of heparin and FGF. siRNAs knockdowns (KDs) were performed in the MCF-7 (lowly invasive and poorly metastatic) and the MDA-MB-231 (highly invasive and metastatic) human BC cell lines. Significantly decreased gene expression of SDC1 and SDC4 was observed in both cell lines following KD. Additionally, via gene expression analysis, downregulation of SDC1/4 decreased the biosynthesis of heparan sulfate modification enzymes and reduced expression of Wnt signalling molecules. Following the enhancement/inhibition of HSPGs via heparin/siRNA treatment, heparin increased the migratory characteristics of MCF-7 cells while KD of SDC1 increased cell migration in both MCF-7 and MDA-MB-231 cells when compared to scramble negative control conditions. Our findings suggest that a niche-specific function exists for SDC1/4 in the BC microenvironment, mediating Wnt signalling cascades and potentially regulating migration of BC cells.

99mTc-labeled peptide targeting interleukin 13 receptor α 2 for tumor imaging in a cervical cancer mouse model.

Pep-1 (CGEMGWVRC) can potently bind to interleukin 13 receptor α 2 (IL-13Rα2), a tumor-restricted receptor found to be expressed in various malignancies. In this study, we intended to prepare a 99mTc-labeled probe and evaluate its in vivo tumor accumulation properties in a cervical cancer xenograft model. The Pep-1 was designed and radiolabeled with 99mTc by conjugation with mercaptoacetyl-triglycine (MAG3). The labeling yield, radiochemical purity and stability were characterized in vitro. Cell uptake assays and fluorescence imaging were conducted for qualitative and quantitative evaluation of the specificity and affinity of Pep-1. Flow cytometry and tissue immunofluorescence were used to confirm the IL-13Rα2 expression in cervical cancer. Biodistribution and in vivo imaging were performed periodically to evaluate the imaging value of 99mTc-MAG3-Pep-1 in cervical cancer xenograft model. 99mTc-MAG3-Pep-1 was successfully prepared with a high labeling yield and radiochemical purity (> 95%). Specific cell uptake was demonstrated by scramble control and unlabeled MAG3-Pep-1 blockade. Flow cytometry and tissue immunofluorescence also confirmed the mild IL-13Rα2 expression of HeLa. In the gamma imaging study and biodistribution, the tumors were imaged clearly at 2-6h after injection of 99mTc-MAG3-Pep-1 and the accumulation of 99mTc-MAG3-Pep-1 in tumor was significantly higher than that in the blocking and scramble controls, demonstrating ligand-receptor binding specificity. This work demonstrated that 99mTc-MAG3-Pep-1 can bind to cervical cancer with high affinity and specificity. MAG3-Pep-1 may be a prospective precursor for IL-13Rα2-expressing cancer therapy.

Prediction of Angiopoietin-like Protein 4-related Signaling Pathways in Cholangiocarcinoma Cells.

Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional signaling protein implicated in carbohydrate metabolism, inflammation, cancer growth and progression, anoikis resistance, angiogenesis, and metastasis. However, signaling pathways of ANGPTL4 in cholangiocarcinoma (CCA) remain unknown. The aim of this study was to explore ANGPTL4-related signaling proteins and pathways associated with CCA biology. ANGPTL4 of CCA cells was silenced by small interfering RNA (siRNA) with scramble control and ANGPTL4-related signaling proteins were investigated using mass spectrometry, bioinformatics tools and molecular docking. Among the 321 differentially expressed proteins, 151 were down-regulated. Among them, bioinformatic analyses revealed that ANGPTL4 interacts with DNA-dependent protein kinase catalytic subunit (PRKDC) and 60S ribosomal protein L21 (RPL21) via AKT serine/threonine kinase 1 (AKT1), mechanistic target of rapamycin kinase (MTOR) and ribosomal protein L5 (RPL5). Online database analysis showed that mRNA and protein expression levels of ANGPTL4-related signaling proteins were significantly higher in CCA than in normal tissues. Moreover, a high mRNA expression level was associated with high tumor grade (p<0.0001) and lymph node metastasis (p<0.0001). The signaling pathway of ANGPTL4 in CCA progression might be regulated by PRKDC and RPL21. Furthermore, high expression of ANGPTL4-related signaling proteins has potential to be used in clinical prognosis.

Abstract 9526: Fto Demethylase Mediates Altered N6-methyladenosine (m6a)-rna Modification in Human Failing Heart and Stressed-Induced Rat Cardiomyocytes

Background: N6-methyladenosine (m6A) methylation is one of the important posttranscriptional modification of RNA, which affects RNA splicing, translation and stability. These modifications are dynamically regulated by writers, readers and erasers, and their alteration affects m6A modification and have shown to play key regulatory role in numerous biological processes. Fat mass- and obesity-associated (FTO) gene, known as a nucleic acid demethylase removes the methyl group from RNA and has been shown to be associated with progression of heart diseases. In this study, we determined m6A methylation in human failing heart, and evaluated if FTO regulates m6A-methylation of transcripts related to inflammation in cultured rat cardiomyocytes under various stress conditions. Methods and Results: m6A RNA-methylation was increased in human failing hearts as compared to heart samples from non-failing subjects. Interestingly, failing hearts were associated with significant decrease in FTO mRNA and protein expression. Furthermore, cultured rat cardiomyoblasts (H9C2) cells exposed to stressors (hypoxia or isoproterenol) for 24hrs showed significant decrease of FTO and an associated increase in m6A-RNA methylation (dot blot analysis). To further test if stress-induced increase in m6A-RNA modification is mediated through FTO, we knocked-down FTO, treated with isoproterenol for 24hrs and evaluated m6A methylation status of RNA. Interestingly, dot blot analysis showed significant increase in m6A methylation levels and proinflammatory cytokines (IL6 and TNFa) in FTO knockdown cells as compared to scramble, both at basal level and after exposure to stress. Furthermore, we evaluated the effect of FTO on methylation of RNA transcripts that encode inflammatory cytokines. MeRIP assay (Methylated RNA immunoprecipitation using m6A antibody and analysis of m6A-transcript by qRT-PCR) demonstrated that the gene expression of proinflammatory cytokines, IL6 and TNF-alpha was significantly increased in FTO knockdown cells as compared to scramble control cells . Conclusion: This data suggests stress-induced loss of FTO in the heart might mediate m6A-RNA methylation and therefore potential leading to upregulation of inflammatory response in the myocardium.

Investigation of miRNA-29b As a Fetal Hemoglobin Inducer in Sickle Cell Disease

Introduction: Therapeutic intervention aimed at inducing fetal hemoglobin (HbF) expression is an effective approach for ameliorating the clinical severity of sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved drug with proven efficacy for inducing HbF in SCD. Recently, DNA methyltransferase (DNMT) inhibitors were shown to reactivate γ-globin gene expression via DNA hypomethylation. However, alternate approaches involve development of small non-coding microRNAs (miR) to silence major repressors of γ-globin transcription. Previous studies from our group showed that miR-29b inhibits DNA methylation by binding the 3' untranslated region of DNMT3A/B (Starlard-Davenport A et al., J Carcinog 12:15; 2013). Subsequent, studies demonstrated that increased levels of miR-29b are associated with high HbF levels in patients with SCD. To gain insights into mechanisms, studies performed in KU812 cells demonstrated DNMT3A/B silencing with ү-globin gene activation. Moreover, miR29b increased HbF expression in erythroid progenitors generated from normal adult CD34 + stem cells (Starlard-Davenport A et al., Br J Haematol 186:91-100; 2019). Therefore, we tested the hypothesis that miR-29b activates γ-globin transcription via DNA hypomethylation in normal and sickle erythroid progenitors and Townes SCD mouse model.Methods: Normal CD34 + cells (n=3) and peripheral blood mononuclear cells (n=2) isolated from SCD patients, were cultured using a two-phase erythroid culturing system. Cells were electroporated with miR-29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 h. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the % HbF positive cells (F-cells). Real-time PCR was used to quantify expression of miR-29b, γ-globin, and β-globin genes, and Western blot was performed to measure DNMT, HbF and HbS protein levels. In vivo studies were performed in Townes SCD mice (3 per group) treated for 28 days by continuous infusion with subcutaneous mini-osmotic pumps. The treatment groups included miR-29b (2mg/kg/day and 3mg/kg/day) and corresponding doses of Scr control mimics.Results: We observed erythroid differentiation of untreated normal and SCD erythroid progenitors at day 7, 12, and 14 of culture assessed by Giemsa stain. Treatment with miR-29b increased the ү/ү+β mRNA ratio by 2.5‐fold (p<0.01) and F‐cell levels increased from ~6.0% (Scr) to 15% with miR-29b (100nM); we observed lower DNMT3 mRNA and protein levels after 100nM miR-29b treatment. To confirm HbF induction by miR-29b in sickle progenitors we treated cells on day 8 with 50nM and 100nM miR-29b and observed >85% increase in %F-cells compared to Scr cells. Subsequent miR-29b treatment was conducted for 28 days in Townes SCD mice, which was well tolerated documented by good weight gain and no deaths. There were minimal effects on hematopoiesis with a decrease in total white blood count and an increase in neutrophils. Total hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a 0.5 fold and 2.3-fold (p<0.005) increase in %F-cells at the 2mg/kg and 3 mg/kg miR-29b doses respectively compared to corresponding Scr controls. By week 4, miR29b increased HbF levels 2.1-fold by Western blot, and sickle cell levels under hypoxic conditions decreased 32% (p<0.01).Conclusions: Our findings support the ability of miR-29b to induce HbF in normal and sickle erythroid progenitors without significant toxicity in vitro and in SCD mice in vivo. This research highlights a novel miRNA-based epigenetic approach to induce HbF supporting discovery of new drugs to expand treatment options for SCD. DisclosuresPace: Imara Inc.: Consultancy.

Targeting USP7 Suppresses Tumor Development By Promoting Ubiquitination of PHF8 and Suppressing SNAI1/Wnt Signaling in T-Cell Acute Lymphoblastic Leukemia

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy with a high unfavorable prognosis. Ubiquitin-specific-processing protease 7 (USP7) is one of the deubiquitinating enzymes attracting concentrated attention in current studies for cancers; it is also involved in regulation of oncogenic transcriptional program in T-ALL, and serves as a potential target to treat T-ALL. USP7 is interacted with PHD Finger protein 8 (PHF8), a histone lysine demethylase in T-ALL. However, the impact of the interaction in T-ALL development is undetermined. In this study, we explored the anti-tumor effect of targeting USP7 and its downstream substrate in T-ALL and underlying mechanism.MethodsIn vitro GST pull-down assay, co-immunoprecipitation assay, protein deubiquitination and stability assays were used to detect the interaction of USP7 with its direct substrate and the ubiquitination level. Gain-of-function(overexpression) and loss-of-function (lentiviral shRNA knockdown) approaches for USP7 were conducted for gene expression, CCK-8 cell proliferation assay, and apoptosis assay with annexin V + PI staining. ChIP-qPCR assay was used to explore the enrichment of histone markers in promoter region of target gene. In vivo human T-ALL xenograft mouse model was developed in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse by tail vein injection of 2.5 × 10 6 /mouse and the leukemia engraftment was evaluated with human CD45+ cells by flow cytometry. The qPCR and western blot were carried out for detection mRNA level and protein level of related genes. The mRNA level of USP7 and its substrate PHF8 was examined by qPCR in 43 newly-diagnosed T-ALL patients with an approval of the Ethics Committee. Microarray datasets of T-ALL patients from GEO database were used to determine the USP7 and its substrate -related genes with web-based platform MEM and Limma.ResultsResults showed that USP7 directly interacted with PHF8; and overexpressed USP7 deubiquitinated and stabilized PHF8, and enhanced cell proliferation of JURKAT T-ALL cells. These data indicates targeting USP7 promotes cell proliferation of T-ALL cells through its substrate PHF8. Moreover, SNAI1 was identified as one of key co-expressed genes with PHF8 in microarray datasets from T-ALL patients, and knock-down of PHF8 suppressed the SNAL1 expression and increased the recruitment of repressive histone marker, H3K9me2 but decreased the enrichment of H3K4me3 in SNAI1 promoter. SNAI1 knockdown significantly induced the cell proliferation arrest and apoptosis of T-ALL cells (Fig.1A & 1B). With in vivo human leukemia xenograft mouse model, we further demonstrated that SNAI1 knockdown significantly reduce the spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity and also the inflammatory cell infiltration compared to the scramble shRNA control (Fig.1C-1F). Moreover, Wnt signaling was identified as SNAI1 interaction partners by the pathway analysis in the SNAI1 interaction genes in the microarray data from T-ALL patients. SNAI1 knockdown significantly suppressed the expression of the Axin2 and Survivn, the key components of Wnt signaling. These data suggested oncogenic role of USP7/PHF8/SNAI1/Wnt signaling in T-ALL. Next, we explored the effect of SNAI1 on USP7 knockdown-induced anti-tumor effect. We found that USP7 knockdown induced cell proliferation arrest and apoptosis, and overexpression of SNAI1 could block the effect in vitro. Furthermore, the in vivo data showed that USP7 knockdown significantly suppressed spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity, inflammatory cell infiltration, and the protein expression of PHF8, SNAI1, Axin2 and Survivin; and SNAI1 overexpression completely rescue the USP7 knockdown-mediated antitumor effect and restored the expression of Axin2 and Survivin in vivo (Fig.2).Conclusion:Our results demonstrated that targeting USP7 by shRNA induces the cell proliferation arrest and apoptosis by promoting ubiquitination of PHF8 and suppressing SNAI1/Wnt signaling in T-ALL. Our data also revealed the oncogenic roles of USP7/PHF8/SNAI1/Wnt signaling in T-ALL and suggested targeting the signaling pathway as potential therapy in T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gene Expression Patterns and Functional Characterization of CD36 in Normal Hematopoietic Cells

Metabolic reprogramming is a hallmark of cancer development and progression. Lipid metabolism is among the most deregulated metabolic pathways in cancer contributing to cell survival and invasion. The fatty acid translocase CD36 has attracted attention in cancer research in recent years due to its role in fatty acid transport as a scavenger receptor. We have recently reported that upregulated APOC2 cooperates with CD36 contributing to leukemia growth by promoting the LYN-ERK signaling mediated metabolic activities of acute myeloid leukemia (AML) cells. Consistently, the knockdown of CD36 or treatment with anti-CD36 antibody reduced the leukemia progression and promoted the overall survival of AML xenograft mice. CD36 is also present on various types of normal cells, including monocytes, macrophages, endothelial cells, adipocytes, and platelets and contribute to their normal functions. Thus, whether targeting CD36 in normal hematopoietic cells poses any toxicity warrants investigation to bring the therapeutic strategy of targeting CD36 in AML to maturity. Here, we leveraged public data to assess the expression patterns of CD36 in normal hematopoietic cells. We found that CD36 expression is several folds higher in dendritic cells, monocytes, and NK cells compared with B cells, eosinophils, neutrophils, and T cells (ANOVA P < 0.001). To investigate the role of Cd36 in normal hematopoietic cells, we carried out gain- and loss-of-function approaches in murine normal hematopoietic primary cells. We engineered a tet-on inducible shRNA lentiviral plasmid targeting two different sequences of the mouse Cd36. In both healthy mouse primary spleen and bone marrow cells, the two pairs of shRNAs reduced the Cd36 mRNA levels compared with scramble control group (~70-90% reduction in mRNA expression). We also engineered two different lentivirus ectopic expression plasmid to overexpress murine Cd36 (PLVX-Cd36 virus and PCDH-Cd36). Lentiviral transduction of both spleen and bone marrow mouse cells confirmed by microscopy visualization of GFP-expressing cells showed several folds increase in Cd36 expression for both plasmids measured by qPCR and western blot. We assessed the effect of Cd36 knockdown on the expansion of spleen and bone marrow hematopoietic cells over five days of cell culture. Relative to the 1 st day of cell count, there was no significant decrease in the mean bone marrow cell and spleen count between the two pair of shRNA compared with scramble control group at day 3 and day 5 post doxycycline treatment (P > 0.05). Cells in the different groups showed about 2 folds expansion on day 5 relative to day 1. Our preliminary data suggest that Cd36 knockdown has limited impact on the survival of normal hematopoietic cells. In vivo experiments assessing the effect of Cd36 gain and loss of function on the engraftment of normal hematopoietic cells in mice are ongoing to further establish the functional impact of Cd36 manipulation on normal hematopoietic cells. Altogether, our studies will demonstrate whether CD36 is dispensable for the survival of normal hematopoietic cells and thus may potentially present a viable therapeutic approach in AML. DisclosuresYaghmour: Jazz: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Astellas: Speakers Bureau; Alexion: Speakers Bureau; BMS: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau.

MicroRNA-497 induced by Clonorchis sinensis enhances the TGF-\u03b2/Smad signaling pathway to promote hepatic fibrosis by targeting Smad7

BackgroundVarious stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-β1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-β/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis.MethodsThe target of miR-497 was determined by bioinformatics analysis combined with a dual-luciferase activity assay. LX-2 cells were transfected with miR-497 inhibitor and then stimulated with TGF-β1 or excretory/secretory products of C. sinensis (CsESPs), and activation of LX-2 was assessed using qPCR or western blot. In vivo, the mice treated with CCl4 were intravenously injected with a single dose of adeno-associated virus serotype 8 (AAV8) that overexpressed anti-miR-497 sequences or their scramble control for 6 weeks. Liver fibrosis and damage were assessed by hematoxylin and eosin (H&E) staining, Masson staining, and qPCR; the activation of the TGF-β/Smad signaling pathway was detected by qPCR or western blot.ResultsIn the present study, the expression of miR-497 was increased in HSCs activated by TGF-β1 or ESPs of C. sinensis. We identified that Smad7 was the target of miR-497 using combined bioinformatics analysis with luciferase activity assays. Transfection of anti-miR-497 into HSCs upregulated the expression of Smad7, leading to a decrease in the level of p-Smad2/3 and subsequent suppression of the activation of HSCs induced by TGF-β1 or CsESPs. Furthermore, miR-497 inhibitor delivered by highly-hepatotropic (rAAV8) inhibited TGF-β/smads signaling pathway by targeting at Smad7 to ameliorate CCL4-induced liver fibrosis.ConclusionsThe present study demonstrates that miR-497 promotes liver fibrogenesis by targeting Smad7 to promote TGF-β/Smad signaling pathway transduction both in vivo and in vitro, which provides a promising therapeutic strategy using anti-miR-497 against liver fibrosis.Graphical