Showing papers on "Small hairpin RNA published in 2020"
TL;DR: This work indicates a novel mechanism for maintaining sorafenib resistance and is a proof-of-concept study for targeting circRNA-SORE in sorAFenib-treated HCC patients as a novel pharmaceutical intervention for advanced HCC.
Abstract: Accumulating evidence suggests that the primary and acquired resistance of hepatocellular carcinoma (HCC) to sorafenib is mediated by multiple molecular, cellular, and microenvironmental mechanisms. Understanding these mechanisms will enhance the likelihood of effective sorafenib therapy. In vitro and in vivo experiments were performed and clinical samples and online databases were acquired for clinical investigation. In this study, we found that a circular RNA, circRNA-SORE, which is up-regulated in sorafenib-resistant HCC cells, was necessary for the maintenance of sorafenib resistance, and that silencing circRNA-SORE substantially increased the efficacy of sorafenib-induced apoptosis. Mechanistic studies determined that circRNA-SORE sequestered miR-103a-2-5p and miR-660-3p by acting as a microRNA sponge, thereby competitively activating the Wnt/β-catenin pathway and inducing sorafenib resistance. The increased level of circRNA-SORE in sorafenib-resistant cells resulted from increased RNA stability. This was caused by an increased level of N6-methyladenosine (m6A) at a specific adenosine in circRNA-SORE. In vivo delivery of circRNA-SORE interfering RNA by local short hairpin RNA lentivirus injection substantially enhanced sorafenib efficacy in animal models. This work indicates a novel mechanism for maintaining sorafenib resistance and is a proof-of-concept study for targeting circRNA-SORE in sorafenib-treated HCC patients as a novel pharmaceutical intervention for advanced HCC.
156 citations
TL;DR: A novel regulatory mechanism in which YTHDF2 mediates the mRNA degradation of the tumor suppressors LHPP and NKX3–1 in m 6 A-dependent way to regulate AKT phosphorylation-induced tumor progression in prostate cancer is proposed.
Abstract: N6-methyladenosine (m6A) is the most abundant modification in mRNA of humans. Emerging evidence has supported the fact that m6A is comprehensively involved in various diseases especially cancers. As a crucial reader, YTHDF2 usually mediates the degradation of m6A-modified mRNAs in m6A-dependent way. However, the function and mechanisms of m6A especially YTHDF2 in prostate cancer (PCa) still remain elusive. To investigate the functions and mechanisms of YTHDF2 in PCa, in vitro, in vivo biofunctional assays and epigenetics experiments were performed. Endogenous expression silencing of YTHDF2 and METTL3 was established with lentivirus-based shRNA technique. Colony formation, flow cytometry and trans-well assays were performed for cell function identifications. Subcutaneous xenografts and metastatic mice models were combined with in vivo imaging system to investigate the phenotypes when knocking down YTHDF2 and METTL3. m6A RNA immunoprecipitation (MeRIP) sequencing, mRNA sequencing, RIP-RT-qPCR and bioinformatics analysis were mainly used to screen and validate the direct common targets of YTHDF2 and METTL3. In addition, TCGA database was also used to analyze the expression pattern of YTHDF2, METTL3 and the common target LHPP in PCa, and their correlation with clinical prognosis. The upregulated YTHDF2 and METTL3 in PCa predicted a worse overall survival rate. Knocking down YTHDF2 or METTL3 markedly inhibited the proliferation and migration of PCa in vivo and in vitro. LHPP and NKX3–1 were identified as the direct targets of both YTHDF2 and METTL3. YTHDF2 directly bound to the m6A modification sites of LHPP and NKX3–1 to mediate the mRNA degradation. Knock-down of YTHDF2 or METTL3 significantly induced the expression of LHPP and NKX3–1 at both mRNA and protein level with inhibited phosphorylated AKT. Overexpression of LHPP and NKX3–1 presented the consistent phenotypes and AKT phosphorylation inhibition with knock-down of YTHDF2 or METTL3. Phosphorylated AKT was consequently confirmed as the downstream of METTL3/YTHDF2/LHPP/NKX3–1 to induce tumor proliferation and migration. We propose a novel regulatory mechanism in which YTHDF2 mediates the mRNA degradation of the tumor suppressors LHPP and NKX3–1 in m6A-dependent way to regulate AKT phosphorylation-induced tumor progression in prostate cancer. We hope our findings may provide new concepts of PCa biology.
125 citations
TL;DR: This work uncovers a critical function of DDX3 and provides a new role in m6 demethylation of RNA and a combination regimen of ketorolac salt with cisplatin deserves further clinical investigation in advanced OSCC.
Abstract: Platinum based drugs alone or in combination with 5FU and docetaxel are common regimen chemotherapeutics for the treatment of advanced OSCC. Chemoresistance is one of the major factors of treatment failure in OSCC. Human RNA helicase DDX3 plays an important role in cell proliferation, invasion, and metastasis in several neoplasms. The potential role of DDX3 in chemoresistance is yet to be explored. Enhanced cancer stem cells (CSCs) population significantly contributes to chemoresistance and recurrence. A recent study showed that m6A RNA regulates self-renewal and tumorigenesis property in cancer. In this study we found genetic (shRNA) or pharmacological (ketorolac salt) inhibition of DDX3 reduced CSC population by suppressing the expression of FOXM1 and NANOG. We also found that m6A demethylase ALKBH5 is directly regulated by DDX3 which leads to decreased m6A methylation in FOXM1 and NANOG nascent transcript that contribute to chemoresistance. Here, we found DDX3 expression was upregulated in both cisplatin-resistant OSCC lines and chemoresistant tumors when compared with their respective sensitive counterparts. In a patient-derived cell xenograft model of chemoresistant OSCC, ketorolac salt restores cisplatin-mediated cell death and facilitates a significant reduction of tumor burdens. Our work uncovers a critical function of DDX3 and provides a new role in m6 demethylation of RNA. A combination regimen of ketorolac salt with cisplatin deserves further clinical investigation in advanced OSCC.
94 citations
TL;DR: CHD1 loss results in chromatin dysregulation, thereby establishing a state of transcriptional plasticity that enables the emergence of antiandrogen resistance through heterogeneous mechanisms.
79 citations
TL;DR: It is found that CMTM6 expression was correlated with increased activity through the Wnt/β-catenin signaling pathway, which is essential for tumorigenesis, maintenance of cancer stem cells (CSC), and the epithelial-to-mesenchymal transition (EMT) characteristic of multiple cancers and may be a promising therapeutic target in the treatment of HNSCC.
Abstract: CMTM6, a regulator of PD-L1 expression, also modulates tumor immunity. Little is known about the function of CMTM6 and its mechanism of action in head and neck squamous cell carcinoma (HNSCC). In this study, we found by IHC analysis that CMTM6 overexpression predicted a poor prognosis for patients with HNSCC. We discovered that CMTM6 expression was correlated with increased activity through the Wnt/β-catenin signaling pathway, which is essential for tumorigenesis, maintenance of cancer stem cells (CSC), and the epithelial-to-mesenchymal transition (EMT) characteristic of multiple cancers. We used short hairpin RNA to eliminate expression of CMTM6, which led, in HNSCC cells, to reduced expression of nuclear β-catenin as well as inhibition of stem cell-like properties, TGFβ-induced EMT, and cell proliferation. Consistent with these results, we identified a significant positive correlation between expression of CMTM6 and EMT- and CSC-related genes in The Cancer Genome Atlas (TCGA). We found positive correlations for both RNA and protein between expression of CMTM6 and immune checkpoint components. CMTM6 silencing-induced PD-L1 downregulation delayed SCC7 tumor growth and increased CD8+ and CD4+ T-cell infiltration. The proportions of PD-1+, TIM-3+, VISTA+, LAG-3+, and B7-H3+ exhausted T cells were decreased significantly in the CMTM6 knockdown group. CMTM6 thus regulates stemness, EMT, and T-cell dysfunction and may be a promising therapeutic target in the treatment of HNSCC.
79 citations
TL;DR: The data suggest a close association of DLD with cystine deprivation- or import inhibition-induced ferroptosis, which is similar to that seen in head and neck cancer.
Abstract: Ferroptosis is a new form of regulated cell death driven by iron-dependent lipid peroxidation. Glutaminolysis and tricarboxylic acid cycle are involved in ferroptosis, but the underlying metabolic process remains unclear. We examined the role of dihydrolipoamide dehydrogenase (DLD) in ferroptosis induction in head and neck cancer (HNC). The effects of cystine deprivation or sulfasalazine treatment and of DLD gene silencing/overexpression were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed with regard to cell death, lipid reactive oxygen species (ROS) and mitochondrial iron production, mitochondrial membrane potential, mRNA/protein expression, and α-ketoglutarate dehydrogenase (KGDH)/succinate/aconitase activities. Cystine deprivation induced ferroptosis via glutaminolysis. Cystine deprivation or import inhibition using sulfasalazine induced cancer cell death and increased lipid ROS and mitochondrial iron levels, which had been significantly decreased by short-interfering RNA (siRNA) or short hairpin RNA (shRNA) targeting DLD (P
78 citations
TL;DR: It is demonstrated that NEAT1 interacted with DNMT1 to regulate cytotoxic T cell infiltration in lung cancer via inhibition of cGAS/STING pathway and provided the novel mechanistic insight into the pathogenesis of lung cancer.
Abstract: Purpose Lung cancer is the main cause of cancer-related mortality worldwide. We report here the biological role of nuclear paraspeckle assembly transcript 1 (NEAT1) in the pathogenesis of lung cancer and the underlying mechanisms. Methods Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to evaluate expression of mRNA and protein. RNA immunoprecipitation (RIP) assay, chromatin immunoprecipitation followed by qPCR analysis, and reporter assay were used to detect DNA/RNA and protein binding. Tumor-infiltrating lymphocytes were assessed with hematoxylin-eosin staining. Cytotoxic T cell infiltration was evaluated with flow cytometric analysis and immunohistochemistry (IHC) staining. The changes of cell viability and cell invasive and migratory ability were analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), colony formation, and Transwell assays, respectively. Syngeneic tumor model was set up to evaluate antitumor effect. Results The results showed that NEAT1 was overexpressed in lung cancer tissues and cancer cell lines. This aberrant expression was closely related with tumor stage and lymph node metastasis. Tumor sample with high CD8+ showed lower NEAT1 expression. In vitro studies displayed that inhibition of NEAT1 with shRNA resulted in suppression of survival and migration/invasion of lung cancer cells. On the other side, NEAT1 was found to promote tumor growth via inhibiting cytotoxic T cell immunity in syngeneic models. Finally, NEAT1 was found to interact with DNMT1, which in turn inhibited P53 and cyclic GMP-AMP synthase stimulator of interferon genes (cGAS/STING) expression. Conclusion Our findings demonstrated that NEAT1 interacted with DNMT1 to regulate cytotoxic T cell infiltration in lung cancer via inhibition of cGAS/STING pathway. The results provided the novel mechanistic insight into the pathogenesis of lung cancer.
64 citations
TL;DR: UBE2C-mediated tumor EMT promotion by estrogen is a novel mechanism for the progression of estrogen-induced endometrial cancer, which could offer new biomarkers for diagnosis and therapy of endometrian cancer in the future.
Abstract: Ubiquitin-conjugating enzyme E2C (UBE2C) plays important roles in tumor progression; nevertheless, its function in endometrial cancer remains unclear. This study elucidated the impact of UBE2C on endometrial cancer and its underlying mechanism. Human endometrial cancer and normal endometrial tissues were acquired from patients at Wuhan Union Hospital and UBE2C expression was detected by Western blotting and qRT-PCR. Endometrial cancer cells were transfected with a UBE2C overexpression plasmid or UBE2C-specific short hairpin RNA (shRNA) to up- or downregulate UBE2C expression, respectively. CCK8 and transwell assays were applied to assess the effects of UBE2C on cell proliferation, migration, and invasion. We found a significant elevation of UBE2C expression in patients with endometrial cancer, and that UBE2C upregulation was associated with advanced histologic grade, FIGO stage, recurrence, and shorter overall survival. UBE2C knockdown inhibited endometrial cancer cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), whereas UBE2C overexpression exerted the opposite effects. UBE2C downregulation increased p53 and its downstream p21 expression, with p53 overexpression reversing the EMT-promoting effects of UBE2C. UBE2C enhanced p53 ubiquitination to facilitate its degradation in endometrial cancer cells. Estradiol (E2) induced UBE2C expression via estrogen receptor α, which binds directly to the UBE2C promoter element. Silencing of UBE2C inhibited E2-promoted migration, invasion, and EMT in vitro and in vivo. IMPLICATIONS: UBE2C-mediated tumor EMT promotion by estrogen is a novel mechanism for the progression of estrogen-induced endometrial cancer, which could offer new biomarkers for diagnosis and therapy of endometrial cancer in the future.
62 citations
TL;DR: The first genome-wide genetic screens in the mammalian central nervous system using both short hairpin RNA (shRNA) and CRISPR libraries are reported, revealing a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases.
55 citations
TL;DR: The data suggest that inhibition of GLRX5 predisposes therapy-resistant head and neck cancer cells to ferroptosis, which was relatively less sensitive in cisplatin-resistant HNC cells.
Abstract: Rationale: Loss of iron-sulfur cluster function predisposes cancer cells to ferroptosis by upregulating iron-starvation response, but the role of glutaredoxin 5 (GLRX5) silencing in ferroptosis remains unknown. We examined the role of GLRX5 functional loss in promoting ferroptosis in cisplatin-resistant head and neck cancer (HNC) cells. Methods: The effects of sulfasalazine treatment and GLRX5 gene silencing were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed concerning cell viability and death, lipid reactive oxygen species (ROS) and mitochondrial iron production, labile iron pool, mRNA/protein expression, and malondialdehyde assays. Results: Cyst(e)ine deprivation, erastin, or sulfasalazine induced ferroptosis in HNC cells, which was relatively less sensitive in cisplatin-resistant HNC cells. Sulfasalazine or cyst(e)ine deprivation-induced ferroptosis resulted from increased lipid peroxidation and intracellular free iron, which were significantly promoted by short-interfering RNA or short hairpin RNA (shRNA) targeting GLRX5 (P<0.05). GLRX5 silencing activated iron-starvation response and boosted up intracellular free iron through the iron-responsive element-binding activity of increased iron regulatory protein (increased transferrin receptor and decreased ferritin). These effects were rescued by resistant GLRX5 cDNA but not by catalytically inactive mutant GLRX5 K101Q. The same results were noted in an in vivo mouse model transplanted with vector or shGLRX5-transduced HNC cells and treated with sulfasalazine. Conclusion: Our data suggest that inhibition of GLRX5 predisposes therapy-resistant HNC cells to ferroptosis.
55 citations
TL;DR: It is identified that nuclear MYH9-induced CTNNB1 expression promotes GC metastasis, which could be inhibited by staurosporine, indicating a novel therapy for GC peritoneal metastasis.
Abstract: Rationale: Peritoneal metastasis predicts poor prognosis of gastric cancer (GC) patients, and the underlying mechanisms are poorly understood. Methods: The 2-DIGE, MALDI-TOF/TOF MS and single-cell transcriptome were used to detect differentially expressed proteins among normal gastric mucosa, primary GC and peritoneal metastatic tissues. Lentiviruses carrying shRNA and transcription activator-like effector nuclease technology were used to knock down myosin heavy chain 9 (MYH9) expression in GC cell lines. Immunofluorescence, immune transmission electron microscopy, chromatin fractionation, co-immunoprecipitation, and assays for chromatin immunoprecipitation, dual luciferase reporter, agarose-oligonucleotide pull-down, flow cytometry and cell anoikis were performed to uncover nuclear MYH9-induced β-catenin (CTNNB1) transcription in vitro. Nude mice and conditional transgenic mice were used to investigate the findings in vivo. Results: We observed that MYH9 was upregulated in metastatic GC tissues and was associated with a poor prognosis of GC patients. Mechanistically, we confirmed that MYH9 was mainly localized in the GC cell nuclei by four potential nuclear localization signals. Nuclear MYH9 bound to the CTNNB1 promoter through its DNA-binding domain, and interacted with myosin light chain 9, β-actin and RNA polymerase II to promote CTNNB1 transcription, which conferred resistance to anoikis in GC cells in vitro and in vivo. Staurosporine reduced nuclear MYH9 S1943 phosphorylation to inhibit CTNNB1 transcription, Wnt/β-catenin signaling activation and GC progression in both orthotropic xenograft GC nude mouse and transgenic GC mouse models. Conclusion: This study identified that nuclear MYH9-induced CTNNB1 expression promotes GC metastasis, which could be inhibited by staurosporine, indicating a novel therapy for GC peritoneal metastasis.
TL;DR: In this paper, the anti-HBV activity of MX2 and functional variants were assessed in transfected and HBV-infected hepatoma cells and primary human hepatocytes, employing multiple assays to analyze the synthesis and decay of HBV nucleic acids.
TL;DR: A novel cellular mechanism was demonstrated that was dependent of the lncRNA‐XIST/miR‐126/IRS1/PI3K/Akt pathway in enhanced glucose metabolism in glioma.
Abstract: Abnormal glucose metabolism may contribute to cancer progression. Glioma represents a cancer resulting from an imbalance between glucose metabolism and tumor growth. However, the molecular mechanisms responsible for dysregulated brain glucose metabolism and lactate accumulation in glioma remain to be elucidated. The present study identified a long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) as a candidate to mediate glucose metabolism in glioma. Cell viability, migration, invasion, and resistance to apoptosis were evaluated in lncRNA-XIST-depleted glioblastoma cells by short hairpin RNA. Glucose uptake, lactate production, as well as levels of glucose transporter 1 (GLUT1) and GLUT3, were measured. Luciferase assay, RNA pull-down, and RNA immunoprecipitation were performed to validate the interactions among lncRNA-XIST, microRNA-126 (miR-126), and insulin receptor substrate 1 (IRS1). An in vivo analysis was carried out in nude mice bearing glioblastoma cell xenografts. The study found that lncRNA-XIST knockdown inhibited cell viability, migration, invasion, resistance to apoptosis, and glucose metabolism of glioblastoma cells. LncRNA-XIST functioned as a competing endogenous RNA of miR-126 and then regulated IRS1/PI3K/Akt pathway in glioblastoma cells. In vivo results demonstrated lncRNA-XIST knockdown reduces the tumorigenicity of glioblastoma cells. Taken together, we demonstrated a novel cellular mechanism that was dependent of the lncRNA-XIST/miR-126/IRS1/PI3K/Akt pathway in enhanced glucose metabolism in glioma.
TL;DR: Tumor-derived exosomal TRIM59 converts macrophages to tumor-promoting functions of macrophage via regulating ABHD5 proteasomal degradation, to activate NLRP3 inflammasome signaling pathway to promote lung cancer progression by IL-1β secretion.
Abstract: Exosomes are emerging as important mediators of the cross-talk between tumor cells and the microenvironment. The communication between tumor-derived exosomes and macrophages has a critical role in facilitating tumor progression. However, the mechanisms by which exosomes modulate tumor development in lung cancer are not fully understood. Short hairpin RNA mediated knockdown or exogenous expression of TRIM59 combined with in vitro and in vivo assays were performed to prove the functional significance of TRIM59. Western blotting, real-time PCR, co-immunoprecipitation, immunofluorescence (IF) staining assays, proximity ligation assay (PLA), ubiquitination assays, lactate secretion and lipid droplets content measurement, and rescue experiments were used to evaluate the mechanism. Lewis lung carcinoma (LLC) cells were injected via subcutaneously or tail vein into C57BL/6 wild-type (WT) and transgenic mice to assess the role of TRIM59 in vivo. We demonstrated that tripartite motif-containing 59 (TRIM59) was expressed in lung cancer cells-derived exosomes, and can be transferred to macrophages through the exosomes. Activated macrophages by TRIM59 promote lung cancer progression in vitro and in vivo. Mechanistic investigations revealed that TRIM59 physically interacts with abhydrolase domain containing 5 (ABHD5) and directly induced the ubiquitination of ABHD5 and led to its proteasome-dependent degradation. ABHD5, an lipolytic co-activator, deficiency induced metabolic reprogramming and enabled NLRP3 inflammasome activation in macrophages. Further studies showed that the exacerbation of NLRP3 inflammasome activation by ABHD5 deficiency, provides a positive feedback loop to promote cancer progression by preferentially secrete the proinflammatory cytokine IL-1β. Collectively, these data indicate that tumor-derived exosomal TRIM59 converts macrophages to tumor-promoting functions of macrophages via regulating ABHD5 proteasomal degradation, to activate NLRP3 inflammasome signaling pathway to promote lung cancer progression by IL-1β secretion. Our findings also indicate that tumor-derived exosomal TRIM59 has an important role in intercellular communication for fostering an inflammatory microenvironment and promoting lung metastasis.
TL;DR: It is suggested that therapeutic targeting of HNF4A-AS1/hnRNPU/CTCF axis inhibits aerobic glycolysis and NB progression.
Abstract: Aerobic glycolysis is a hallmark of metabolic reprogramming that contributes to tumor progression. However, the mechanisms regulating expression of glycolytic genes in neuroblastoma (NB), the most common extracranial solid tumor in childhood, still remain elusive. Crucial transcriptional regulators and their downstream glycolytic genes were identified by integrative analysis of a publicly available expression profiling dataset. In vitro and in vivo assays were undertaken to explore the biological effects and underlying mechanisms of transcriptional regulators in NB cells. Survival analysis was performed by using Kaplan-Meier method and log-rank test. Hepatocyte nuclear factor 4 alpha (HNF4A) and its derived long noncoding RNA (HNF4A-AS1) promoted aerobic glycolysis and NB progression. Gain- and loss-of-function studies indicated that HNF4A and HNF4A-AS1 facilitated the glycolysis process, glucose uptake, lactate production, and ATP levels of NB cells. Mechanistically, transcription factor HNF4A increased the expression of hexokinase 2 (HK2) and solute carrier family 2 member 1 (SLC2A1), while HNF4A-AS1 bound to heterogeneous nuclear ribonucleoprotein U (hnRNPU) to facilitate its interaction with CCCTC-binding factor (CTCF), resulting in transactivation of CTCF and transcriptional alteration of HNF4A and other genes associated with tumor progression. Administration of a small peptide blocking HNF4A-AS1-hnRNPU interaction or lentivirus-mediated short hairpin RNA targeting HNF4A-AS1 significantly suppressed aerobic glycolysis, tumorigenesis, and aggressiveness of NB cells. In clinical NB cases, high expression of HNF4A-AS1, hnRNPU, CTCF, or HNF4A was associated with poor survival of patients. These findings suggest that therapeutic targeting of HNF4A-AS1/hnRNPU/CTCF axis inhibits aerobic glycolysis and NB progression.
TL;DR: In this paper, the role of NEAT1 in regulating macrophage polarization and its potential for alleviating inflammatory responses during sepsis pathogenesis was investigated in mouse RAW264.7 cells.
Abstract: The lncRNA nuclear enriched abundant transcript 1 (NEAT1) promotes sepsis-inflammatory responses and acute kidney injury (AKI), but little known about the underlying mechanisms. This study aims to investigate the roles of NEAT1 in regulating macrophage polarization and its potential for alleviating inflammatory responses during sepsis pathogenesis. Mouse RAW264.7 macrophages were treated with lipopolysaccharide (LPS) as a cellular inflammatory model. NEAT1 shRNA, miR-125a-5p mimics, and TRAF6-overexpressing vector were used to transfect RAW264.7 cells. NEAT1, miR-125a-5p, and mRNA levels of functional genes were detected by quantitative RT-PCR. Protein abundances were analyzed by western blotting. Macrophage polarization was evaluated by flow cytometry. The bindings of miR-125a-5p with NEAT1 or TRAF6 gene were validated by dual luciferase reporter assay. LPS treatment promoted NEAT1 and suppressed miR-125a-5p expression in mouse macrophage cells. NEAT1 silencing by shRNAs promoted macrophage M2 polarization under LPS treatment, which upregulated miR-125a-5p expression, repressed TRAF6 expression and TAK1 protein phosphorylation in macrophages. These cellular and molecular changes induced by NEAT1 shRNAs were abrogated by miR-125a-5p inhibitors. Moreover, miR-125a-5p mimics suppressed TRAF6 expression and TAK1 protein phosphorylation in LPS-treated macrophages, thus causing macrophage M2 polarization under LPS treatment. TRAF6 overexpression abrogated the miR-125a-5p mimics-induced macrophage M2 polarization. miR-125a-5p could directly bind to NEAT1 or TRAF6 gene in macrophages. lncRNA NEAT1 knockdown ameliorates LPS-induced inflammation by promoting macrophage M2 polarization via miR-125a-5p/TRAF6/TAK1 axis.
TL;DR: The potential of RNAi in the field of organ transplantation will be discussed, and studies using RNAi to target genes related to IRI in liver, kidney, lung, and heart transplantation are discussed.
TL;DR: Several lncRNAs that significantly change during DPSC differentiation are revealed, including SNHG7, which reveals new targets for dentin‐pulp complex regeneration and tissue engineering.
Abstract: Dentin-pulp regeneration requires dental pulp stem cells (DPSCs), but the role of long noncoding RNAs (lncRNAs) during this process remains unclear. Here, we cultured human DPSCs in osteogenic/odontogenic medium for 14 days and analyzed cells via RNA-sequencing. The data were validated by quantitative reverse transcription-polymerase chain reaction and lncRNA-microRNA (miRNA)-messenger RNA (mRNA) networks were constructed to reveal the potential competing endogenous RNA regulatory role of lncRNAs. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis were performed. One lncRNA, SNHG7, was identified and validated by genetic shRNA silencing. A total of 89 lncRNAs, 1,636 mRNAs, and 113 miRNAs were differentially expressed after differentiation. Bioinformatics identified an array of affected signaling pathways including phosphoinositide-3-kinase-protein kinase B, transforming growth factor-β, and Wnt. mRNAs were enriched in cell migration, cell differentiation, stem cell development, ossification, and skeletal development. One lncRNA, SNHG7, was indentified to inhibit the odonto/osteogenic differentiation of DPSCs when silenced. In summary, we reveal several lncRNAs that significantly change during DPSC differentiation, including SNHG7. This reveals new targets for dentin-pulp complex regeneration and tissue engineering.
TL;DR: The results suggested that lncRNA TUG1 may function as a competing endogenous RNA (ceRNA) for miR‐145 to induce cell damage, possibly providing a new therapeutic target in cerebral ischaemia/reperfusion injury.
Abstract: Emerging studies have shown that long noncoding RNA (lncRNA) TUG1 (taurine-up-regulated gene 1) plays critical roles in multiple biological processes. However, the expression and function of lncRNA TUG1 in cerebral ischaemia/reperfusion injury have not been reported yet. In this study, we found that LncRNA TUG1 expression was significantly up-regulated in cultured MA-C cells exposed to OGD/R injury, while similar results were also observed in MCAO model. Mechanistically, knockdown of TUG1 decreased lactate dehydrogenase levels and the ratio of apoptotic cells and promoted cell survival in vitro. Moreover, knockdown of TUG1 decreased AQP4 (encoding aquaporin 4) expression to attenuate OGD/R injury. TUG1 could interact directly with miR-145, and down-regulation of miR-145 could efficiently reverse the function of TUG1 siRNA on AQP4 expression. Finally, the TUG1 shRNA reduced the infarction area and cell apoptosis in I/R mouse brains in vivo. In summary, our results suggested that lncRNA TUG1 may function as a competing endogenous RNA (ceRNA) for miR-145 to induce cell damage, possibly providing a new therapeutic target in cerebral ischaemia/reperfusion injury.
TL;DR: It is demonstrated that circHIPK3 contributes to hyperglycemia and insulin resistance by sponging miR-192-5p and up-regulating FOXO1.
Abstract: It has been shown that circular RNAs, a class of non-coding RNA molecules, play an important role in the regulation of glucose and lipid homeostasis. In the present study, we sought to investigate the function of circular RNA HIPK3 (circHIPK3) in diabetes-associated metabolic disorders, including hyperglycemia and insulin resistance. Results show that oleate stimulated circHIPK3 increase, and that circHIPK3 enhanced the stimulatory effect of oleate on adipose deposition, triglyceride (TG) content, and cellular glucose content in HepG2 cells. MiR-192-5p was the potential target of circHIPK3, since circHIPK3 significantly decreased miR-192-5p mRNA level, whereas anti-circHIPK3 significantly increased miR-192-5p mRNA level. Further study shows that transcription factor forkhead box O1 (FOXO1) was a downstream regulator of miR-192-5p, since miR-192-5p significantly decreased FOXO1 expression, whereas circHIPK3 significantly increased FOXO1 expression. Notably, the inhibitory effect of miR-192-5p was significantly reversed by circHIPK3. In vivo study shows that anti-miR-192-5p significantly increased blood glucose content, which was significantly inhibited by FOXO1 shRNA. MiR-192-5p significantly decreased adipose deposition and TG content in HepG2 cells, which was significantly reversed by the co-treatment with circHIPK3. Forskolin/dexamethasone (FSK/DEX) significantly increased cellular glucose, mRNA level of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), and this stimulatory effect of FSK/DEX was significantly inhibited by miR-192-5p. In the presence of circHIPK3, however, the inhibitory effect of miR-192-5p was totally lost. In summary, the present study demonstrated that circHIPK3 contributes to hyperglycemia and insulin resistance by sponging miR-192-5p and up-regulating FOXO1.
TL;DR: A novel established regulatory way of LINC00174/miR-138-5p/SOX9 axis was systematically studied, which may provide a new manner for glioma therapy and reveal that LINC0174 shRNA can serve as a tumor suppressor through down-regulating SOX9 in gliomas.
Abstract: Temozolomide (TMZ) is one of the most common drugs selected for glioma chemotherapy, but the therapeutic effect of glioma treatment is usually limited due to its resistance. Long non-coding RNA (lncRNA) is gradually found to be a vital regulator in numerous physiological and pathological processes. Lately, it was revealed that LINC00174 could promote CRC cell growth. However, the function and potential regulatory manner of LINC00174 in glioma remain unclear. Our results demonstrated that the expression level of LINC00174 was higher in glioma tissues, and LINC00174 down-regulation could remarkably prevent cell proliferation and promote cell apoptosis in both glioma cells and TMZ-resistant glioma cells. Mechanistic studies revealed that LINC00174 can sponge microRNA-138-5p (miR-138-5p) and down-regulate its expression, thereby up-regulating the protein level of miR-138-5p's target, sex-determining region Y (SRY)-box9 protein (SOX9). Additionally, in vivo experiments revealed that LINC00174 shRNA can serve as a tumor suppressor through down-regulating SOX9 in glioma. In this study, a novel established regulatory way of LINC00174/miR-138-5p/SOX9 axis was systematically studied, which may provide a new manner for glioma therapy.
TL;DR: This study reveals that GAS5 downregulates M MP9 expression through recruiting EZH2 to MMP9 promoter region and alleviates the progression of renal fibrosis in DN rats, which sheds new light on the therapeutic potential of GAS 5‐targeted therapies in combating that disease.
Abstract: Diabetic nephropathy (DN) is a frequently occurred microvascular complication associated with type I and type II diabetes mellitus. The participation of long noncoding RNAs (lncRNAs) in diabetes-related microvascular complications has been reported extensively. We attempted to unveil the possible regulatory mechanism of lncRNA growth arrest-specific transcript 5 (GAS5) and matrix metalloproteinase 9 (MMP9), an important inflammatory protein, in the progression of DN. A rat DN model was induced by streptozocin (STZ). The low expression of GAS5 and high expression of MMP9 in DN rats with DN was then determined by RT-qPCR and western blot analysis, and lentivirus-mediated GAS5 overexpression was shown to ameliorate STZ-induced renal interstitial fibrosis (RIF) and inflammatory reaction in the kidney of DN rats. Moreover, MMP9 was found to be upregulated in STZ-induced DN, while MMP9 silencing induced by lentivirus expressing shRNA against MMP9 reduced RIF and suppressed inflammation in the kidney of DN rats. RIP, RNA pull-down, and ChIP assays demonstrated that GAS5 downregulated MMP9 via recruiting enhancer of zeste homolog 2 (EZH2) in the promoter region of MMP9. Overall, our study reveals that GAS5 downregulates MMP9 expression through recruiting EZH2 to MMP9 promoter region and alleviates the progression of renal fibrosis in DN rats, which sheds new light on the therapeutic potential of GAS5-targeted therapies in combating that disease.
TL;DR: TIPE promotes angiogenesis in CRC by regulating the expression of VEGFR2, which may be a target for antiangiogenic cancer therapy and is found to be highly expressed in CRC and act as oncogenes.
Abstract: Background: Metastasis is the leading cause of death in colorectal cancer (CRC) patients. It is regulated mainly by tumor cell angiogenesis, and angiogenesis is caused by the binding of vascular endothelial growth factor (VEGF) to vascular endothelial growth factor receptor 2 (VEGFR2). Tumor necrosis factor-α-induced protein 8 (TNFAIP8, hereto after TIPE) plays an important role in tumorigenesis, development, and prognosis. However, the relationship between TIPE and VEGFR2 in CRC angiogenesis and the mechanism of action remain unknown. Method: In this study, we used quantitative real-time PCR, Western blotting and immunohistochemistry to detect TIPE and VEGFR2 expression in 55 specimens from CRC patients. We also used HCT116 CRC cells and human umbilical vein endothelial cells (HUVECs) for in vitro experiments by stably transducing shTIPE and shRNA control lentivirus into HCT116 cells, detecting VEGFR2 expression after TIPE knockdown and repurposing the culture supernatant as conditioned medium to stimulate angiogenesis of HUVECs. In vivo experiments with chicken chorioallantoic membranes (CAMs) and a nude mouse matrix subcutaneous tumor model were performed to validate the effects of TIPE on angiogenesis. Additionally, we analyzed the expression and phosphorylation levels of PDK1 and blocked PDK1 expression using inhibitors to determine whether TIPE-induced changes in VEGFR2-mediated angiogenesis acted via the PI3K-Akt pathway. Results: We found that TIPE and VEGFR2 are highly expressed in CRC and act as oncogenes. TIPE knockdown also downregulated VEGFR2 expression, which resulted in simultaneous inhibition of cell proliferation, cell migration and angiogenesis. Then, in vivo experiments further demonstrated that TIPE promotes angiogenesis in CRC. Finally, we found that TIPE promotes VEGFR2-mediated angiogenesis by upregulating PDK1 expression and phosphorylation and that blocking PDK1 expression can inhibit this process. Conclusion: TIPE promotes angiogenesis in CRC by regulating the expression of VEGFR2, which may be a target for antiangiogenic cancer therapy.
TL;DR: Mechanism investigation indicated that YTHDF1 might promote the aggressive phenotypes by facilitating epithelial–mesenchymal transition (EMT) and activating AKT/glycogen synthase kinase (GSK)-3β/β-catenin signaling.
Abstract: Purpose: N6-methyladenosine (m6A) RNA methylation has been implicated in various malignancies. This study aimed to identify the m6A methylation regulator-based prognostic signature for hepatocellular carcinoma (HCC) as well as provide candidate targets for HCC treatment. Methods: The least absolute shrinkage and selection operator (LASSO) analyses were performed to identify a risk signature in The Cancer Genome Atlas (TCGA) datasets. The risk signature was further validated in International Cancer Genome Consortium (ICGC) and Pan-Cancer Analysis of Whole Genomes (PCAWG) datasets. Following transfection of short hairpin RNA (shRNA) targeting YTHDF1, the biological activities of HCC cells were evaluated by Cell Counting Kit-8 (CCK-8), wound-healing, Transwell, flow cytometry, and xenograft tumor assays, respectively. The potential mechanisms mediated by YTHDF1 were predicted by overrepresentation enrichment analysis (ORA)/gene set enrichment analysis (GSEA) and validated by Western blotting. Results: Overexpression of m6A RNA methylation regulators was correlated with malignant clinicopathological characteristics of HCC patients. The Cox regression and LASSO analyses identified a risk signature with five m6A methylation regulators (KIAA1429, ZC3H13, YTHDF1, YTHDF2, and METTL3). In accordance with HCC cases in TCGA, the prognostic value of risk signature was also determined in ICGC and PCAWG datasets. Following analyzing the expression and clinical implications in TCGA and Gene Expression Omnibus (GEO), YTHDF1 was chosen for further experimental validation. Knockdown of YTHDF1 significantly inhibited the proliferation, migration, and invasion of HCC cells, as well as enhanced the apoptosis in vitro. Moreover, silencing YTHDF1 repressed the growth of xenograft tumors in vivo. Mechanism investigation indicated that YTHDF1 might promote the aggressive phenotypes by facilitating epithelial-mesenchymal transition (EMT) and activating AKT/glycogen synthase kinase (GSK)-3β/β-catenin signaling. Conclusion: The current study identified a robust risk signature consisting of m6A RNA methylation regulators for HCC prognosis. In addition, YTHDF1 was a potential molecular target for HCC treatment.
TL;DR: The novelty of this thermosensitive hydrogel to be an excellent depot for the co-delivery of anticancer drugs and siRNA is demonstrated and will not only provide extended drug release but also combine the advantages offered by the chitosan-based copolymer structure for siRNA delivery to broaden treatment modalities in cancer therapy.
Abstract: In this study, we aimed to develop a multifunctional drug/gene delivery system for the treatment of glioblastoma multiforme by combining the ligand-mediated active targeting and the pH-triggered drug release features of graphene oxide (GO). Toward this end, we load irinotecan (CPT-11) to cetuximab (CET)-conjugated GO (GO-CET/CPT11) for pH-responsive drug release after endocytosis by epidermal growth factor receptor (EGFR) over-expressed U87 human glioblastoma cells. The ultimate injectable drug/gene delivery system was designed by co-entrapping stomatin-like protein 2 (SLP2) short hairpin RNA (shRNA) and GO-CET/CPT11 in thermosensitive chitosan-g-poly(N-isopropylacrylamide) (CPN) polymer solution, which offers a hydrogel depot for localized, sustained delivery of the therapeutics after the in situ formation of CPN@GO-CET/CPT11@shRNA hydrogel. An optimal drug formulation was achieved by considering both the loading efficiency and loading content of CPT-11 on GO-CET. A sustained and controlled release behavior was found for CPT-11 and shRNA from CPN hydrogel. Confocal microscopy analysis confirmed the intracellular trafficking for the targeted delivery of CPT-11 through interactions of CET with EGFR on the U87 cell surface. The efficient transfection of U87 using SLP2 shRNA was achieved using CPN as a delivery milieu, possibly by the formation of shRNA/CPN polyplex after hydrogel degradation. In vitro cell culture experiments confirmed cell apoptosis induced by CPT-11 released from acid organelles in the cytoplasm by flow cytometry, as well as reduced SLP2 protein expression and inhibited cell migration due to gene silencing. Finally, in vivo therapeutic efficacy was demonstrated using the xenograft of U87 tumor-bearing nude mice through non-invasive intratumoral delivery of CPN@GO-CET/CPT11@shRNA by injection. Overall, we have demonstrated the novelty of this thermosensitive hydrogel to be an excellent depot for the co-delivery of anticancer drugs and siRNA. The in situ forming hydrogel will not only provide extended drug release but also combine the advantages offered by the chitosan-based copolymer structure for siRNA delivery to broaden treatment modalities in cancer therapy.
TL;DR: CAR T-derived IL-6 is one of the most important initiators to amplify release of IL- 6 from monocytes that further drive sCRS development and shRNA technology provide a promising strategy to improve the safety of CAR T cell therapy.
Abstract: T cells expressing a chimeric antigen receptor (CAR) engineered to target CD19 can treat leukemia effectively but also increase the risk of complications such as cytokine release syndrome (CRS) and CAR T cell related encephalopathy (CRES) driven by interleukin-6 (IL-6). Here, we investigated whether IL-6 knockdown in CART-19 cells can reduce IL-6 secretion from monocytes, which may reduce the risk of adverse events. Supernatants from cocultures of regular CART-19 cells and B lymphoma cells were added to monocytes in vitro, and the IL-6 levels in monocyte supernatants were measured 24 h later. IL-6 expression was knocked down in regular CART-19 cells by adding a short hairpin RNA (shRNA) (termed ssCART-19) expression cassette specific for IL-6 to the conventional CAR vector. Transduction efficiency and cell proliferation were measured by flow cytometry, and cytotoxicity was measured by evaluating the release of lactate dehydrogenase into the medium. Gene expression was assessed by qRT-PCR and RNA sequencing. A xenograft leukemia mouse model was established by injecting NOD/SCID/γc-/- mice with luciferase-expressing B lymphoma cells, and then the animals were treated with regular CART-19 cells or ssCART-19. Tumor growth was assessed by bioluminescence imaging. Both recombinant IL-6 and CART-19 derived IL-6 significantly triggered IL-6 release by monocytes. IL-6 knockdown in ssCART-19 cells dramatically reduced IL-6 release from monocytes in vitro stduy. In vivo study further demonstrated that the mice bearing Raji cells treated with ssCART-19 cells showed significant lower IL-6 levels in serum than those treated with regular CART-19 cells, but comparable anti-tumor efficacy between the animal groups. CAR T-derived IL-6 is one of the most important initiators to amplify release of IL-6 from monocytes that further drive sCRS development. IL-6 knockdown in ssCART-19 cells by shRNA technology provide a promising strategy to improve the safety of CAR T cell therapy.
TL;DR: MALAT1 regulates miR-206/IGF-1 axis, thereby modulating trophoblast cells migration and invasion through PI3K/Akt signal pathway, and shows light on the underlying mechanisms of PE and provides potential targets for PE therapy.
Abstract: Preeclampsia (PE) is a pregnancy-specific syndrome that substantially leads to maternal and fetal mortality. Multiple factors contribute to the disease, but the exact pathogenesis still remains elusive. Here we explored the roles of lncRNA MALAT1 and miR-206 in PE. qRT-PCR was applied to measure mRNA levels of MALAT1 and miR-206 in the placenta of PE patients. Scratch wound healing assay and transwell invasion assay were conducted to test the effects of MALAT1 and miR-206 on migration and invasion of trophoblast cells. In addition, we validated MALAT1/miR-206 and miR-206/IGF-1 interactions with dual luciferase reporter assay. Western bot was used to detect protein expressions of IGF-1, p-PI3K, PI3K, p-Akt and Akt. We found that MALAT1 was decreased but miR-206 was increased in the placenta of patients with PE. Inhibition of MALAT1, knockdown IGF-1, or miR-206 mimics suppressed the trophoblast cells migration and invasion, while overexpression of MALAT1, IGF-1 or miR-206 inhibitors exhibited opposite effects. Further, miR-206 was confirmed as a direct target of MALAT1. Besides, miR-206 inhibited IGF-1 expression by directly binding to the 3'UTR. Mechanistically, our study demonstrated that MALAT1 regulates IGF-1/PI3K/Akt signaling via miR-206. Together, these results suggest that MALAT1 and miR-206 play important roles in PE. MALAT1 regulates miR-206/IGF-1 axis, thereby modulating trophoblast cells migration and invasion through PI3K/Akt signal pathway. These results show light on the underlying mechanisms of PE and provide potential targets for PE therapy.Abbreviations: PE: Preeclampsia; lncRNA: Long-non-coding RNA; MALAT1: Metastasis-associated lung adenocarcinoma transcript 1; IGF-1: Insulin-like growth factor 1; PI3k: Phosphatidylinositol-4, 5-bisphosphate 3-kinase; Akt: Protein kinase B; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; qRT-PCR: Quantitative Reverse Transcription polymerase chain reaction; shRNA: Short hairpin RNA; siRNA: Small interfering RNA; EMT: Epithelial-mesenchymal transition.
TL;DR: The results show that IL-6 is involved in immunosuppression triggered by human mesenchymal stromal cells, and data indicate thatIL-6 signals in hMSCs by a previously undescribed intracellular mechanism.
Abstract: Interleukin (IL)-6 is a pleiotropic cytokine involved in the regulation of hematological and immune responses. IL-6 is secreted chiefly by stromal cells, but little is known about its precise role in the homeostasis of human mesenchymal stromal cells (hMSCs) and the role it may play in hMSC-mediated immunoregulation. We studied the role of IL-6 in the biology of bone marrow derived hMSC in vitro by silencing its expression using short hairpin RNA targeting. Our results show that IL-6 is involved in immunosuppression triggered by hMSCs. Cells silenced for IL-6 showed a reduced capacity to suppress activated T-cell proliferation. Moreover, silencing of IL-6 significantly blocked the capacity of hMSCs to proliferate. Notably, increasing the intracellular level of IL-6 but not recovering the extracellular level could restore the proliferative impairment observed in IL-6-silenced hMSC. Our data indicate that IL-6 signals in hMSCs by a previously undescribed intracellular mechanism.
TL;DR: It is demonstrated that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.
Abstract: In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.
TL;DR: MMP-1 may promote malignant behavior in colorectal cancer via EMT and the Akt signaling pathway through which it was significantly related to lymphatic metastasis as well as TNM stage.
Abstract: The present study aimed to investigate the role of matrix metalloproteinase‑1 (MMP‑1) in the development of colorectal cancer and reveal the mechanism underlying this progression. Bioinformatics methods and a public dataset were first used to analyze MMP‑1 gene expression in a public dataset. MMP‑1 expression in colorectal cancer patients was assessed by immunohistochemistry; its association with clinicopathological parameters and its significance for prognosis were analyzed. Then proliferation, scratch and Transwell assays and a xenograft model were used to assess the change in malignant behavior in cells transfected with an MMP‑1 shRNA. Changes involved in epithelial‑mesenchymal transition (EMT) and the Akt signaling pathway were detected by western blotting. According to the results, MMP‑1 expression was higher in colorectal cancer tissues than it was in matched adjacent noncancerous tissues, and its high expression was significantly related to lymphatic metastasis as well as TNM stage (P<0.05). Downregulation of MMP‑1 expression inhibited the progression of colorectal cancer in vitro and in vivo. Furthermore, after the cells were stably transfected with MMP‑1 shRNA, the expression of N‑cadherin, vimentin and Twist1 decreased while that of E‑cadherin increased. The expression of p‑Akt and c‑Myc also decreased. In conclusion, MMP‑1 may promote malignant behavior in colorectal cancer via EMT and the Akt signaling pathway.