[Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells].

OBJECTIVE: To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function. METHODS: Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1ß, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR. RESULTS: Knockdown of HuR significantly up- regulated the expressions of IL-1ß, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05). CONCLUSION: circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells / 南方医科大学学报

OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

ALKBH5-Modified HMGB1-STING Activation Contributes to Radiation Induced Liver Disease via Innate Immune Response.

PURPOSE: Radiation therapy, which is vital for the treatment of primary liver cancer, comes with unavoidable liver injury, which limits its implementation. N6-methyladenosine (m6A) methylation is involved in many molecular functions. However, its role in radiation-induced liver diseases (RILD) remains unknown. Herein, we investigate the role of m6A methylation in RILD. METHODS AND MATERIALS: Methylated RNA-immunoprecipitation sequencing and RNA transcriptome sequencing were used to reveal the methylation pattern of human hepatic stellate cells (HSCs) exposed to irradiation. C3H/HeN mice and stimulator of interferon genes (STING)-deficient mice underwent x-ray irradiation of 24 Gy in 3 fractions. The m6A methylation of the high-mobility group box 1 (HMGB1) transcript was validated using methylated RNA immunoprecipitation, RNA immunoprecipitation, luciferase assays, and a messenger RNA decay assay. RESULTS: Human hepatic stellate cells showed significant differences in methylation patterns after 8 Gy of x-ray irradiation. Irradiation recruited AlkB homolog 5 (ALKBH5) to demethylate m6A residues in the 3' untranslated region of HMGB1, which resulted in the activation of STING-interferon regulatory factor 3 signaling. Changes in the transcription of the 3' untranslated region of HMGB1 occurred after the knockdown of ALKBH5, which were eliminated after m6A residue mutation. Strikingly, ALKBH5 deficiency or HMGB1 silencing both attenuated type I interferon production and decreased hepatocyte apoptosis. In vivo depletion of ALKBH5 abolished the upregulation of HMGB1-mediated STING signaling and decreased liver inflammation, which was consistent with STING-/- mice treated with irradiation. Notably, YTHDF2 (m6A reader protein) directly bound to HMGB1 m6A-modified sites and promoted its degradation. CONCLUSIONS: ALKBH5-dependent HMGB1 expression mediates STING-interferon regulatory factor 3 innate immune response in RILD.

Circular RNA TUBD1 Acts as the miR-146a-5p Sponge to Affect the Viability and Pro-Inflammatory Cytokine Production of LX-2 Cells through the TLR4 Pathway.

The functions and molecular mechanism of circRNAs in the development of radiation-induced liver disease (RILD) remain largely unknown. The goal of this study was to explore the expression and potential role of a new circular RNA, named circTUBD1, in irradiated and lipopolysaccharide (LPS)-stimulated human hepatic stellate cell (HSC) line LX-2 cells. The expression of circTUBD1 was significantly upregulated in irradiated and LPS-stimulated LX-2 cells compared to non-treated LX-2 cells. To explore the functions of circTUBD1, small interfering RNAs targeting circTUBD1 were designed. Silencing circTUBD1 inhibited proliferation, promoted apoptosis of LX-2 cells, and significantly decreased the expression level of pro-inflammatory cytokines, including IL-1ß, IL-6 and TNF-α in irradiated and LPS-stimulated LX-2 cells. Mechanistic analysis suggested that circTUBD1 acted as the miR-146a-5p sponge to affect pro-inflammatory cytokine production through regulating expression of Toll-like receptor 4 (TLR4), interleukin receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor-6 (TRAF6), and phosphorylation of nuclear factor-kappa B (pNF-κB) in irradiated and LPS-stimulated LX-2 cells. To our knowledge, this is the first study to show that circTUBD1 acts as a miR-146a-5p sponge to affect the viability and pro-inflammatory cytokine production of LX-2 cells through the TLR4 pathway, suggesting that circTUBD1 is a potential target for RILD therapy.

Therapeutic modulators of hepatic stellate cells for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary tumor in the liver and is a leading cause of cancer-related death worldwide. Activated hepatic stellate cells (HSCs) are key components of the HCC microenvironment and play an important role in the onset and progression of HCC through the secretion of growth factors and cytokines. Current treatment modalities that include chemotherapy, radiotherapy and ablation are able to activate HSCs and remodel the tumor microenvironment. Growing evidence has demonstrated that the complex interaction between activated HSCs and tumor cells can facilitate cancer chemoresistance and metastasis. Therefore, therapeutic targeting of activated HSCs has emerged as a promising strategy to improve treatment outcomes for HCC. This review summarizes the molecular mechanisms of HSC activation triggered by treatment modalities, the function of activated HSCs in HCC, as well as the crosstalk between tumor cells and activated HSCs. Pathways of activated HSC reduction are discussed, including inhibition, apoptosis, and reversion to the inactivated state. Finally, we outline the progress and challenges of therapeutic approaches targeting activated HSCs in the development of HCC treatment.

Circular RNA RSF1 promotes inflammatory and fibrotic phenotypes of irradiated hepatic stellate cell by modulating miR-146a-5p.

The role of circular RNA (circRNA) in radiation-induced liver disease (RILD) remains largely unknown. In this study, Ras-related C3 botulinum toxin substrate 1 (RAC1) was elevated in irradiated human hepatic stellate cell (HSC) line LX2, the important effector cell mediating RILD. Overexpression of RAC1 promotes cell proliferation, proinflammatory cytokines production, and α-smooth muscle actin expression, which were blocked by microRNA (miR)-146a-5p mimics. CircRNA RSF1 (circRSF1) was upregulated in irradiated LX2 cells and predicted to harbor binding site for miR-146a-5p. Biotinylated-RNA pull down and dual-luciferase reporter detection confirmed the direct interaction of circRSF1 and miR-146a-5p. Enforced expression of circRSF1 increased RAC1 expression by acting as miR-146a-5p sponge to inhibit miR-146a-5p activity, and thus enhanced the cell viability, and promoted inflammatory and fibrotic phenotype of irradiated LX2 cells. These findings indicate a functional regulatory axis composing of circRSF1, miR-146a-5p, and RAC1 in irradiated HSC, which may provide attractive therapeutic targets for RILD.

MicroRNA-146a-5p Attenuates Fibrosis-related Molecules in Irradiated and TGF-beta1-Treated Human Hepatic Stellate Cells by Regulating PTPRA-SRC Signaling.

MicroRNAs (miRNAs) have been shown to play a pivotal role in the pathogenesis and maintenance of liver fibrosis by altering expression of their downstream target genes. However, their role in radiation-induced liver fibrosis has not been assessed in detail. Here, we investigated the role of miR-146a-5p and the target gene in regulation of fibrosis-related markers in the human hepatic stellate cell line LX2. LX2 cells were stimulated with 8 Gy of X rays and various concentrations of TGF-ß1 (0-5 ng/ml). Expression of α-SMA, collagen 1 and miR-146a-5p was evaluated. The MiR-146a-5p target gene predictions were performed using bioinformatics analysis and confirmed by dual-luciferase reporter experiment. The effect of miR-146a-5p and the involved target gene on the expression of these fibrogenic molecules was also assessed. Expression of α-SMA and collagen 1 were upregulated in response to radiation and/or TGF-ß1 treatment and miR-146a-5p levels were altered in LX2 cells. Restoration of miR-146a-5p expression suppressed expression of α-SMA and collagen 1 in irradiated and TGF-ß1-treated LX2 cells. Subsequent mechanism experiments revealed that miR-146a-5p overexpression inhibited PTPRA expression by binding to its 3'-untrans-lated region and reduced SRC activation. In addition, enhancement of PTPRA partially reversed the suppressive effect of miR-146a-5p on α-SMA and collagen 1 expression in LX2 cells. In conclusion, miR-146a-5p may negatively regulate the PTPRA-SRC signaling to inhibit expression of fibrosis-related markers in irradiated and TGF-ß1-stimulated LX2 cells.

Pinealectomy or light exposure exacerbates biliary damage and liver fibrosis in cholestatic rats through decreased melatonin synthesis.

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.

Proteomic Profiling of Human Hepatic Stellate Cell Line LX2 Responses to Irradiation and TGF-ß1.

Hepatic stellate cells (HSCs) are the main target of radiation damage and primarily contribute to the development of radiation-induced liver fibrosis. However, the molecular events underlying the radiation-induced activation of HSCs are not fully elucidated. In the present study, human HSC line LX2 was treated with X-ray irradiation and/or TGF-ß1, and profibrogenic molecules were evaluated. The iTRAQ LC-MS/MS technology was performed to identify global protein expression profiles in LX2 following exposure to different stimuli. Irradiation or TGF-ß1 alone increased expression of α-SMA, collagen 1, CTGF, PAI-1, and fibronectin. Irradiation and TGF-ß1 cooperatively induced expression of these profibrotic markers. In total, 102, 137, 155 dysregulated proteins were identified in LX2 cell samples affected by irradiation, TGF-ß1, or cotreatment, respectively. Bioinformatic analyses showed that the three differentially expressed protein sets were commonly associated with cell cycle and protein processing in endoplasmic reticulum. The expression of a set of proteins was properly validated: CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3 were upregulated upon irradiation or irradiation and TGF-ß1 costimulation, whereas SPARC and THBS1 were elevated by TGF-ß1 or TGF-ß1 plus irradiation treatment. Furthermore, CDC20 inhibition suppressed expression of profibrotic markers in irradiated and TGF-ß1-stimulated LX2 cells. Detailed data on potential molecular mechanisms causing the radiation-induced HSC activation presented here would be instrumental in developing radiotherapy strategies that minimize radiation-induced liver fibrosis.

Diosmin attenuates radiation-induced hepatic fibrosis by boosting PPAR-γ expression and hampering miR-17-5p-activated canonical Wnt-ß-catenin signaling.

BACKGROUND: Liver fibrosis is one of the major complications from upper right quadrant radiotherapy. MicroRNA-17-5p (miR-17-5p) is hypothesized to act as a regulator of hepatic stellate cell (HSCs) activation by activation of the canonical Wnt-ß-catenin pathway. Diosmin (Dios), a citrus bioflavonoid, is known to possess potent antioxidant, anti-inflammatory, and anti-apoptotic properties. PURPOSE: To explore the molecular mechanisms that underlie radiation-induced liver fibrosis, and to evaluate the possible influence of Dios on the miR-17-5p-Wnt-ß-catenin signaling axis during fibrogenesis provoked by irradiation (IRR) in rats. Also, the effect of Dios on hepatic peroxisome proliferator activated receptor-γ (PPAR-γ) expression as a regulator for HSC activation was considered. METHODS: We administered 100 mg·(kg body mass)-1·day-1 (per oral) of Dios were administered to IRR-exposed rats (overall dose of 12 Gy on 6 fractions of 2 Gy each) for 6 successive weeks. RESULTS: Data analysis revealed that Dios treatment mitigated oxidative stress, enhanced antioxidant defenses, alleviated hepatic inflammatory responses, abrogated pro-fibrogenic cytokines, and stimulated PPAR-γ expression. Dios treatment repressed the miR-17-5p activated Wnt-ß-catenin signaling induced by IRR. Moreover, Dios treatment restored the normal hepatic architecture and reversed pathological alterations induced by IRR. CONCLUSION: We hypothesize that the stimulation of PPAR-γ expression and interference with miR-17-5p activated Wnt-ß-catenin signaling mediates the antifibrotic properties of Dios.

Dicliptera Chinensis polysaccharides target TGF-ß/Smad pathway and inhibit stellate cells activation in rats with dimethylnitrosamine-induced hepatic fibrosis.

This study aims to study impact of Dicliptera chinensis polysaccharide (DCP) on hepatic fibrosis (HF) and activation of hepatic stellate cells (HSCs). Liver fibrosis model was induced by intraperitoneal injection of dimethyl nitrosamines (DMN) in rat. Rats in treatment group were administrated with different concentrations of DCP (0, 100, 300 mg/kg) by intraperitoneal injection. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to assess histo-pathological change. α-SMA, TGF-ß1 and pSmad 2/3 were assayed by immuno-histochemistry. HSC-T6 cells were stimulated by recombined rat TGF-ß1 (1 ng/mL) to simulate an activating model in vitro and then interfered with DCP (concentration of 0, 25, 50, 100, 200, 400 µg/ml). MTT assay was used to determine cell proliferation and western blotting was used to detect α-SMA and pSmad 2/3 expression. Results demonstrated that DCP alleviated DMN-induced liver fibrosis in rat and significantly down-regulated TGF-ß1 expression, pSmad2/3 and α-SMA in liver tissue in a dose-dependent way. DCP inhibited proliferation and activation of TGF-ß1-stimulated HSC-T6 in vitro and significantly down-regulated α-SMA and pSmad2/3 expression. In conclusion, this study revealed that DCP attenuates progression of liver fibrosis through suppressing TGF-ß/Smad pathway. DCP is a potential botanical polysaccharide to management liver fibrosis.

Low-level laser therapy ameliorates CCl4-induced liver cirrhosis in rats.

This study investigated the effects of low-level laser therapy (LLLT) in the liver function, structure and inflammation in a experimental model of carbon tetrachloride (CCl(4))-induced liver cirrhosis. Wistar rats were divided into Control, LLLT, CCl(4) and CCl(4) +LLLT groups. CCl(4) groups received CCl(4) (0.4 g kg(-1); i.p.), three times a week, for 12 weeks. A 830 nm LLLT was performed with a continuous wave, 35 mW, 2.5 J cm(-2) per point, applied to four points of the liver (right and left upper and lower extremities, in the four lobes of the liver) for 2 weeks. Liver structure and inflammation (cirrhotic areas, collagen deposition, inflammation, density of Kupffer and hepatic stellate cells) and function (aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase, lactate dehydrogenase, total proteins and globulins) were evaluated. LLLT significantly reduced CCl(4)-increased aspartate aminotransferase (P < 0.001), alkaline phosphatase (P < 0.001), gamma-glutamyl transferase (P < 0.001) and lactate dehydrogenase (P < 0.01) activity, as well as total proteins (P < 0.05) and globulins (P < 0.01). LLLT also reduced the number of cirrhotic areas, the collagen accumulation and the hepatic inflammatory infiltrate. Of note, LLLT reduced CCl(4)-increased number of Kupffer cells (P < 0.05) and hepatic stellate cells (P < 0.05). We conclude that LLLT presents beneficial effects on liver function and structure in an experimental model of CCl(4)-induced cirrhosis.

Hedgehog signaling regulates the repair response in mouse liver damaged by irradiation.

Radiotherapy is commonly used in treating many kinds of cancers that cannot be cured by other therapeutic strategies. However, radiation-induced fibrosis in the treatment of intrahepatic cancer is a major obstacle. Hedgehog pathway is known to regulate the fibrotic process and proliferation of progenitor cells. Hedgehog ligands act as a profibrotic factor and hedgehog-responsive cells undergo epithelial-to-mesenchymal transition (EMT), eventually contributing to the fibrogenic process. Herein, we investigated whether the hedgehog pathway was associated with radiation-induced hepatic fibrosis. Female mice were irradiated with a single dose of 20 Gy and were sacrificed 1 week postirradiation, to obtain the livers for biochemical and histological analysis. Hematoxylin and eosin and Sirius Red staining were used in evaluating liver morphology and fibrosis, respectively. Immunochemical staining for active caspase 3 and CD44 was used to examine the repair response of the irradiated livers. Immunoblot analysis was performed to detect the expression of hedgehog molecules and fibrogenic markers. Fat accumulation in hepatocytes and increased apoptosis were observed in liver sections from mice treated with radiation. Expression of hedgehog ligand, Indian hedgehog, and hedgehog target gene, Gli2, were significantly up-regulated in the liver of mice treated with radiation. Levels of transforming growth factor-ß (inducer of fibrosis) and α-smooth muscle actin (marker of myofibroblastic hepatic stellate cells) were also greatly increased in the damaged liver compared to the normal liver. The EMT marker, laminin-ß3, showed a great increase, whereas EMT inhibitor, bmp7, was significantly decreased in mouse liver postirradiation. Furthermore, CD44-positive progenitors were shown to accumulated in the injured liver. These results suggest that increased expression of hedgehog signaling promotes proliferation of myofibroblastic hepatic stellate cells and progenitors, and thereby contributes to the repair response after irradiation.