Development of di(2-ethylhexyl) phthalate-containing thioglycolic acid immobilized chitosan mucoadhesive gel as an alternative hormone therapy for menopausal syndrome.

Menopausal syndrome includes the symptoms that most women experience owing to hormone changes after menopause. Although hormone replacement therapy is a common treatment for menopausal syndrome, there are still many side effects and challenges hindering research. In this study, thioglycolic acid (TGA)-immobilized chitosan mucoadhesive gel was synthesized by a new method of low concentration of 1,4-butanediol diglycidyl ether (BDDE) would encapsulate di(2-ethylhexyl) phthalate (DEHP) as an alternative hormone replacement therapy for menopausal syndrome. The efficacies of the DEHP-containing TGA-chitosan gel (CT-D) were confirmed and evaluated by materials characterization and in vitro study. Results showed that CT-D was not cytotoxic and had better mucoadhesive ability than chitosan. The animal model was constructed 1 month after bilateral ovariectomy in SD rats. CT-D was administered intravaginally every 3 days. Bodyweight, wet weight of the uterus and vagina, vaginal smears, histology, blood element analysis, and serological analysis was used to assess the ability of the material to relieve menopausal syndrome. The results indicated that the combination of the sustained release of DEHP and mucoadhesive TGA-immobilized chitosan allows the developed CT-D to relieve the menopausal syndrome through low concentrations of DEHP, which falls in the safety level of the tolerable daily intake of DEHP.

Engineered cell-laden thermosensitive poly(N-isopropylacrylamide)-immobilized gelatin microspheres as 3D cell carriers for regenerative medicine.

Several studies have focused on using cell carriers to solve the problem of mesenchymal stem cell expansion on regenerative medicine. However, the disadvantages of using prolonged enzymatic treatment and low cell harvest efficiency still trouble researchers. In this study, PNIPAAm-immobilized gelatin microspheres (abbreviated as GNMS) were synthesized using a simple power-driven flow-focusing microinjection system. The developed thermosensitive GNMS can allow easier harvesting of cells from the microspheres, requiring only 10 â€‹min of low-temperature treatment and 5 â€‹min of trypsin treatment. The developed GNMS was characterized by Fourier-transform infrared spectroscopy, optical microscopy, and scanning electron microscopy. Further, live/dead staining, F-actin staining, and PrestoBlue cell viability assays were used to evaluate cytotoxicity, cell morphology, cell proliferation, and harvest efficiency. The gene expression of stem cell markers was determined by real-time quantitative PCR (Q-PCR) analysis to investigate the stemness and phenotypic changes in Wharton's jelly-derived mesenchymal stem cells. The results showed that the engineered cell-laden thermosensitive GNMS could significantly increase the cell harvest rate with over 99% cell survival rate and no change in the cell phenotype. Thus, the described strategy GNMS could be the suitable 3D cell carriers in the therapeutic application and opens new avenues for regenerative medicine.

The Production of Fat-Containing Cultured Meat by Stacking Aligned Muscle Layers and Adipose Layers Formed From Gelatin-Soymilk Scaffold.

Tissue engineered cultured meat has been proposed as an emerging innovative process for meat production to overcome the severe consequences of livestock farming, climate change, and an increasing global population. However, currently, cultured meat lacks organized tissue structure, possesses insufficient fat content, and incurs high production costs, which are the major ongoing challenges. In this study, a developed scaffold was synthesized using gelatin and soymilk to create a friendly environment for myogenesis and adipogenesis in C2C12 and 3T3-L1 cells, respectively. The fat containing cultured meat was fabricated with an aligned muscle-like layer and adipose-like layer by stacking these layers alternately. The muscle-like layer expressing myosin and the adipose-like layer abundant in fat were sandwiched to form fat containing muscle tissue. The cytotoxicity and cell survival rate were evaluated using the WST-1 assay and live/dead staining. Myogenesis was confirmed by the expression of myogenin and myosin. The myotubes, myofibrils, and sarcomeres were observed under an inverted microscope, fluorescence microscope, and scanning electron microscope. Adipogenesis was evaluated by protein expression of the peroxisome proliferator-activated receptor γ, and oil droplet accumulation was determined by fluorescence microscopy with Nile Red stain. Extracellular matrix secretion was examined by safranin-O staining. In this study, the cultured meat was prepared with muscle-like texture with the addition of pre-adipocyte, where the multilayered muscle-like tissues with fat content would produce juicy cultured meat.

The establishment of CDK9/RNA PolII/H3K4me3/DNA methylation feedback promotes HOTAIR expression by RNA elongation enhancement in cancer.

Long non-coding RNA HOX Transcript Antisense RNA (HOTAIR) is overexpressed in multiple cancers with diverse genetic profiles. Importantly, since HOTAIR heavily contributes to cancer progression by promoting tumor growth and metastasis, HOTAIR becomes a potential target for cancer therapy. However, the underlying mechanism leading to HOTAIR deregulation is largely unexplored. Here, we performed a pan-cancer analysis using more than 4,200 samples and found that intragenic exon CpG island (Ex-CGI) was hypermethylated and was positively correlated to HOTAIR expression. Also, we revealed that Ex-CGI methylation promotes HOTAIR expression through enhancing the transcription elongation process. Furthermore, we linked up the aberrant intragenic tri-methylation on H3 at lysine 4 (H3K4me3) and Ex-CGI DNA methylation in promoting transcription elongation of HOTAIR. Targeting the oncogenic CDK7-CDK9-H3K4me3 axis downregulated HOTAIR expression and inhibited cell growth in many cancers. To our knowledge, this is the first time that a positive feedback loop that involved CDK9-mediated phosphorylation of RNA Polymerase II Serine 2 (RNA PolII Ser2), H3K4me3, and intragenic DNA methylation, which induced robust transcriptional elongation and heavily contributed to the upregulation of oncogenic lncRNA in cancer has been demonstrated. Targeting the oncogenic CDK7-CDK9-H3K4me3 axis could be a novel therapy in many cancers through inhibiting the HOTAIR expression.

The Synthesis of Europium-Doped Calcium Carbonate by an Eco-Method as Free Radical Generator Under Low-Intensity Ultrasonic Irradiation for Body Sculpture.

Body sculpture is a common method to remove excessive fat. The diet and exercise are the first suggestion to keep body shape; however, those are difficult to keep adherence. Ultrasound has been developed for fat ablation; however, it could only serve as the side treatment along with liposuction. In the study, a sonosensitizer of europium-doped calcium carbonate (CaCO3: Eu) would be synthesized by an eco-method and combined with low-intensity ultrasound for lipolysis. The crystal structure of CaCO3: Eu was identified by x-ray diffractometer (XRD). The morphology of CaCO3: Eu was analyzed by scanning electron microscope (SEM). The chemical composition of CaCO3: Eu was evaluated by energy-dispersed spectrophotometer (EDS) and inductively coupled plasma mass spectrometer (ICP-MS). The electronic diffraction pattern was to further check crystal structure of the synthesized individual grain by transmission electron microscope (TEM). The particle size was determined by Zeta-sizer. Water-soluble tetrazolium salt (WST-1) were used to evaluate the cell viability. Chloromethyl-2',7'-dichlorofluorescein diacetate (CM-H2DCFDA) and live/dead stain were used to evaluate feasibility in vitro. SD-rat was used to evaluate the safety and efficacy in vivo. The results showed that CaCO3: Eu had good biocompatibility and could produce reactive oxygen species (ROS) after treated with low-intensity ultrasound. After 4-weeks, the CaCO3: Eu exposed to ultrasound irradiation on SD rats could significantly decrease body weight, waistline, and subcutaneous adipose tissue. We believe that ROS from sonoluminescence, CO2-bomb and locally increasing Ca2+ level would be three major mechanisms to remove away adipo-tissue and inhibit adipogenesis. We could say that the combination of the CaCO3: Eu and low-intensity ultrasound would be a non-invasive treatment for the body sculpture.

Glucose deprivation-induced aberrant FUT1-mediated fucosylation drives cancer stemness in hepatocellular carcinoma.

Rapidly growing tumors often experience hypoxia and nutrient (e.g., glucose) deficiency because of poor vascularization. Tumor cells respond to the cytotoxic effects of such stresses by inducing molecular adaptations that promote clonal selection of a more malignant tumor-initiating cell phenotype, especially in the innermost tumor regions. Here, we report a regulatory mechanism involving fucosylation by which glucose restriction promotes cancer stemness to drive drug resistance and tumor recurrence. Using hepatocellular carcinoma (HCC) as a model, we showed that restricted glucose availability enhanced the PERK/eIF2α/ATF4 signaling axis to drive fucosyltransferase 1 (FUT1) transcription via direct binding of ATF4 to the FUT1 promoter. FUT1 overexpression is a poor prognostic indicator for HCC. FUT1 inhibition could mitigate tumor initiation, self-renewal, and drug resistance. Mechanistically, we demonstrated that CD147, ICAM-1, EGFR, and EPHA2 are glycoprotein targets of FUT1, in which such fucosylation would consequently converge on deregulated AKT/mTOR/4EBP1 signaling to drive cancer stemness. Treatment with an α-(1,2)-fucosylation inhibitor sensitized HCC tumors to sorafenib, a first-line molecularly targeted drug used for advanced HCC patients, and reduced the tumor-initiating subset. FUT1 overexpression and/or CD147, ICAM-1, EGFR, and EPHA2 fucosylation may be good prognostic markers and therapeutic targets for cancer patients.

LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Gemcitabine resistance is rapidly acquired by pancreatic ductal adenocarcinoma (PDAC) patients. Novel approaches that predict the gemcitabine response of patients and enhance gemcitabine chemosensitivity are important to improve patient survival. We aimed to identify genes as novel biomarkers to predict the gemcitabine response and the therapeutic targets to attenuate chemoresistance in PDAC cells. METHODS: Genome-wide RNA interference screening was conducted to identify genes that regulated gemcitabine chemoresistance. A cell proliferation assay and a tumor formation assay were conducted to study the role of lethal giant larvae homolog 1 (LLGL1) in gemcitabine chemoresistance. Levels of LLGL1 and its regulating targets were measured by immunohistochemical staining in tumor tissues obtained from patients who received gemcitabine as a single therapeutic agent. A gene-expression microarray was conducted to identify the targets regulated by LLGL1. RESULTS: Silencing of LLGL1 markedly reduced the gemcitabine chemosensitivity in PDAC cells. Patients had significantly shorter survival (6 months) if they bore tumors expressing low LLGL1 level than tumors with high LLGL1 level (20 months) (hazard ratio, 0.1567; 95% CI, 0.05966-0.4117). Loss of LLGL1 promoted cytokine receptor oncostatin M receptor (OSMR) expression in PDAC cells that led to gemcitabine resistance, while knockdown of OSMR effectively rescued the chemoresistance phenotype. The LLGL1-OSMR regulatory pathway showed great clinical importance because low LLGL1 and high OSMR expressions were observed frequently in PDAC tissues. Silencing of LLGL1 induced phosphorylation of extracellular signal-regulated kinase 2 and specificity protein 1 (Sp1), promoted Sp1 (pThr453) binding at the OSMR promoter, and enhanced OSMR transcription. CONCLUSIONS: LLGL1 possessed a tumor-suppressor role as an inhibitor of chemoresistance by regulating OSMR-extracellular signal-regulated kinase 2/Sp1 signaling. The data sets generated and analyzed during the current study are available in the Gene Expression Omnibus repository (ID: GSE64681).

A study of Drynaria fortunei in modulation of BMP­2 signalling by bone tissue engineering

Background/aim: Drynaria fortunei (Gusuibu; GSB) is a popular traditional Chinese medicine used for bone repair. An increasing number of studies have reported that GSB induces osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). These results provide insight into the application of GSB for bone tissue engineering techniques used to repair large bone defects. However, few studies have described the molecular mechanisms of GSB. Materials and methods: In the present study, the effects of GSB and naringin, a marker compound, on the binding of BMP-2 to BMPR and BMP-2-derived signal transduction were investigated using surface plasmon resonance (SPR) and coculturing with BMPR- expressed cell line, C2C12, respectively. Furthermore, naringin was also used to prepare naringin contained scaffolds for bone tissue engineering. The physical and chemical properties of these scaffolds were analysed using scanning electron microscopy (SEM) and highperformance liquid chromatography (HPLC). These scaffolds were cocultured with rabbit BMSCs in vitro and implanted into rabbit calvarial defects for bone repair assessment. Results: The results showed that GSB and naringin affect the binding of BMP and BMPR in SPR experiments. GSB is a subtle BMP modulator that simultaneously inhibits the binding of BMP-2 to BMPR-1A and enhances its binding to BMPR-1B. In contrast, naringin inhibited BMP-2 binding to BMPR-1A. In vitro studies involving the phosphorylation of signals downstream of BMPR and Smad showed that GSB and naringin affected stem cell differentiation by inhibiting BMPR-1A signalling. When using GSB for bone tissue engineering, naringin exhibited a higher capacity for slow and gradual release from the scaffold, which promotes bone formation via osteoinduction. Moreover, control and naringin scaffolds were implanted into rabbit calvarial defects for 4 weeks, and naringin enhanced bone regeneration in vivo significantly. Conclusions: GSB and its marker compound (naringin) could inhibit the binding of BMP-2 and BMPR-1A to control cell differentiation by blocked BMPR-1A signalling and enhanced BMPR-1B signalling. GSB and naringin could be good natural BMP regulators for bone tissue engineering.

Ectopic HOTTIP expression induces noncanonical transactivation pathways to promote growth and invasiveness in pancreatic ductal adenocarcinoma.

HOXA transcript at the distal tip (HOTTIP), a long noncoding RNA, is upregulated in pancreatic ductal adenocarcinoma (PDAC), but the HOTTIP-mediated oncogenic pathway is not fully understood. We identified canonical HOTTIP-HOXA13 targets, CYP26B1, CLIC5, CHI3L1 and UCP2-responsible for cell growth and cell invasion. Genome-wide analysis revealed that 38% of HOTTIP-regulated genes contain H3K4me3 and HOTTIP enrichment at their promoters, without HOXA13 binding. HOTTIP complexes with WDR5-MLL1 to trans-activate oncogenic proteins CYB5R2, SULT1A1, KIF26A, SLC1A4, and TSC22D1 by directly inducing H3K4me3 at their promoters. The WDR5, MLL1, and H3K4me3 levels at their promoters and their expression levels are sensitive to HOTTIP expression. These results indicate the importance of the noncanonical trans-acting HOTTIP-WDR5-MLL1 pathway in the HOTTIP regulatory mechanism by promoting oncogenic protein expression. Furthermore, HOTTIP is regulated by miR-497 in PDAC cells, but HOTTIP is negatively correlated with miR-497 levels in PDAC tissues. In conclusion, HOTTIP is upregulated in PDAC due to the loss of the inhibitory miR-497; HOTTIP promotes PDAC progression through the canonical HOTTIP-HOXA13 axis. A novel noncanonical trans-acting HOTTIP-WDR5-MLL1-H3K4me3 pathway is also delineated.

Genome-Wide Screening and Functional Analysis Identifies Tumor Suppressor Long Noncoding RNAs Epigenetically Silenced in Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNA) play critical roles in the development of cancer, including hepatocellular carcinoma (HCC). However, the mechanisms underlying their deregulation remain largely unexplored. In this study, we report that two lncRNAs frequently downregulated in HCC function as tumor suppressors and are epigenetically silenced by histone methyltransferase EZH2. lncRNAs TCAM1P-004 and RP11-598D14.1 were inhibited by EZH-mediated trimethylation of H3K27me3 at their promoters. Downregulation of TCAM1P-004 and RP11-598D14.1 was frequently observed in HCC tumors compared with adjacent normal tissues. Both lncRNAs inhibited cell growth, cell survival, and transformation in HCC cells in vitro as well as tumor formation in vivo. Using RNA pull-down and mass spectrometry, we demonstrated that TCAM1P-004 bound IGF2BP1 and HIST1H1C, whereas RP11-598D14.1 bound IGF2BP1 and STAU1. These lncRNA-protein interactions were critical in regulating p53, MAPK, and HIF1α pathways that promoted cell proliferation in HCC. Overexpression of EZH2 was critical in repressing TCAM1P-004 and RP11-598D14.1, and EZH2-TCAM1P-004/RP11-598D14.1-regulated pathways were prevalent in human HCC. Aberrant suppression of TCAM1P-004 and RP11-598D14.1 led to loss of their tumor-suppressive effects by disrupting the interaction with IGF2BP1, HIST1H1C, and STAU1, which in turn promoted HCC development and progression. Collectively, these findings demonstrate the role of TCAMP1P-004 and RP11-598D14.1 in suppressing tumor growth and suggest that EZH2 may serve as a therapeutic target in HCC. SIGNIFICANCE: EZH2-mediated loss of lncRNAs TCAM1P-004 and RP11-598D14.1 hinders the formation of tumor suppressor lncRNA-protein complexes and subsequently promotes HCC growth.

Berberine induces miR-373 expression in hepatocytes to inactivate hepatic steatosis associated AKT-S6 kinase pathway.

Berberine is a Chinese herbal medicine extracted from rhizoma coptidis that functions to improve insulin resistance, hyperlipidemia, hepatosteatosis and inflammation. Berberine can modify the activity of cell metabolism and signaling pathways by regulating expression of genes. However, the roles and effects of differential microRNA (miRNA) expression induced by berberine treatment are largely unexplored. It is believed that miRNAs expression modified by berberine contributes to its therapeutic effects to diseases such as hepatosteatosis and non-alcoholic fatty liver disease. By identifying novel miRNAs and their putative gene targets associated with abnormal hepatic lipid deposition, the underlying mechanism of these diseases could be established and effective therapies against the diseases could be developed. Here, we used the immortalized hepatocyte cell line MIHA as a model to study the effect of berberine on global miRNA expression profile of hepatocytes. Global miRNA expression levels were measured in berberine-treated MIHA cells by quantitative reverse transcription PCR miRNA panel, and the potential berberine regulated miRNAs were then validated in MIHA and HepG2 cells. MicroRNA-373 (MiR-373) was consistently upregulated in both cell lines upon berberine treatments. Gene expression microarray showed that berberine upregulated Early Growth Response 1 (EGR1) level which functioned to transactivate miR-373 expression. Subsequently, we showed that upregulation of miR-373 depleted its target gene AKT serine/threonine kinase 1 (AKT1) mRNA level, which led to the inhibition of AKT-mTOR-S6K signaling pathway in hepatocytes that was critical in the development of hepatosteatosis. Study of the therapeutic effect of manipulating miR-373 against abnormal lipid deposition in liver is warranted.

Phenylephrine effects on the morphology of osteoblasts and expression of nicotinamide phosphoribosyltransferase under oxidative stress / 中国组织工程研究

BACKGROUND: Phenylephrine has been proved to exert a protective effect on radiant-induced salivary gland and epithelial cell injuries, but its effect on hydrogen peroxide (H2O2)-induced oxidative stress in osteoblasts are not fully understood. OBJECTIVE: To explore the effect of phenylephrine on H2O2-induced oxidative stress in osteoblasts, and to explore the mechanism underlying the regulation by the expression level of nicotinamide phosphoribosyltransferase (Nampt). METHODS: Primary osteoblasts were cultured and randomly divided into four groups: blank control group, H2O2group, phenylephrine group, and combination group (0.5 hour pretreatment of 1×10-5mol/L phenylephrine, and then given 300 μmol/L H2O2). The morphology of osteoblasts was observed at different time points. Osteoblasts were collected after 24-hour culture, and total RNA and protein were then extracted to detect the mRNA and protein expression levels of Nampt by RT-PCR and western blot assay, respectively. RESULTS AND CONCLUSION: Compared with the blank control group, reduced osteoblasts and evident cell shrinks were observed in the H2O2group, while the number of osteoblasts significantly increased in the combined group compared with the H2O2group at 12, 24 and 48 hours of culture. RT-PCR results showed that the mRNA level of Nampt in the H2O2group was reduced by 31.23% of that in the blank control group, while the mRNA level of Nampt in the combination group was dramatically increased by 206.20% of that in the H2O2group at 24 hours of culture (both P < 0.05). Furthermore, western blot assay findings revealed that the protein level of Nampt in the H2O2group was reduced by 67.98% of that in the blank control group, while the protein level of Nampt in the combination group was increased by 152.25% of that in the H2O2group at 24 hours of culture (both P < 0.05). Our results indicate that phenylephrine can alleviate the shrink and atrophy of osteoblasts caused by H2O2, thereby exerting protective effect by up-regulating the mRNA and protein levels of Nampt that may be a regulatory gene.

Active fraction (HS7) from Taiwanofungus camphoratus inhibits AKT-mTOR, ERK and STAT3 pathways and induces CDK inhibitors in CL1-0 human lung cancer cells.

BACKGROUND: The non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. In NSCLC, the oncogenic AKT-mTOR, ERK and STAT3 pathways are commonly dysregulated and have emerged as attractive targets for therapeutic developments. In a relatively limited subset of NSCLC, these pathways driven by mutant EGFR can be treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy. However, for the most NSCLC, more novel targeted agents are imperatively needed. Therefore, we investigated the inhibitory effects of the active fraction HS7 from Taiwanofungus camphoratus, a unique medicinal fungus in Taiwan, on these pathways in CL1-0 EGFR wild-type human NSCLC cells. METHODS: The active fraction HS7 was prepared by n-hexane extraction of T. camphoratus followed by silica gel chromatography. Its effects on the cell viabilities were determined by sulforhodamine B colorimetric assay. Flow cytometry was used to analyze cell-cycle regulation and apoptosis induction. The changes in cellular protein levels were examined by Western blot. RESULTS: The active fraction HS7 vigorously inhibits AKT-mTOR, ERK and STAT3 signaling pathways in CL1-0 cells. At dose of 25 µg/mL, these signaling pathways were almost completely inhibited by HS7, accompanied with induction of cyclin-dependent kinase inhibitors such as p15, p21 and p27. Accordingly, the AKT-mTOR downstream targets p-p70S6K and HIF-1α were also suppressed as well. At this dose, the cell proliferation was profoundly suppressed to 23.4% of control and apoptosis induction was observed. CONCLUSIONS: The active fraction HS7 from n-hexane extract of T. camphoratus exerts multi-targeting activity on the suppression of AKT-mTOR, ERK and STAT3 pathways and induction of p15, p21 and p27 in EGFR wild-type NSCLC cells. This multi-targeting activity of HS7 suggests its potential as an alternative medicine for the treatment of EGFR TKIs resistant NSCLC.

Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom.

Assessing the neutralization capability of nonlethal but medically relevant toxins in venom has been a challenging task. Nowadays, neutralization efficacy is evaluated based simply on the survival rates of animals injected with antivenom together with a predefined dose of venom, which can determine potency against neurotoxicity but not validate the capability to neutralize cytotoxin-induced complications. In this study, a high correlation with in-vivo and in-vitro neutralization assays was established using the immunoreactive peptides identified from short-chain neurotoxin and cytotoxin A3. These peptides contain conserved residues associated with toxin activities and a competition assay indicated that these peptides could specifically block the antibody binding to toxin and affect the neutralization potency of antivenom. Moreover, the titers of peptide-specific antibody in antivenoms or mouse antisera were determined by enzyme-linked immunosorbent assay (ELISA) simultaneously, and the results indicated that Taiwanese bivalent antivenom (BAV) and Vietnamese snake antivenom-Naja (SAV-Naja) exhibited superior neutralization potency against the lethal effect of short-chain neurotoxin (sNTX) and cytotoxicity of cardiotoxin/cytotoxin (CTX), respectively. Thus, the reported peptide ELISA shows not only its potential for antivenom prequalification use, but also its capability of justifying the cross-neutralization potency of antivenoms against Naja atra venom toxicity.

EZH2 coupled with HOTAIR to silence MicroRNA-34a by the induction of heterochromatin formation in human pancreatic ductal adenocarcinoma.

MicroRNA-34a (miR-34a) is frequently downregulated in pancreatic ductal adenocarcinoma (PDAC) cells, however, the silencing mechanism remains unclear. Enhancer of zeste homolog 2 (EZH2) is overexpressed in PDAC, and our previous miRNA profiling showed that inhibition of EZH2 in PDAC cells led to the re-expression of a group of tumor suppressor miRNAs including miR-34a. Here, we studied the effect of ectopic EZH2 expression to the silencing of miR-34a, and identified HOTAIR as an interacting partner to induce heterochromatin formation during miR-34a repression. We identified EZH2 as a major player in silencing miR-34a. Inhibition of EZH2 upregulated miR-34a expression in PDAC cells, while EZH2 overexpression in human pancreatic ductal epithelial (HPDE) cells repressed miR-34a expression and decreased the miR-34a promoter activity. We then showed that HOTAIR played a critical role in EZH2-mediated repression of miR-34a, as knockdown of HOTAIR attenuated the miR-34a inhibition effect in EZH2-overexpressing HPDE cells. HOTAIR physically interacted with miR-34a promoter, and the EZH2-interacting region located at 5' HOTAIR RNA was essential in repressing miR-34a and promoting cell proliferation. More importantly, we showed that the interaction between EZH2 and HOTAIR underlay the silencing of miR-34a through induction of heterochromatin formation. We first showed that manipulation of EZH2 level interfered the occupancy of heterochromatin markers H3K9me2, heterochromatin associated protein 1α and 1γ in PDAC cells. In turn, we showed that knockdown of HOTAIR reduced the occupancy of EZH2 at miR-34a promoter. The identification of HOTAIR-guided miR-34a silencing opened a new avenue in miR-34a-oriented therapy against PDAC.

A small-molecule modulator of the tumor-suppressor miR34a inhibits the growth of hepatocellular carcinoma.

Small molecules that restore the expression of growth-inhibitory microRNAs (miRNA) downregulated in tumors may have potential as anticancer agents. miR34a functions as a tumor suppressor and is downregulated or silenced commonly in a variety of human cancers, including hepatocellular carcinoma (HCC). In this study, we used an HCC cell-based miR34a luciferase reporter system to screen for miR34a modulators that could exert anticancer activity. One compound identified as a lead candidate, termed Rubone, was identified through its ability to specifically upregulate miR34a in HCC cells. Rubone activated miR34a expression in HCC cells with wild-type or mutated p53 but not in cells with p53 deletions. Notably, Rubone lacked growth-inhibitory effects on nontumorigenic human hepatocytes. In a mouse xenograft model of HCC, Rubone dramatically inhibited tumor growth, exhibiting stronger anti-HCC activity than sorafenib both in vitro and in vivo. Mechanistic investigations showed that Rubone decreased expression of cyclin D1, Bcl-2, and other miR34a target genes and that it enhanced the occupancy of p53 on the miR34a promoter. Taken together, our results offer a preclinical proof of concept for Rubone as a lead candidate for further investigation as a new class of HCC therapeutic based on restoration of miR34a tumor-suppressor function.

Hepatitis B virus X protein promotes hepatocellular carcinoma transformation through interleukin-6 activation of microRNA-21 expression.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic hepatitis B virus (HBV) infection is the major risk factor of HCC. The virus encodes HBV X (HBx) protein that plays a critical role in the development of HCC. Studies have revealed numerous HBx-altered genes and signalling pathways that heavily contribute to tumourigenesis of non-tumour hepatocytes. However, the role of HBx in regulating other critical gene regulators such as microRNAs is poorly understood, which impedes the exploration of a complete HBx-associated carcinogenic network. Besides, critical microRNAs that drive the transformation of non-tumour hepatocytes are yet to be identified. Here, we overexpressed C-terminal truncated HBx protein in a non-tumour hepatocyte cell line MIHA, and measured a panel of cancer-associated miRNAs. We observed that oncogenic miR-21 was upregulated upon ectopic expression of this viral protein variant. HBx-miR-21 pathway was prevalent in HCC cells as inhibition of HBx in Hep3B and PLC/PRF/5 cells significantly suppressed miR-21 expression. Subsequently, we showed that the upregulation of miR-21 was mediated by HBx-induced interleukin-6 pathway followed by activation of STAT3 transcriptional factor. The high dependency of miR-21 expression to HBx protein suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression. Importantly, miR-21 was essential in the HBx-induced transformation of non-tumour hepatocytes. Inhibition of miR-21 effectively attenuated anchorage-independent colony formation and subcutaneous tumour growth of MIHA cells. Our study suggested that overexpression of miR-21 was critical to promote early carcinogenesis of hepatocytes upon HBV infection.

A potential antitumor ellagitannin, davidiin, inhibited hepatocellular tumor growth by targeting EZH2.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is the third most common cause of cancer-related deaths. Currently available treatment options for HCC patients are scarce resulting in an urgent need to develop a novel effective cure. Polygonum capitatum is a medicinal herb which has been used to treat inflammatory diseases in Miao nationality of China. We recently isolated a pure compound davidiin from P. capitatum extract. Four HCC cell lines were treated with davidiin. Cell viability was recorded by MTT assay. siRNAs targeting enhancer of zeste homolog 2 (EZH2) were applied to modulate the expression of EZH2. Established xenograft mice models of HCC were applied to evaluate the in vivo anticancer activity of davidiin. We investigated the anticancer activity and the underlying mechanism of davidiin. The compound inhibited HCC cell growth and also suppressed tumor growth in xenografted HCC mouse. Such inhibition was facilitated by specifically downregulation on EZH2. The compound possesses anticancer activity both in vitro and in vivo which warrants further clinical investigation as a potential anti-HCC agent.

Targeting long non-coding RNAs in cancers: progress and prospects.

Pervasive transcription occurs in the human genome to generate thousands of RNA transcripts, and accumulating evidence suggested that the RNA molecules, without protein coding ability, have important roles in diverse biological functions. Long non-coding RNA (lncRNA), with size larger than 200 nt, is a new class of the non-coding RNA that contributes to cancer development and progression. Roles for several lncRNAs in cancers have been characterized and strategies targeting them have inhibitory effects to malignant cells in vitro and in vivo. These findings point to the potential of lncRNAs as prospective novel therapeutic targets in cancers. Recent advance in biological drugs, led by nucleic acid drugs (i.e. siRNAs, antisense oligonucleotides), suggest directions for the development of cancer therapies targeting lncRNAs. Here, we discuss the characteristics of lncRNAs regarding their synthesis, stability and functional role in cells, and emphasize their unique properties that determine their molecular functions. We then discuss the association of lncRNAs with cancers, and illustrate the anticancer effects induced upon modulating the level and function of lncRNAs. We also revisit established methods for targeting RNA molecules and discuss new agents and strategies to attenuate lncRNAs in cancer.