Synergistic induction of mitotic pyroptosis and tumor remission by inhibiting proteasome and WEE family kinases.

Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.

LINC00998-encoded micropeptide SMIM30 promotes the G1/S transition of cell cycle by regulating cytosolic calcium level.

The biological functions of short open reading frame (sORF)-encoded micropeptides remain largely unknown. Here, we report that LINC00998, a previously annotated lncRNA, was upregulated in multiple cancer types and the sORF on LINC00998 encoded a micropeptide named SMIM30. SMIM30 was localized in the membranes of the endoplasmic reticulum (ER) and mitochondria. Silencing SMIM30 inhibited the proliferation of hepatoma cells in vitro and suppressed the growth of tumor xenografts and N-nitrosodiethylamine-induced hepatoma. Overexpression of the 5'UTR-sORF sequence of LINC00998, encoding wild-type SMIM30, enhanced tumor cell growth, but this was abolished when a premature stop codon was introduced into the sORF via single-base deletion. Gain- and loss-of-function studies revealed that SMIM30 peptide but not LINC00998 reduced cytosolic calcium level, increased CDK4, cyclin E2, phosphorylated-Rb and E2F1, and promoted the G1/S phase transition and cell proliferation. The effect of SMIM30 silencing was attenuated by a calcium chelator or the agonist of sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. These findings suggest a novel function of micropeptide SMIM30 in promoting G1/S transition and cell proliferation by enhancing SERCA activity and reducing cytosolic calcium level.

A long non-coding RNA-based signature predicts early recurrence in hepatocellular carcinoma.

BACKGROUND: Early recurrence is the main obstacle for long-term survival of hepatocellular carcinoma (HCC) patients after curative resection. OBJECTIVE: We aimed to develop a long non-coding RNA (lncRNA) based signature to predict early recurrence. METHODS: Using bioinformatics analysis and quantitative reverse transcription PCR (RT-qPCR), we screened for lncRNA candidates that were abnormally expressed in HCC. The expression levels of candidate lncRNAs were analyzed in HCC tissues from 160 patients who underwent curative resection, and a risk model for the prediction of recurrence within 1 year (early recurrence) of HCC patients was constructed with linear support vector machine (SVM). RESULTS: An lncRNA-based classifier (Clnc), which contained nine differentially expressed lncRNAs including AF339810, AK026286, BC020899, HEIH, HULC, MALAT1, PVT1, uc003fpg, and ZFAS1 was constructed. In the test set, this classifier reliably predicted early recurrence (AUC, 0.675; sensitivity, 72.0%; specificity, 63.1%) with an odds ratio of 4.390 (95% CI, 2.120-9.090). Clnc showed higher accuracy than traditional clinical features, including tumor size, portal vein tumor thrombus (PVTT) in predicting early recurrence (AUC, 0.675 vs 0.523 vs 0.541), and had much higher sensitivity than Barcelona Clinical Liver Cancer (BCLC; 72.0% vs 50.0%), albeit their AUCs were comparable (0.675 vs 0.678). Moreover, combining Clnc with BCLC significantly increased the AUC, compared with Clnc or BCLC alone in predicting early recurrence (all P< 0.05). Finally, logistic and Cox regression analyses suggested that Clnc was an independent prognostic factor and associated with the early recurrence and recurrence-free survival of HCC patients after resection, respectively (all P= 0.001). CONCLUSIONS: Our lncRNA-based classifier Clnc can predict early recurrence of patients undergoing surgical resection of HCC.

LncRNA SNHG17 interacts with LRPPRC to stabilize c-Myc protein and promote G1/S transition and cell proliferation.

Oncogenic c-Myc is a master regulator of G1/S transition. Long non-coding RNAs (lncRNAs) emerge as new regulators of various cell activities. Here, we found that lncRNA SnoRNA Host Gene 17 (SNHG17) was elevated at the early G1-phase of cell cycle. Both gain- and loss-of function studies disclosed that SNHG17 increased c-Myc protein level, accelerated G1/S transition and cell proliferation, and consequently promoted tumor cell growth in vitro and in vivo. Mechanistically, the 1-150-nt of SNHG17 physically interacted with the 1035-1369-aa of leucine rich pentatricopeptide repeat containing (LRPPRC) protein, and disrupting this interaction abrogated the promoting role of SNHG17 in c-Myc expression, G1/S transition, and cell proliferation. The effect of SNHG17 in stimulating cell proliferation was attenuated by silencing c-Myc or LRPPRC. Furthermore, silencing SNHG17 or LRPPRC increased the level of ubiquitylated c-Myc and reduced the stability of c-Myc protein. Analysis of human hepatocellular carcinoma (HCC) tissues revealed that SNHG17, LRPPRC, and c-Myc were significantly upregulated in HCC, and they showed a positive correlation with each other. High level of SNHG17 or LRPPRC was associated with worse survival of HCC patients. These data suggest that SNHG17 may inhibit c-Myc ubiquitination and thus enhance c-Myc level and facilitate proliferation by interacting with LRPPRC. Our findings identify a novel SNHG17-LRPPRC-c-Myc regulatory axis and elucidate its roles in G1/S transition and tumor growth, which may provide potential targets for cancer therapy.

Functional long non-coding RNAs in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent human malignancy with high morbidity worldwide. Hepatocarcinogenesis is a complex multistep process, and its underlying molecular mechanisms remain largely unknown. Recently, long non-coding RNAs (lncRNAs), a class of newly discovered molecules, have been revealed as essential regulators in the development of HCC. HCC-associated lncRNAs affect multiple malignant phenotypes by modulating gene expression or protein activity. Moreover, the dysregulation of lncRNAs in the liver is also associated with diseases predisposing to HCC, such as chronic viral infection, nonalcoholic steatohepatitis, and liver fibrosis/cirrhosis. A deeper understanding of the lncRNA regulatory network in the multistep processes of HCC development will provide new insights into the diagnosis and treatment of HCC. In this review, we introduce the biogenesis and function of lncRNAs and summarize recent knowledge on how lncRNAs regulate the malignant hallmarks of HCC, such as uncontrolled cell proliferation, resistance to cell death, metabolic reprogramming, immune escape, angiogenesis, and metastasis. We also review emerging insights into the role of lncRNAs in HCC-associated liver diseases. Finally, we discuss the potential applications of lncRNAs as early diagnostic biomarkers and therapeutic targets.

A p53/lnc-Ip53 Negative Feedback Loop Regulates Tumor Growth and Chemoresistance.

Acetylation is a critical mechanism to modulate tumor-suppressive activity of p53, but the causative roles of long non-coding RNAs (lncRNAs) in p53 acetylation and their biological significance remain unexplored. Here, lncRNA LOC100294145 is discovered to be transactivated by p53 and is thus designated as lnc-Ip53 for lncRNA induced by p53. Furthermore, lnc-Ip53 impedes p53 acetylation by interacting with histone deacetylase 1 (HDAC1) and E1A binding protein p300 (p300) to prevent HDAC1 degradation and attenuate p300 activity, resulting in abrogation of p53 activity and subsequent cell proliferation and apoptosis resistance. Mouse xenograft models reveal that lnc-Ip53 promotes tumor growth and chemoresistance in vivo, which is attenuated by an HDAC inhibitor. Silencing lnc-Ip53 inhibits the growth of xenografts with wild-type p53, but not those expressing acetylation-resistant p53. Consistently, lnc-Ip53 is upregulated in multiple cancer types, including hepatocellular carcinoma (HCC). High levels of lnc-Ip53 is associated with low levels of acetylated p53 in human HCC and mouse xenografts, and is also correlated with poor survival of HCC patients. These findings identify a novel p53/lnc-Ip53 negative feedback loop in cells and indicate that abnormal upregulation of lnc-Ip53 represents an important mechanism to inhibit p53 acetylation/activity and thereby promote tumor growth and chemoresistance, which may be exploited for anticancer therapy.

Investigating the Efficacy of Hydrocolloid Dressing for Preventing Nasotracheal Tube-Related Pressure Injury in the PICU.

OBJECTIVES: To investigate the efficacy of hydrocolloid dressing in reducing the occurrence rate and severity of nasotracheal tube-related pressure injury. DESIGN: Randomized controlled trial. SETTING: A PICU in a tertiary medical center in southern China. PATIENTS: Pediatric patients received invasive mechanical ventilation via nasotracheal tubes. INTERVENTIONS: The hydrocolloid dressing was cut into an optimal square size, which should cover the area from the nasal columella to the ala. MEASUREMENTS AND MAIN RESULTS: Eligible participants were randomly allocated to the control group and the experimental group. The participants in the experimental group received hydrocolloid dressing to protect nasal skin from the beginning of nasotracheal intubation, while the participants in the control group received the current care procedure (without hydrocolloid dressing) unless pressure injuries occurred. The hydrocolloid dressing was changed daily to assess the nasal skin. The pressure injury staging system that was redefined and updated by the National Pressure Ulcer Advisory Panel in 2016 was used. The mean duration of nasotracheal intubation was 150.10 ± 117.09 hours in the experimental group and 161.75 ± 120.72 hours in the control group. Forty-five participants had nasotracheal tube-related pressure injuries in control group, whereas 26 patients had in experimental group (72.6% vs 43.3%; absolute difference, 29.3%, 95% CI, 12.5-46%; p = 0.001). The median survival times of the nasal skin integrity were 95.5 hours in the control group and 219.5 hours in the experimental group (p < 0.001). CONCLUSIONS: Hydrocolloid dressing can not only reduce the occurrence rate of nasotracheal tube-related pressure injury in the child with long-term nasotracheal intubation but also improve the endurance of the nasal skin significantly.

LncRNA GOLGA2P10 is induced by PERK/ATF4/CHOP signaling and protects tumor cells from ER stress-induced apoptosis by regulating Bcl-2 family members.

Elevated endoplasmic reticulum (ER) stress is frequently observed in cancers, whereas sustained ER stress may trigger apoptosis. How cancer cells escape from ER stress-induced apoptosis remain unclear. Here, we found that a pseudogene-derived lncRNA, Golgin A2 pseudogene 10 (GOLGA2P10), was frequently upregulated in HCC tissues and significantly elevated in hepatoma cells treated with ER stress inducers, such as tunicamycin and thapsigargin. Higher GOLGA2P10 level was correlated with shorter recurrence-free survival of HCC patients. Upon ER stress, CHOP directly bound to the promoter of GOLGA2P10 and induced its transcription via the PERK/ATF4/CHOP pathway. Interestingly, the ER stress inducer-stimulated apoptosis was promoted by silencing GOLGA2P10 but was antagonized by overexpressing GOLGA2P10. Both gain- and loss-of-function analyses disclosed that GOLGA2P10 increased BCL-xL protein level, promoted BAD phosphorylation, and conferred tumor cells with resistance to ER stress-induced apoptosis. Moreover, BCL-xL overexpression or BAD knockdown abrogated the apoptosis-promoting effect of GOLGA2P10 silencing. Consistently, the Ser75Ala mutation in BAD, which caused phosphorylation-resistance, further enhanced the promoting effect of BAD in tunicamycin-induced apoptosis. These results suggest that ER stress induces GOLGA2P10 transcription through the PERK/ATF4/CHOP pathway, and upregulation of GOLGA2P10 protects tumor cells from the cytotoxic effect of persistent ER stress in tumor microenvironment by regulating Bcl-2 family members, which highlight GOLGA2P10 as a potential target for anticancer therapy.

[Characteristics of Phosphorus Adsorption in Aqueous Solution By Ca/Mg-Loaded Biogas Residue Biochar].

In this study, we synthesized Ca/Mg biochar sorbents (CMZZs) by pyrolysis of biogas residue (ZZs) impregnated with calcium chloride and magnesium chloride and investigated their potential to adsorb P from water. The results showed that the content of calcium and magnesium in the modified biochar was 1.3 and 15.4 times, respectively, what they were before the modification. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) patterns showed that the type of chemical functional groups on the surface of biochar had not changed, but several new peaks appeared, indicating that Mg (OH)2 and MgO might be present on the surface of the CMZZs. The biochar equilibrium data were well described by the Freundlich model, and the adsorption reached equilibrium after 100 min. The adsorption kinetics followed a pseudo-second-order model, and the maximum adsorption capacity of the CMZZs was 76.92 mg·g-1 when the pH was 9 and the temperature 303 K. The results revealed that a soaking method can effectively load Ca2+ and Mg2+ onto the surface of ZZ, and CMZZs offer a promising adsorbent for P removal with a high adsorption capacity for P from wastewater.

Lnc-UCID Promotes G1/S Transition and Hepatoma Growth by Preventing DHX9-Mediated CDK6 Down-regulation.

Although thousands of long noncoding RNAs (lncRNAs) have been annotated, only a limited number of them have been functionally characterized. Here, we identified an oncogenic lncRNA, named lnc-UCID (lncRNA up-regulating CDK6 by interacting with DHX9). Lnc-UCID was up-regulated in hepatocellular carcinoma (HCC), and a higher lnc-UCID level was correlated with shorter recurrence-free survival of HCC patients. Both gain-of-function and loss-of function studies revealed that lnc-UCID enhanced cyclin-dependent kinase 6 (CDK6) expression and thereby promoted G1/S transition and cell proliferation. Studies from mouse xenograft models revealed that tumors derived from lnc-UCID-silenced HCC cells had a much smaller size than those from control cells, and intratumoral injection of lnc-UCID small interfering RNA suppressed xenograft growth. Mechanistically, the 850-1030-nt domain of lnc-UCID interacted physically with DEAH (Asp-Glu-Ala-His) box helicase 9 (DHX9), an RNA helicase. On the other hand, DHX9 post-transcriptionally suppressed CDK6 expression by binding to the 3'-untranslated region (3'UTR) of CDK6 mRNA. Further investigation disclosed that lnc-UCID enhanced CDK6 expression by competitively binding to DHX9 and sequestering DHX9 from CDK6-3'UTR. In an attempt to explore the mechanisms responsible for lnc-UCID up-regulation in HCC, we found that the lnc-UCID gene was frequently amplified in HCC. Furthermore, miR-148a, whose down-regulation was associated with an increase of lnc-UCID in HCC, could bind lnc-UCID and inhibit its expression. Conclusion: Up-regulation of lnc-UCID, which may result from amplification of its gene locus and down-regulation of miR-148a, can promote HCC growth by preventing the interaction of DHX9 with CDK6 and subsequently enhancing CDK6 expression. These findings provide insights into the biological functions of lncRNAs, the regulatory network of cell cycle control, and the mechanisms of HCC development, which may be exploited for anticancer therapy.

MicroRNA-125b Promotes Hepatic Stellate Cell Activation and Liver Fibrosis by Activating RhoA Signaling.

miR-125b is frequently dysregulated in different diseases. Activation of hepatic stellate cells (HSCs) is a critical event during liver fibrogenesis. However, the function and its underlying mechanism of miR-125b in HSC activation and liver fibrosis are still unknown. Here, we showed that miR-125b was upregulated in HSCs, but not in hepatocytes, during hepatic fibrogenesis in vivo and upon culture activation in vitro. Inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor ß (TGF-ß)-induced alpha-smooth muscle actin (α-SMA) expression and cell contraction of the immortalized HSC cell line. In contrast, ectopic expression of miR-125b promoted α-SMA expression and HSC contraction. Moreover, antagonizing miR-125b in vivo significantly alleviated liver fibrosis in CCl4-treated mice. Mechanistically, overexpression of miR-125b in HSCs enhanced RhoA activity by directly targeting StAR-related lipid transfer (START) domain containing 13 (Stard13), a RhoA-specific GTPase-activating protein, whereas knockdown of miR-125b abrogated RhoA activation. Furthermore, inhibition of RhoA or its downstream molecules, Mrtf-A and Srf, attenuated the miR-125b-induced α-SMA expression and HSC contraction. Therefore, our findings identify a miR-125b-Stard13-RhoA-α-SMA signaling cascade in HSCs and highlight its importance in hepatic fibrosis.

Identification of a TGF-ß-miR-195 positive feedback loop in hepatocytes and its deregulation in hepatoma cells.

Resistance to TGF-ß-induced growth repression is prevalent in various cancer cells, but the underlying mechanisms remain unclear. In this study, we showed that activation of TGF-ß signaling caused Sma- and Mad-related family (Smad) 2 and Smad3 to bind directly to the promoter region of miR-195, and then activated miR-195 transcription in normal hepatocytes. Conversely, miR-195 inhibited the expression of Smad7 by binding to its 3'-UTR, thereby strengthening TGF-ß-Smad signaling. These data identify a novel TGF-ß-miR-195 positive regulatory circuitry in normal hepatocytes. Further investigation revealed that HDAC1, a histone deacetylase that was abnormally overexpressed in hepatocellular carcinoma, could bind to the miR-195 promoter via Smad3 and cause hypoacetylation in the histones associated with the miR-195 promoter in hepatoma cells. This resulted in transcriptional repression of miR-195 and, subsequently, disruption of the TGF-ß-miR-195 regulatory loop and evasion of TGF-ß-mediated growth inhibition. Moreover, silencing HDAC1 in hepatoma cells restored TGF-ß-mediated growth suppression, but this effect was attenuated if miR-195 expression decreased. These findings suggest that HDAC1-induced miR-195 down-regulation is an important mechanism for tumor cells to resist the cytostatic activity of TGF-ß, and highlight the importance of TGF-ß-Smad2/3-miR-195-Smad7 circuitry in preventing uncontrolled cell proliferation.-Wang, R., Fu, T., You, K., Li, S., Zhao, N., Yang, J., Zhuang, S.-M. Identification of a TGF-ß-miR-195 positive feedback loop in hepatocytes and its deregulation in hepatoma cells.

Vascular mimicry formation is promoted by paracrine TGF-ß and SDF1 of cancer-associated fibroblasts and inhibited by miR-101 in hepatocellular carcinoma.

Vascular mimicry (VM) describes the phenomenon that tumor cells but not endothelial cells form vascular-like channels, which provide blood perfusion for tumor tissues. VM is associated with tumor growth, metastasis and worse survival of different cancers. The mechanisms of VM formation remain largely unknown. We showed that the conditioned medium of cancer-associated fibroblast (CM-CAF) promoted tumor cells to form capillary-like structure in vitro. Consistently, co-implantation of CAFs with tumor cells significantly enhanced VM formation in mouse xenografts, and higher amount of CAFs was found in VM+ human HCC tissues compared to VM- ones. However, the CM-CAF-promoted VM formation was attenuated when TGF-ß or SDF1 signaling was abrogated. Similar to CM-CAF, recombinant TGF-ß1 and SDF1 induced VM formation. We further disclosed that the CAF-secreted TGF-ß and SDF1 enhanced the expression of VE-cadherin, MMP2 and laminin5γ2 via TGF-ßR1 and CXCR4 in tumor cells, thereby promoted VM formation. Moreover, tumor cells with high activity of self-sustaining TGF-ß signaling displayed strong capability of VM formation. Subsequent investigations showed that miR-101, which was down-regulated in both tumor cells and CAFs, suppressed the CAF-promoted VM formation in vitro and in vivo. Gain- and loss-of-function analyses revealed that miR-101 attenuated TGF-ß signaling transduction by targeting TGF-ßR1 and Smad2 in tumor cells, and simultaneously abrogated SDF1 signaling by suppressing SDF1 expression in CAFs and inhibiting VE-cadherin expression in tumor cells. Our findings suggest that the miR-101-TGF-ß/SDF1-VE-cadherin/MMP2/LAMC2 networks regulate VM formation and represent the potential targets for cancer therapy.

Expression of microRNA-195 is transactivated by Sp1 but inhibited by histone deacetylase 3 in hepatocellular carcinoma cells.

MiR-195 expression is frequently reduced in various cancers, but its underlying mechanisms remain unknown. To explore whether abnormal transcription contributed to miR-195 downregulation in hepatocellular carcinoma (HCC), we characterized the -2165-bp site upstream of mature miR-195 as transcription start site and the -2.4 to -2.0-kb fragment as the promoter of miR-195 gene. Subsequent investigation showed that deletion of the predicted Sp1 binding site decreased the miR-195 promoter activity; Sp1 silencing significantly reduced the miR-195 promoter activity and the endogenous miR-195 level; Sp1 directly interacted with the miR-195 promoter in vitro and in vivo. These data suggest Sp1 as a transactivator for miR-195 transcription. Interestingly, miR-195 expression was also subjected to epigenetic regulation. Histone deacetylase 3 (HDAC3) could anchor to the miR-195 promoter via interacting with Sp1 and consequently repress the Sp1-mediated miR-195 transactivation by deacetylating histone in HCC cells. Consistently, substantial increase of HDAC3 protein was detected in human HCC tissues and HDAC3 upregulation was significantly correlated with miR-195 downregulation, suggesting that HDAC3 elevation may represent an important cause for miR-195 reduction in HCC. Our findings uncover the mechanisms underlying the transcriptional regulation and expression deregulation of miR-195 in HCC cells and provide new insight into microRNA biogenesis in cancer cells.

Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells.

Metastasis is responsible for rapid recurrence of hepatocellular carcinoma (HCC) and poor survival of HCC patients. Here we showed that miR-100 downregulation in HCC tissues was significantly associated with venous invasion, advanced TNM stage, tumor nodule without complete capsule, poorer cell differentiation, and shorter recurrence-free survival. Both gain- and loss-of-function studies showed that miR-100 dramatically suppressed the ability of HCC cells to migrate and to invade through Matrigel in vitro. Analyses using mouse orthotopic xenograft model further revealed that xenografts of miR-100-stable-expressing HCC cells displayed a significant reduction in pulmonary metastasis, compared with control group. Subsequent investigations revealed that miR-100 directly inhibited the expression of isoprenylcysteine carboxyl methyltransferase (ICMT) and ras-related C3 botulinum toxin substrate 1 (Rac1) by binding to their 3'-UTRs, and in turn suppressed lamellipodia formation and matrix metallopeptidase 2 (MMP2) activation. Furthermore, knockdown of ICMT and Rac1 phenocopied the anti-metastasis effect of miR-100, whereas overexpression of the constitutively active Rac1 (Q61L) antagonized the function of miR-100. Taken together, miR-100 represses metastasis of HCC cells by abrogating the ICMT-Rac1 signaling. Downregulation of miR-100 contributes to HCC metastasis and the restoration of miR-100 is a potential strategy for cancer therapy.

A novel AP-1/miR-101 regulatory feedback loop and its implication in the migration and invasion of hepatoma cells.

MicroRNA-101 (miR-101) is frequently downregulated in various cancers. To date, the regulatory networks of miR-101 remain obscure. In this study, we demonstrated that miR-101 was mainly transcribed from human miR-101-2 and mouse miR-101bgene loci. Subsequent analyses revealed that activator protein-1 (AP-1) directly binded to the -17.4 to -16.4 k region upstream of pre-miR-101-2 and activated the expression of miR-101. On the other hand, miR-101 could inhibit the expression of ERK2 and c-Fos, two key factors of the AP-1 pathway, by binding to their 3'-UTRs. Furthermore, reintroduction of miR-101 efficiently suppressed the AP-1 activity and pri-miR-101-2 transcription. These data thus suggest a novel AP-1/miR-101 regulatory circuitry, that is, AP-1 promotes the transcription of miR-101, whereas the expression of miR-101 reduces the level of ERK2 and c-Fos and thereby attenuates the AP-1 signaling. Further investigation disclosed that the AP-1 activator TPA-induced MMP9 activity and the TPA-promoted migration and invasion of hepatoma cells were significantly attenuated by miR-101 but were enhanced by miR-101 inhibitor. Our results suggest that the AP-1/miR-101 feedback loop may prevent the excessive activation of metastatic signals imposed by ERK2/AP-1 and highlight the biological significance of miR-101 downregulation in cancer metastasis.

MicroRNA-100 promotes the autophagy of hepatocellular carcinoma cells by inhibiting the expression of mTOR and IGF-1R.

We found that restoration of miR-100 expression resulted in accumulation of LC3B-II and decrease of p62 in hepatocellular carcinoma (HCC) cells, whereas antagonism of miR-100 reduced the level of LC3B-II. Moreover, a significant correlation between miR-100 downregulation and p62 upregulation was observed in human HCC tissues, suggesting an autophagy-promoting effect of miR-100. Subsequent investigations disclosed that knockdown of Atg7 but not Beclin-1 attenuated the miR-100-induced LC3B-II elevation. Furthermore, miR-100 overexpression caused massive cell death, which was abrogated by both the Atg7 silencing and chloroquine treatment. Simultaneously, miR-100 expression led to increased fraction of cells with Annexin V-staining and loss of mitochondrial potential, implying that miR-100 may promote the Atg7-dependent autophagy and subsequent apoptotic cell death. Consistently, mouse xenograft models revealed that miR-100 inhibited the in vivo growth of HCC cells. We further showed that miR-100 suppressed the expression of mTOR and IGF-1R by binding to their 3' untranslated region, and knockdown of mTOR or IGF-1R phenocopied the pro-autophagy effect of miR-100, indicating that miR-100 may promote autophagy by reducing mTOR and IGF-1R level. Collectively, our data uncover a new regulatory mechanism of autophagy and a novel function of miR-100, and provide a potential therapeutic target for HCC.

Flaxseed oil and alpha-lipoic acid combination ameliorates hepatic oxidative stress and lipid accumulation in comparison to lard.

BACKGROUND: Intake of high-fat diet is associated with increased non-alcoholic fatty liver disease (NAFLD). Hepatic lipid accumulation and oxidative stress are key pathophysiological mechanisms in NAFLD. Both flaxseed oil (FO) and α-lipoic acid (LA) exert potential benefit to NAFLD. The aim of this study was to determine the effect of the combination of FO and LA on hepatic lipid accumulation and oxidative stress in rats induced by high-fat diet. METHODS: LA was dissolved in flaxseed oil to a final concentration of 8 g/kg (FO + LA). The rodent diet contained 20% fat. One-fifth of the fat was soybean oil and the others were lard (control group), or 75% lard and 25% FO + LA (L-FO + LA group), or 50% lard and 50% FO + LA (M-FO + LA group), or FO + LA (H-FO + LA group). Male Sprague-Dawley rats were fed for 10 weeks and then killed for liver collection. RESULTS: Intake of high-fat lard caused a significant hepatic steatosis. Replacement with FO + LA was effective in reducing steatosis as well as total triglyceride and total cholesterol contents in liver. The combination of FO and LA also significantly elevated hepatic antioxidant defense capacities, as evaluated by the remarkable increase in the activities of SOD, CAT and GPx as well as the level of GSH, and the significant decline in lipid peroxidation. CONCLUSION: The combination of FO and LA may contribute to prevent fatty livers such as NAFLD by ameliorating hepatic lipid accumulation and oxidative stress.

Micronutrients-fortified rapeseed oil improves hepatic lipid accumulation and oxidative stress in rats fed a high-fat diet.

Intake of high-fat diet is associated with increased fatty livers. Hepatic lipid accumulation and oxidative stress are key pathophysiological mechanisms in this disease. Micronutrients polyphenols, tocopherols and phytosterols in rapeseed exert potential benefit to hepatoprotection, but most of these micronutrients are removed by the traditional refining process. The purpose of the present study was to determine whether rapeseed oil fortified with these micronutrients can decrease hepatic lipid accumulation and oxidative stress induced by high-fat diet. Sprague-Dawley rats received rodent diet contained 20% fat whose source was refined rapeseed oil (RRO) or fortified RRO with low, middle and high quantities of these micronutrients for 10 weeks. Intake of RRO caused a remarkable hepatic steatosis. Micronutrients supplementation was effective in reducing steatosis as well as total triglyceride and total cholesterol contents in liver. These micronutrients also significantly increased hepatic antioxidant defense capacities, as evaluated by the significant elevation in the activities of SOD and GPx as well as the level of GSH, and the significant decline in lipid peroxidation. These findings suggest that rapeseed oil fortified with micronutrients polyphenols, tocopherols and phytosterols may contribute to prevent fatty livers such as nonalcoholic fatty liver disease by ameliorating hepatic lipid accumulation and oxidative stress.

MicroRNA-195 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF, VAV2, and CDC42.

UNLABELLED: Hepatocellular carcinoma (HCC) is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. There is frequent down-regulation of miR-195 expression in HCC tissues. In this study, the role of miR-195 in HCC angiogenesis and metastasis was investigated with in vitro capillary tube formation and transwell assays, in vivo orthotopic xenograft mouse models, and human HCC specimens. Reduction of miR-195 in HCC tissues was significantly associated with increased angiogenesis, metastasis, and worse recurrence-free survival. Both gain-of-function and loss-of-function studies of in vitro models revealed that miR-195 not only suppressed the ability of HCC cells to promote the migration and capillary tube formation of endothelial cells but also directly repressed the abilities of HCC cells to migrate and invade extracellular matrix gel. Based on mouse models, we found that the induced expression of miR-195 dramatically reduced microvessel densities in xenograft tumors and repressed both intrahepatic and pulmonary metastasis. Subsequent investigations disclosed that miR-195 directly inhibited the expression of the proangiogenic factor vascular endothelial growth factor (VEGF) and the prometastatic factors VAV2 and CDC42. Knockdown of these target molecules of miR-195 phenocopied the effects of miR-195 restoration, whereas overexpression of these targets antagonized the function of miR-195. Furthermore, we revealed that miR-195 down-regulation resulted in enhanced VEGF levels in the tumor microenvironment, which subsequently activated VEGF receptor 2 signaling in endothelial cells and thereby promoted angiogenesis. Additionally, miR-195 down-regulation led to increases in VAV2 and CDC42 expression, which stimulated VAV2/Rac1/CDC42 signaling and lamellipodia formation and thereby facilitated the metastasis of HCC cells. CONCLUSION: miR-195 deregulation contributes to angiogenesis and metastasis in HCC. The restoration of miR-195 expression may be a promising strategy for HCC therapy.