Increased Small Ubiquitin-like Modifier-Activating Enzyme SAE1 Promotes Hepatocellular Carcinoma by Enhancing mTOR SUMOylation.

SUMOylation, one of the most important posttranslational modifications of proteins, plays an essential role in various biological processes; however, enzymes that control SUMOylation in hepatocellular carcinoma (HCC) are still unclear. Comprehensive exploration of the expression and clinical significance of SUMO enzymes in HCC would be of great value. Here, we obtained the gene expression profile of each small ubiquitin-like modifier (SUMO) protein and the corresponding clinical information from The Cancer Genome Atlas. We found that all SUMO enzymes were significantly increased in HCC tissues compared with that in adjacent nontumorous tissues. We identified a 6-gene prognostic signature, including SAE1, PIAS2, PIAS3, SENP3, SENP5, and UBC9, that could effectively predict the overall survival in patients with HCC. Specifically, SAE1 was the most valuable prognostic indicator. In 282 clinical samples, we found that SAE1 was closely related to the clinicopathologic parameters and prognosis of patients with HCC. In vitro and in vivo studies showed that SAE1 knockdown inhibits the proliferation, migration, and invasion of HCC cells. Mechanistically, we confirmed that SAE1 plays a role in driving HCC progression, which is largely dependent on the SUMOylation of mTOR signaling. In conclusion, our study revealed that the expression of SUMO enzymes, especially SAE1, is highly associated with HCC development and acts as a promising prognostic predictor.

Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2.

BACKGROUND: Hepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages. METHODS: Constitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3LCKO& IL-6-/-) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells. RESULTS: We observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC. CONCLUSION: In summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

Mitochondrial Dysfunction in Metabolic Dysfunction Fatty Liver Disease (MAFLD).

Nonalcoholic fatty liver disease (NAFLD) has become an increasingly common disease in Western countries and has become the major cause of liver cirrhosis or hepatocellular carcinoma (HCC) in addition to viral hepatitis in recent decades. Furthermore, studies have shown that NAFLD is inextricably linked to the development of extrahepatic diseases. However, there is currently no effective treatment to cure NAFLD. In addition, in 2020, NAFLD was renamed metabolic dysfunction fatty liver disease (MAFLD) to show that its pathogenesis is closely related to metabolic disorders. Recent studies have reported that the development of MAFLD is inextricably associated with mitochondrial dysfunction in hepatocytes and hepatic stellate cells (HSCs). Simultaneously, mitochondrial stress caused by structural and functional disorders stimulates the occurrence and accumulation of fat and lipo-toxicity in hepatocytes and HSCs. In addition, the interaction between mitochondrial dysfunction and the liver-gut axis has also become a new point during the development of MAFLD. In this review, we summarize the effects of several potential treatment strategies for MAFLD, including antioxidants, reagents, and intestinal microorganisms and metabolites.

Facile Recrystallization Process for Tuning the Crystal Morphology and Thermal Safety of Industrial Grade PYX.

In this study, the crystal appearance of industrial grade 2,6-diamino-3,5-dinitropyridine (PYX) was mostly needle-shaped or rod-shaped with an average aspect ratio of 3.47 and roundness of 0.47. According to national military standards, the explosion percentage of impact sensitivity s about 40% and friction sensitivity is about 60%. To improve loading density and pressing safety, the solvent-antisolvent method was used to optimize the crystal morphology, i.e., to reduce the aspect ratio and increase the roundness value. Firstly, the solubility of PYX in DMSO, DMF, and NMP was measured by the static differential weight method, and the solubility model was established. The results showed that the Apelblat equation and Van't Hoff equation could be used to clarify the temperature dependence of PYX solubility in a single solvent. Scanning electron microscopy (SEM) was used to characterize the morphology of the recrystallized samples. After recrystallization, the aspect ratio of the samples decreased from 3.47 to 1.19, and roundness increased from 0.47 to 0.86. The morphology was greatly improved, and the particle size decreased. The structures before and after recrystallization were characterized by infrared spectroscopy (IR). The results showed that no chemical structure changes occurred during recrystallization, and the chemical purity was improved by 0.7%. According to the GJB-772A-97 explosion probability method, the mechanical sensitivity of explosives was characterized. After recrystallization, the impact sensitivity of explosives was significantly reduced from 40% to 12%. A differential scanning calorimeter (DSC) was used to study the thermal decomposition. The thermal decomposition temperature peak of the sample after recrystallization was 5 °C higher than that of the raw PYX. The thermal decomposition kinetic parameters of the samples were calculated by AKTS software, and the thermal decomposition process under isothermal conditions was predicted. The results showed that the activation energy (E) of the samples after recrystallization was higher by 37.9~527.6 kJ/mol than raw PYX, so the thermal stability and safety of the recrystallized samples were improved.

Rapamycin facilitates differentiation of regulatory T cells via enhancement of oxidative phosphorylation.

We explored the interplay between energy metabolism and the impact of rapamycin (Rapa) on regulatory T cell (Treg) differentiation. Naïve CD4+ T cells were stimulated under Treg-polarizing conditions with or without Rapa. Rapa promoted Treg induction, as the expression of Foxp3 and Treg phenotypic markers were enhanced. Rapa disrupts glycolysis while favoring mitochondrial metabolism in induced Tregs (iTregs). Metabolic profiling showed reduced glycolytic metabolites in Rapa-treated iTregs, in line with the downregulation of glucose uptake and the expression of glycolytic enzymes. Conversely, Rapa increased the ratios of ATP/ADP and ATP/AMP, the production of mitochondrial ATP, and the expression of ATP5A. Treatment with oxidative phosphorylation inhibitors suppressed Foxp3 expression in Rapa-treated cells. Moreover, Rapa decreased oleic acid and palmitoleic acid levels and increased l-carnitine and acetylcarnitine levels and CPT1A expression in iTregs, indicative of augmented fatty acid oxidation. In conclusion, Rapa induces metabolic reprogramming in Tregs, affecting their differentiation.

A single nucleotide mutation drastically increases the expression of tumor-homing NGR-TNFα in the E. coli M15-pQE30 system by improving gene transcription.

Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated. KEY POINTS : • A single nucleotide mutation increased the expression of NGR-TNFα by two orders. • Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity.

Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling.

The motility of mesenchymal stem cells (MSCs) is highly related to their homing in vivo, a critical issue in regenerative medicine. Our previous study indicated copper (Cu) might promote the recruitment of endogenous MSCs in canine esophagus defect model. In this study, we investigated the effect of Cu on the motility of bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanism in vitro. Cu supplementation could enhance the motility of BMSCs, and upregulate the expression of hypoxia-inducible factor 1α (Hif1α) at the protein level, and upregulate the expression of rho family GTPase 3 (Rnd3) at messenger RNA and protein level. When Hif1α was silenced by small interfering RNA (siRNA), Cu-induced Rnd3 upregulation was blocked. When Rnd3 was silenced by siRNA, the motility of BMSCs was decreased with or without Cu supplementation, and Cu-induced cytoskeleton remodeling was neutralized. Furthermore, overexpression of Rnd3 also increased the motility of BMSCs and induced cytoskeleton remodeling. Overall, our results demonstrated that Cu enhanced BMSCs migration through, at least in part, cytoskeleton remodeling via Hif1α-dependent upregulation of Rnd3. This study provided an insight into the mechanism of the effect of Cu on the motility of BMSCs, and a theoretical foundation of applying Cu to improve the recruitment of BMSCs in tissue engineering and cytotherapy.

Improved TDM scheme and data extracting algorithm for polymerization evaluation.

To quickly evaluate holographic photopolymers with different formulations, the most effective method is to record a volume holographic grating in the samples and detect the grating's diffraction in real time. Since the volume grating is highly sensitive to incident angle, existing schemes need to precisely control many space-related parameters. This study proposes an improved scheme, in which two different sized spots are used to reduce the requirements for the overlap of the two spots and the installation precision of the samples. Transmittances, diffractive efficiencies and diffractive asymmetries are obtained at a high sampling rate, through a specifically designed algorithm with the data from uncalibrated high-speed photodiodes. The experimental results show that the proposed scheme performance well in evaluating holographic photopolymer.

Selective Photokilling of Colorectal Tumors by Near-Infrared Photoimmunotherapy with a GPA33-Targeted Single-Chain Antibody Variable Fragment Conjugate.

Antibody-based near-infrared photoimmunotherapy (NIR-PIT) is an attractive strategy for cancer treatment. Tumor cells can be selectively and efficiently killed by the targeted delivery of an antibody-photoabsorber complex followed by exposure to NIR light. Glycoprotein A33 antigen (GPA33) is highly expressed in most human colorectal cancers (CRCs) and is an ideal diagnostic and therapeutic target. We previously produced a single-chain fragment of a variable antibody against GPA33 (A33scFv antibody). Here, we investigate the efficacy of NIR-PIT by combining A33scFv with the NIR photoabsorber IR700 (A33scFv-IR700). In vitro, recombinant A33scFv displayed specific binding and delivery of an NIR dye to GPA33-positive tumor cells. Furthermore, A33scFv-IR700-mediated NIR-PIT was successful in rapidly and specifically killing GPA33-positive colorectal tumor cells. NIR-PIT treatment induced the release of lactate dehydrogenase from tumor cells, followed by cell necrosis, rather than apoptosis, through the promotion of reactive oxygen species accumulation in tumor cells. In mice bearing LS174T tumor grafts, A33scFv selectively accumulated in GPA33-positive tumors. Following only a single injection of the conjugate and subsequent illumination, A33scFv-IR700-mediated NIR-PIT induced a significant increase in therapeutic response in LS174T-tumor mice compared with that in the non-NIR-PIT groups (p < 0.001). Because the GPA33 antigen is specifically expressed in CRC tumors, A33scFv-IR700 might be a promising antibody fragment-photoabsorber conjugate for NIR-PIT of CRC.

Customizing a Tridomain TRAIL Variant to Achieve Active Tumor Homing and Endogenous Albumin-Controlled Release of the Molecular Machine In Vivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive antitumor drug candidate for precision cancer therapy due to its superior selective cytotoxicity in a variety of tumor cells. However, the clinical application of TRAIL in cancer therapy has been limited by its poor tumor-homing capacities and short half-life. Herein, we designed a tridomain TRAIL variant, Z-ABD-TRAIL, by sequentially fusing the platelet-derived growth factor receptor beta (PDGFRß)-specific affibody ZPDGFRß and an albumin-binding domain (ABD) to the N-terminus of TRAIL. The fusion protein Z-ABD-TRAIL was produced as a soluble protein with high yield in Escherichia coli (E. coli). The ZPDGFRß domain provided Z-ABD-TRAIL with PDGFRß-binding properties and thus promoted its tumor homing via the engagement of PDGFRß-expressing pericytes on tumor microvessels. ABD-mediated binding of Z-ABD-TRAIL to albumin in the blood endowed TRAIL with long-lasting (>72 h for Z-ABD-TRAIL vs <0.5 h for TRAIL) abilities to kill tumor cells. Although the in vitro cytotoxicity of Z-ABD-TRAIL in tumor cells was similar to that of the parent TRAIL, the in vivo tumor uptake, apoptosis-inducing ability, and antitumor effect of Z-ABD-TRAIL were much greater than those of TRAIL, indicating that ZPDGFRß-mediated tumor homing and ABD-introduced albumin binding significantly improved the pharmacodynamics of TRAIL. In addition, repeated injection of high-dose Z-ABD-TRAIL showed no obvious acute toxicity in mice. These results demonstrate that the newly designed tridomain Z-ABD-TRAIL is a promising agent for precision cancer therapy.

[Comparison Between Different Mitochondrial Staining Methods in Cell and Tissue Samples].

OBJECTIVE: To compare the effects of mitochondria staining between specific mitochondrial fluorescent probes and anti-mitochondrial protein antibody in cell and tissue samples. METHODS: The HepG2 cells fixed by 4% paraformaldehyde were stained with MitoTracker Deep Red (100 nmol/L) or anti-Grp75 antibody (75 nmol/L or 100 nmol/L). The human healthy liver tissue samples fixed by 4% paraformaldehyde were stained with 150 nmol/L MitoTracker Deep Red or anti-Grp75 antibody. The above stained cell and tissue samples were observed using confocal microscopy. RESULTS: We found non-specific staining in HeLa cells and obscure mitochondrial image using MitoTracker Deep Red probes, while clear tubular and punctate distribution using anti-Grp75 antibody. In contrast, we observed more specific and better effects of MitoTracker Deep Red probes-stained liver tissue samples as compared to the antibody. CONCLUSION: To visualize mitochondria, the anti-Grp75 antibody staining worked better on cells and the MitoTracker Deep Red probes are more suitable for tissue samples.

SNR enhancement in in-line particle holography with the aid of off-axis illumination.

The digital in-line particle holography suffers from speckle noise, for which considerable efforts have been devoted in order to mitigate it by designing post-processing algorithms. This paper proposes a novel approach, which mitigates the speckle noise by increasing the signal-to-noise ratio (SNR). It involves the joint design of optical systems and post-processing algorithms, and enhances the SNR by combining several holograms captured under different illumination angles. The experimental results show that the proposed scheme performs better than the normal scheme in terms of SNR and false-detection-ratio.

Self-calibrated and SNR-enhanced particle holography.

In-line particle holography suffers from speckle noise, and great effort has been made to alleviate it by designing post-processing algorithms. Recently, we proposed a novel approach which mitigates it by increasing the signal-to-noise ratio (SNR). The approach enhances the SNR by combining several holograms captured under different illumination angles. Accurate knowledge of the illumination angles is essential for a high-quality reconstruction, and thus requires system calibration which is often time- and labor-intensive. Here, to eliminate the need for intensive pre-calibration, we propose a self-calibrated approach that estimates the illumination angles directly from experimentally collected holograms without requiring any additional hardware or captured images. We demonstrate our method for correcting the illumination misalignment with both numerical simulation and experimental data.

[Expression and Significance of HIF-3α in Mitochondria].

OBJECTIVE: To investigate the mitochondrial translocation of hypoxia inducible factor-3α (HIF-3α) under normoxia and hypoxia and its physiological and pathological meanings. METHODS: ① After hypoxic (1%O2) or DMOG, CoCl2 treatments mimicking the hypoxic treatment, Western blot and immunofluorescence were used to examine the HIF-3α expression in mitochondria of HeLa and ACHN cells, respectively. ②The protease sensitivity experiment was used to explore the sub-organelle localization of HIF-3α in mitochondria. ③Western blot was used to examine mitochondrial HIF-3α in the normal mouse tissues and human liver carcinoma tissues. RESULTS: ① In HeLa and ACHN cells, HIF-3α translocated to mitochondria under normoxia and hypoxia, and its mitochondrial expression was higher under hypoxia; ②The protease sensitivity of HIF-3α was similar to proteins locating in the mitochondrial outer membrane; ③Mitochondrial HIF-3α expressed in multiple normal mouse tissues; The expression of mitochondrial HIF-3α was higher in human liver carcinoma tissues than the normal and adjacent tissues. CONCLUSIONS: HIF-3α translocated to mitochondrial outer membrane under both normoxia and hypoxia, and hypoxia could up-regulated HIF-3α mitochondrial translocation. Meanwhile, the phenomenon may be involved in the process of liver carcinoma.

Celastrol mediates Th17 and Treg cell generation via metabolic signaling.

A T helper 17 (Th17) cell/regulatory T (Treg) cell imbalance is involved in many immune disorders and diseases. Celastrol, a Chinese herbal compound that has anti-inflammatory and immunosuppressive properties, has been indicated to suppress T cell proliferation and Th17 cell induction, while facilitating Forkhead box P3 (Foxp3) expression and Treg cell generation. In this study, we explored the impact and mechanism of celastrol on Th17 cell/induced Treg (iTreg) cell induction. CD4+CD25- T cells were purified, stimulated with anti-CD3 and anti-CD28 antibodies, and polarized in vitro to generate Th17 or iTreg cells in the presence or absence of celastrol. Initially, we determined that Interleukin (IL)-17 expression by celastrol-treated Th17 was significantly decreased compared with untreated cells; however, the frequency of Foxp3+ cells was increased in celastrol-treated cells. We verified that celastrol inhibited phospho-STAT3 expression in cultured Th17 cells and up-regulated phospho-STAT5 expression in iTreg cells. Furthermore, T cells treated with celastrol were more likely to participate in FAO metabolism instead of glycolysis. Celastrol suppressed the expression of glucose transporter, Glut1, and the rate-limiting enzyme, HK2, in addition to mTOR, HIF-1α, c-Myc and Akt expression in Th17 cells. Conversely, celastrol promoted FAO of lipids by up-regulating CPT1A and AMPKα expression in iTreg cells. Our results suggest that celastrol suppresses Th17 cell induction, while promoting the generation of iTreg cells. We found that celastrol inhibits glycolysis in Th17 cells and promotes FAO by iTreg cells, suggesting that celastrol could mediate the metabolism of Th17 and iTreg cells.

One new linear C14 polyacetylene glucoside with antiadipogenic activities on 3T3-L1 cells from the capitula of Coreopsis tinctoria.

Based on characteristic UV spectrum of the ene-diyne chromophore, one new polyacetylene glucoside and three known polyacetylene glucosides have been isolated from the EtOH extract of Coreopsis tinctoria. Their chemical structures were determined by detailed spectroscopic analysis and by comparison with literature data. Compounds 1-2 were tested for their antiadipogenic effects on 3T3-L1 adipocytes, and both of them reduced lipid accumulation dose-dependently in 3T3-L1 adipocytes.

Analytical model of optical fluence inside multiple cylindrical inhomogeneities embedded in an otherwise homogeneous turbid medium for quantitative photoacoustic imaging.

We present an analytical model of optical fluence for multiple cylindrical inhomogeneities embedded in an otherwise homogeneous turbid medium. The model is based on the diffusion equation and represents the optical fluence distribution inside and outside inhomogeneities as a series of modified Bessel functions. We take into account the interplay between cylindrical inhomogeneities by introducing new boundary conditions on the surface of inhomogeneities. The model is compared with the numerical solution of the diffusion equation with NIRFAST software. The fluences inside the inhomogeneities obtained by the two methods are in close agreement. This permits the use of the model as a forward model for quantitative photoacoustic imaging.

Gene expression profiles of various cytokines in mesenchymal stem cells derived from umbilical cord tissue and bone marrow following infection with human cytomegalovirus.

Mesenchymal stem cells (MSCs) have both multi-lineage differentiation potential and immunosuppressive properties, making them ideal candidates for regenerative medicine. However, their immunosuppressive properties potentially increase the risk of cancer progression and opportunistic infections. In this study, MSCs isolated from human umbilical cord blood (UCMSCs) and adult bone marrow (BMMSCs) were infected with human cytomegalovirus (HCMV). Cytopathic changes were observed 10 days post infection. PCR products amplified from genomic DNA and cDNA were used to confirm the HCMV infection of the UCMSCs and BMMSCs. Real-time PCR was conducted to quantify the expression of immunomodulatory molecules, including cytokines, chemokines, growth factors, adhesion molecules and cancer-related genes. Our results indicate high upregulation of the majority of these molecules, including many growth factors, tumor necrosis factor alpha, interleukin-8, interleukin-6 and interferon gamma. Adhesion molecules (VCAM-1, TCAM-1 and selectin-E) were downregulated in the infected UCMSCs and BMMSCs. Antibody chip array evaluation of cell culture media indicated that the growth factor secretion by UCMSCs and BMMSCs was greatly influenced (p < 0.001) by HCMV. The stimulation of MSCs with HCMV led to the activation of downstream signaling pathways, including pSTAT3 and Wnt2. Our results show that HCMV can significantly alter the functions of both UCMSCs and BMMSCs, although not in the same way or to the same extent. In both cases, there was an increase in the expression of proangiogenic factors in the microenvironment following HMCV infection. The discrepancy between the two cell types may be explained by their different developmental origin, although further analysis is necessary. Future studies should decipher the underlying mechanism by which HCMV controls MSCs, which may lead to the development of new therapeutic treatments.

Mechanisms of cyclosporine-induced renal cell apoptosis: a systematic review.

BACKGROUND/AIMS: Chronic cyclosporine A (CsA) nephrotoxicity (CCN) is an important cause of chronic renal dysfunction with no effective clinical intervention. To further elucidate the mechanisms of renal cell apoptosis in CCN, all relevant in vivo studies on this subject were analyzed. METHODS: We searched for in vivo studies on the mechanisms of CsA-induced renal cell apoptosis in Medline (1966-July 2010), Embase (1980-July 2010) and ISI (1986-July 2010). The studies were evaluated for their quality according to a set of in vivo standards, data extracted according to PICOS, and then synthesized. RESULTS: Renal cell apoptosis was an important feature of CCN and an important factor of renal dysfunction. First, CsA could upregulate Fas/Fas ligand, downregulate Bcl-2/Bcl-XL, and increase caspase-1 and caspase-3. Second, it could induce oxidative stress and damage the antioxidant defense system. Third, it could increase endoplasmic reticulum stress protein in a dose- and time-dependent manner. Fourth, CsA could impair the urine concentration and decrease the expression of hypertonicity-induced genes. Fifth, CsA-induced renal cell apoptosis was significantly decreased by blocking the angiotensin II type 1 receptor using losartan. CONCLUSIONS: The in vivo mechanisms for CCN are more complex than those found in vitro. CsA can induce renal cell apoptosis using five pathways in vivo and activated caspases might be the ultimate intersection of these pathways and the common intracellular pathway mediating apoptosis. These data provide new potential points for intervention and need to be confirmed by further studies.

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis.

BACKGROUND: Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α. RESULTS: Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells. CONCLUSIONS: Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.