Effects of polysaccharides on colonic targeting and colonic fermentation of ovalbumin-ferulic acid based emulsion.

Rutin is a polyphenol with beneficial pharmacological properties. However, its bioavailability is often compromised due to low solubility and poor stability. Encapsulation technologies, such as emulsion systems, have been proven to be promising delivery vehicles for enhancing the bioavailability of bioactive compounds. Thus, this study was proposed and designed to investigate the colonic targeting and colonic fermentation characteristics of rutin-loaded ovalbumin-ferulic acid-polysaccharide (OVA-FA-PS) complex emulsions. The results indicate that OVA-FA-PS emulsion effectively inhibits the degradation of rutin active substances and facilitates its transport of rutin to the colon. The analysis revealed that the OVA-FA-κ-carrageenan emulsion loaded with rutin exhibited superior elasticity and colon targeting properties compared to the OVA-FA-hyaluronic acid or OVA-FA-sodium alginate emulsions loaded with rutin in the composite emulsion. Additionally, it was observed that the rutin loaded within the OVA-FA-κ-carrageenan emulsion underwent degradation and was converted to 4-hydroxybenzoic acid during colonic fermentation.

Newly formed phenolics selectively bound to the graded polysaccharides of lychee pulp during heat pump drying using UPLC-ESI-QqQ-TOF-MS/MS.

Our study investigated heat pump drying (HPD) effects on phenolic-polysaccharide adducts of three lychee pulp grades, their composition and bound phenolic contents. During HPD, the hexose content in water soluble polysaccharide (WSP) increased continuously, and the pentose and glucuronic acid contents in WSP and dilute alkali soluble pectin (ASP) together with the hexose content in ASP increased initially and then decreased due to polysaccharide hydrolases pectinase, polygalacturonase and cellulase. After HPD, the bound phenolic content in WSP, ASP and water unextractable polysaccharide (WUP) significantly increased. Protocatechualdehyde and 3,4-dihydroxybenzeneacetic acid were newly generated phenolics and the former combined with all the three polysaccharide grades, while the latter selectively combined with only WSP. During HPD, WSP and ASP surface structures were gradually broken and became loose, but WUP surface structure was a complete and rough sheet structure. Alkaline hydrolysis caused sparser, more porous surfaces of the three polysaccharide grades. The polyphenol selectivity could be related to substrate selectivity of endogenous oxidases and the type of phenolic compounds.

Newly generated and increased bound phenolic in lychee pulp during heat-pump drying detected by UPLC-ESI-triple-TOF-MS/MS.

BACKGROUND: During the thermal processing of fruit, it has been observed for phenolic compounds to either degrade, polymerize, or transfer into macromolecules. In this study, the bound and free phenolic compound composition, content, and phenolic-related enzyme activity of lychee pulp were investigated to determine whether the free phenolic had converted to bound phenolic during heat-pump drying (HPD). RESULTS: It was found that after HPD, when compared with the fresh lychee pulp (control), the content of bound phenolics of dried lychee pulp had increased by 62.69%, whereas the content of free phenolics of dried lychee pulp decreased by 22.26%. It was also found that the antioxidant activity of bound phenolics had also increased after drying. With the use of high-performance liquid chromatography-tandem mass spectrometry, it was identified that (+)-gallocatechin, protocatechuic aldehyde, isorhamnetin-3-O-rutoside, 3,4-dihydroxybenzeneacetic acid, and 4-hydroxybenzoic acid were newly generated during HPD, when compared with the control sample. After drying, the contents of gallic acid, catechin, 4-hydroxybenzoic acid, vanillin, syringic acid, and quercetin in bound phenolics had also increased, and polyphenol oxidase and peroxidase still showed enzyme activity, which could be related to the conversion of free phenolics to bound phenolics. CONCLUSION: Overall, during the thermal processing of lychee pulp, the free phenolics weres found to be converted into bound phenolics, new substances were generated, and antioxidant activity was increased. Hence, it was concluded that HPD improved the bound phenolics content of lychee pulp, thus providing theoretical support for the lychee processing industry. © 2021 Society of Chemical Industry.

Structural elucidation of flavonoids from Shatianyu (Citrus grandis L. Osbeck) pulp and screening of key antioxidant components.

The Citrus genus is a good source of dietary flavonoids, which have many health benefits. As a representative citrus fruit, the flavonoids composition in Shatianyu (Citrus grandis L. Osbeck) pulp remains to be investigated. In the present study, 11 flavonoids were isolated and identified from Shatianyu pulp flavonoid extracts (SPFEs). Among them, 4 flavonoids were previously undescribed and 2 flavonoids were firstly isolated from pummelo. The cellular antioxidant activity (CAA) and oxygen radical absorbance capacity (ORAC) of isolated compounds were evaluated. Naringin and rhoifolin showed the highest ORAC activity, and the presence of a 3-hydroxy-3-methylglutaryl or a 4'-glucose decreased the ORAC activity of flavonoids. The contribution of isolated flavonoids to the holistic antioxidant activity of SPFEs was determined by an online knockout method. Melitidin, bergamjuicin and naringin contributed most to ORAC activity, while bergamjuicin, melitidin and apigenin-4'-O-ß-d-glucopyranosyl-7-O-α-l-rhamnopyranosyl-(1 â†’ 2)-[6″-O-(3- hydroxy-3-methylgltaryl)]-ß-d-glucopyranoside contributed most to CAA activity.

Microvascular Changes in Macular Area After Phacoemulsification and Its Influencing Factors Assessed by Optical Coherence Tomography Angiography.

OBJECTIVE: To investigate macular vascular changes and underlying influencing factors using optical coherence tomography angiography (OCTA) after phacoemulsification in patients with cataracts. METHODS: Patients diagnosed with cataracts at the First Affiliated Hospital of Anhui Medical University from March to September 2019 were included. The macular retinal thickness, the microvascular density in the foveal, parafoveal, and perifoveal area, and the area of the foveal avascular zone (FAZ) were measured using OCTA at baseline, 1-week, 1-month, and 3-month post-operation. RESULTS: Forty-seven cataract patients (58 eyes) were eligible for this study. Compared with baseline thickness, the foveal, parafoveal, and perifoveal thicknesses, particularly in the inner retina, significantly increased at 1- and 3-month post-operation (P<0.05). There was a nonsignificant difference in the microvascular density of the foveal and perifoveal areas at 1-week, 1-month, and 3-month post-operation (P>0.05). At 1-month post-operation, the deep vessel density at the perifovea significantly increased (P<0.05). The FAZ area significantly diminished at 1- and 3-month post-operation compared with the baseline data (P<0.05). CONCLUSION: Phacoemulsification offers a satisfactory efficacy response, with an increased macular thickness, reduced FAZ area, and nonsignificant changes in the microvascular density at the perifovea after surgery.

Structural elucidation, distribution and antioxidant activity of bound phenolics from whole grain brown rice.

Chemical profiles, distribution, and antioxidant activity of bound phenolics from brown rice were investigated. Four new dehydrodiferulic acid dimers (DFA) along with eighteen known phenolics were isolated from brown rice bound phenolic extracts and their structures were determined by multiple spectroscopic methods. Among them, ferulic acid and 8-5' DFA were the most abundant monomeric and dimeric bound phenolics in brown rice, rice bran and polished rice. In whole brown rice, polished rice contributed more than 50% of three phenolic monomers and six phenolic dimers, while rice bran contributed more than half of the other thirteen phenolics including eight monomers, four dimers, and one trimer. All the isolated compounds exhibited oxygen radical absorbance capacity. Thomasidioic acid, caffeic acid, methyl caffeate, and 8-5' DC DFA displayed potent peroxyl radical scavenging capacity, and the last three compounds also showed moderate cellular antioxidant activity.

[Prokaryotic expression and purification of mouse recombinant myelin proteolipid protein polypeptide].

Objective To establish and optimize the prokaryotic expression method for the recombinant mouse myelin proteolipid protein (PLP, 139-208 aa) which is a critical immunogenic polypeptide of PLP. Methods The sequence coding for PLP139-208 polypeptide was cloned into pET-32a(+) vector. Afterwards, the expression vector prepared in this research was transformed into E. coli BL21, and the recombinant PLP polypeptide was induced to express by isopropyl-ß-D-thiogalactoside (IPTG). Two key prokaryotic expression conditions, IPTG's induction length and temperature, were analyzed for further optimization. The recombinant PLP polypeptide was induced to express by the expression method under the optimal expression conditions, and then was purified by Ni-NTA agarose and amylose resin. Finally, the gain of PLP139-208 polypeptide was verified by Western blot analysis. Results The results in the combinatorial optimization revealed that the expression of PLP139-208 was obtained at a satisfactory level when it was incubated at 23DegreesCelsius for 20 hours with the IPTG concentration of 0.5 mmol/L. Conclusion The optimized prokaryotic expression method for the recombinant mouse PLP139-208 was successfully established and effectively performed. This will shed light on the further researches on the improved preparation for experimental autoimmune encephalitis (EAE, an animal model of multiple sclerosis) and the underlying mechanism underlying PLP-induced autoimmune demyelination.

Effects of simulated digestion on the phenolic composition and antioxidant activity of different cultivars of lychee pericarp.

BACKGROUND: Lychee pericarp is rich in phenolic and has good antioxidant activity. The effects of simulated gastric (SGF) and intestinal fluid (SIF) digestion on the contents, composition, and antioxidant activities of the phenolic substances in the pericarp of different lychee cultivars (cv Jizui, Lizhiwang, Guiwei, Yuhe, Nuomici and Guihong) were investigated. RESULTS: Compared with distilled water (DW) treatment, the total phenolic content (TPC) and total flavonoid content (TFC) in the pericarp of different lychee cultivars decreased after SGF digestion; especially, the TFC in "Lizhiwang" decreased by 41.5%. The TPC and TFC of lychee pericarp also decreased after SIF digestion. However, the TPC in "Jizui", "Guiwei" and "Yuhe" increased. The SGF and SIF also had different effects on the FRAP and ABTS antioxidant activities of different lychee cultivars. The SGF digestion decreased the ABTS antioxidant capacity of lychee pericarp but enhanced the FRAP value of some lychee cultivars. However, the SIF digestion decreased the FRAP antioxidant activity of different lychee cultivar pericarps but enhanced the ABTS antioxidant capacity of lychee. The HPLC results showed that lychee pericarp had relatively high contents of procyanidin B2 and procyanidin A2. After SIF digestion, caffeic acid and isoquercitrin could not be detected in any of the lychee varieties. However, quercetin-3-rutinose-7-rhamnoside and isoquercitrin were increased after SGF digestion. CONCLUSIONS: Lychee pericarp could be used as an inexpensive functional food ingredient.

Citrus peel flavonoids improve lipid metabolism by inhibiting miR-33 and miR-122 expression in HepG2 cells.

Citrus plants are rich in flavonoids and beneficial for lipid metabolism. However, the mechanism has not been fully elucidated. Both citrus peel flavonoid extracts (CPFE) and a mixture of their primary flavonoid compounds, namely, nobiletin, tangeretin and hesperidin, citrus flavonoid purity mixture (CFPM), were found to have lipid-lowering effects on oleic acid-induced lipid accumulation in HepG2 cells. The carnitine palmitoyltransferase 1α (CPT1α) gene was markedly increased, while the fatty acid synthase (FAS) gene was significantly decreased by both CPFE and CFPM in oleic acid-treated HepG2 cells. Flavonoid compounds from citrus peel suppressed miR-122 and miR-33 expression, which were induced by oleic acid. Changes in miR-122 and miR-33 expression, which subsequently affect the expression of their target mRNAs FAS and CPT1α, are most likely the principal mechanisms leading to decreased lipid accumulation in HepG2 cells. Citrus flavonoids likely regulate lipid metabolism by modulating the expression levels of miR-122 and miR-33.

Persistence of Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria bassiana (Hypocreales: Clavicipitaceae) in Tobacco Soils and Potential as Biocontrol Agents of Spodoptera litura (Lepidoptera: Noctuidae).

Entomopathogenic fungi (EPF), such as Metarhizium spp. and Beauveria bassiana, are widely used in the biocontrol of many species of insect pests. Tobacco is an economically important crop in Guangdong Province of China, but insect pests, such as Spodoptera litura Fabricius, are a major threat to production. Here, we tested the persistence of five Metarhizium species and B. bassiana in glasshouse pot and field experiments and assessed their long-term efficacy against S. litura. We found that the colony forming units of these EPF decreased by c. 93% by 180 d in the pot soils declines tended to be exponential. In contrast, declines of c. 99% in field soils were more gradual (linear), occurring throughout the 360 d experiment. Metarhizium anisopliae Ma09 had the longest estimated half-life of 41 d, while that of B. bassiana was the shortest (9 d). Fungal density in the upper soil layer (0-5 cm) decreased rapidly and was undetectable after 150 d, whereas density was consistently greatest in the mid-layer (10-15 cm). At 180 d after inoculation, strain Ma09 elicited highest rates of mortality in S. litura. We conclude that soils in Guangdong Province are all suitable for the use of Metarhizium as a biocontrol agent, where M. anisopliae Ma09 offers greatest residual activity.

A Comparison of the Chemical Composition, In Vitro Bioaccessibility and Antioxidant Activity of Phenolic Compounds from Rice Bran and Its Dietary Fibres.

The composition, in vitro bioaccessibility and antioxidant activities of the phenolic compounds in defatted rice bran (DRB) and its soluble and insoluble dietary fibres were systematically evaluated in this study. The total phenolic content of insoluble dietary fibre from DRB (IDFDRB) was much higher than that of the soluble dietary fibre from DRB (SDFDRB) but was 10% lower than that of DRB. Bound phenolics accounted for more than 90% of the total phenolics in IDFDRB, whereas they accounted for 34.2% and 40.5% of the total phenolics in DRB and SDFDRB, respectively. Additionally, the phenolic profiles and antioxidant activities were significantly different in DRB, SDFDRB and IDFDRB. The phenolic compounds in IDFDRB were much less bioaccessibility than those in DRB and SDFDRB due to the higher proportion of bound phenolics in IDFDRB. Considering that bound phenolics could be released from food matrices by bacterial enzymes in the large intestine and go on to exert significant beneficial health effects in vivo, further studies on IDFDRB are needed to investigate the release of the phenolics from IDFDRB via gut microbiota and the related health benefits.

Optimized ultra-high-pressure-assisted extraction of procyanidins from lychee pericarp improves the antioxidant activity of extracts.

To establish optimal ultra-high-pressure (UHP)-assisted extraction conditions for procyanidins from lychee pericarp, a response surface analysis method with four factors and three levels was adopted. The optimum conditions were as follows: 295 MPa pressure, 13 min pressure holding time, 16.0 mL/g liquid-to-solid ratio, and 70% ethanol concentration. Compared with conventional ethanol extraction and ultrasonic-assisted extraction methods, the yields of the total procyanidins, flavonoids, and phenolics extracted using the UHP process were significantly increased; consequently, the oxygen radical absorbance capacity and cellular antioxidant activity of UHP-assisted lychee pericarp extracts were substantially enhanced. LC-MS/MS and high-performance liquid chromatography quantification results for individual phenolic compounds revealed that the yield of procyanidin compounds, including epicatechin, procyanidin A2, and procyanidin B2, from lychee pericarp could be significantly improved by the UHP-assisted extraction process. This UHP-assisted extraction process is thus a practical method for the extraction of procyanidins from lychee pericarp.

Prognostic Value of Pre-Operative Platelet to Lymphocyte Ratio in Patients with Resected Primary Hepatocellular Carcinoma.

BACKGROUND: The platelet to lymphocyte ratio (PLR) is a readily available cancer biomarker but its prognostic value for patients with resected primary hepatocellular carcinoma (HCC) is uncertain. Thus, we investigated the relationship between PLR and survival of patients with resected primary HCC in the Fujian area, a high occurrence area of HCC in China. METHODS: This retrospective study included 337 patients with primary HCC who underwent surgical removal of primary liver cancer between 2004 and 2012 in Fuzhou General Hospital in Fujian, China. Pre-operative peripheral blood PLR was measured by platelet counts divided by lymphocyte counts. PLR and clinical factors were associated with a X2 test and data were analyzed with a Kaplan-Meier plus log rank analysis. Independent prognostic factors related to survival were assessed with multivariable analysis. RESULTS: Subjects were classified by a median PLR value of 91 (low: ≤ 91, n = 169 and high: > 91, n = 168). HighPLR patients died more often (57.7% vs. 35.5%, p = 4.3 x 10-5 and 39 months vs. 88 months, p < 0.001, respectively) and high PLR was associated with a nearly 2-fold elevated death risk (hazard ratio [HR]:1.815, 95% confidence interval (CI): 1.298 - 2.537, p = 4.85 x 10-4). Serum AFP, TNM stage and tumor size were also independent prognostic factors for patients with resected primary HCC according to multivariable analysis. CONCLUSIONS: PLR may be useful as a biomarker for assessing survival of resected primary HCC patients.

Antioxidant and antiproliferative activities of polysaccharide fractions from litchi pulp.

Three litchi polysaccharide fractions (LPFs), LP-4, LP-6 and LP-8, were obtained by fractional precipitation using 40%, 60% and 80% ethanol, respectively. The physicochemical properties, chemical antioxidant, and cellular antioxidant and antiproliferative activities of the three polysaccharide fractions were compared. LP-6 contained the highest contents of uronic acid and binding protein among the three fractions, whereas LP-8 contained the least. Amino acid composition analysis of the binding protein revealed that LP-6 contained the most acidic and aromatic amino acids. However, LP-8 contained more galactose and mannose than LP-4 and LP-6. LP-6 exhibited the highest chemical antioxidant activities, with an oxygen radical absorbance capacity of 28.14 µmol TE per g DW. LP-8 exhibited higher cellular antioxidant activity and a greater inhibitory effect on the proliferation of A549, HepG2 and MGC-803 cells at a concentration of 100-800 µg mL(-1) than LP-4 and LP-6. In summary, the different LPFs exhibited different antioxidant and antiproliferative activities with differential physicochemical properties.

IL-1ß-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9.

BACKGROUND: Interleukin-1ß (IL-1ß) has been implicated in the progression of gastric adenocarcinoma (GA); however, the molecular mechanisms of action of IL-1ß in GA are poorly characterized. P38 and JNK are the major MAPK family members that regulate IL-1ß signaling pathways. Here, we investigated the role of both p38 and JNK in IL-1ß-induced GA cell migration, invasion and metastatic potential. METHODS: The effects of IL-1ß-induced p38 and JNK activation in GA cells were determined using in vitro Transwell migration and invasion assays of MKN-45 and AGS cells, or an in vivo metastasis assay in nude mice. The IL-1ß-induced p38 signaling pathway was further characterized in GA cells. Activation of the IL-1ß/p38 signaling pathway was also assessed in human primary GA tissues by immunohistochemistry. RESULTS: IL-1ß-induced activation of p38 increased GA cell migration and invasion in vitro and promoted the metastatic potential of GA cells in vivo; these effects were attenuated by p38 siRNA or the p38 inhibitor SB202190. MMP2 or MMP9 siRNAs and the MMP2/9 inhibitor BiPS also inhibited IL-1ß-induced GA cell migration and invasion in vitro. IL-1ß-induced p38 activation significantly increased MMP2 and MMP9 mRNA and protein expression and activity. Luciferase reporter assays demonstrated that the activator protein-1 (AP-1) and the AP-1 binding sites of the MMP9 promoter (-670/MMP9) were activated by IL-1ß-induced p38 activation. Phospho-p38 was significantly upregulated in human GA tissues (compared to matched non-neoplastic tissues), and significantly associated with lymph node metastasis, and invasion beyond the serosa. Expression of phospho-p38 significantly correlated with IL-1ß, MMP2, MMP9, and c-fos expression in both human GA tissues and GA cell metastases in the lungs of nude mice. IL-1ß was also capable of activating JNK in GA cells, but activation of JNK was not associated with GA cell migration and invasion. Therefore, IL-1ß-induced the migration and invasion in GA cells were regulated by p38, but not by JNK. CONCLUSIONS: IL-1ß-induced p38 activation and the IL-1ß/p38/AP-1(c-fos)/MMP2 & MMP9 pathway play an important role in metastasis in GA; this pathway may provide a novel therapeutic target for GA.

Antiviral activity of FNC, 2'-deoxy-2'-ß-fluoro-4'-azidocytidine, against human and duck HBV replication.

BACKGROUND: New drugs are needed to combat HBV infection. We investigated the anti-HBV activity of the deoxycytidine analogue FNC, which has anticancer activity and has been found to inhibit HCV replication. METHODS: In this study, a human hepatoma HepG2.2.15 cell culture system and duck HBV (DHBV) infection model were used as the in vitro and in vivo models to evaluate the anti-HBV activity of FNC. RESULTS: In the cell model, FNC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner, with 50% effective concentration values of 0.037 µM for hepatitis B surface antigen and 0.044 µM for hepatitis B e antigen on day 9. Consistent with the HBV antigen reduction, FNC also reduced the HBV DNA level by 92.31% and 93.90% intracellularly and extracellularly, respectively. DHBV DNA levels were markedly reduced after treatment with the FNC at 0.5, 1.0 and 2.0 mg/kg•day dosages. The inhibition rate of FNC at the dose of 2.0 mg/kg•day reached 91.68% and 81.96%, in duck serum and liver, respectively, on day 10. Furthermore, significant liver histology restoration after FNC treatment was observed, as evaluated by the histopathological analysis. CONCLUSIONS: FNC can evidently inhibit the replication of HBV in the HepG2.2.15 cell line in vitro and inhibits DHBV replication in ducks in vivo. It could be potentially developed into a new anti-HBV drug.

Akt2 kinase suppresses glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-mediated apoptosis in ovarian cancer cells via phosphorylating GAPDH at threonine 237 and decreasing its nuclear translocation.

Protein kinase B (Akt) plays important roles in regulation of cell growth and survival, but while many aspects of its mechanism of action are known, there are potentially additional regulatory events that remain to be discovered. Here we detected a 36-kDa protein that was co-immunoprecipitated with protein kinase Bß (Akt2) in OVCAR-3 ovarian cancer cells. The protein was identified to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by MALDI-TOF/TOF MS, and the interaction of Akt2 and GAPDH was verified by reverse immunoprecipitation. Our further study showed that Akt2 may suppress GAPDH-mediated apoptosis in ovarian cancer cells. Overexpression of GAPDH increased ovarian cancer cell apoptosis induced by H(2)O(2), which was inhibited by Akt2 overexpression and restored by the PI3K/Akt inhibitor wortmannin or Akt2 siRNA. Akt2 phosphorylated Thr-237 of GAPDH and decreased its nuclear translocation, an essential step for GAPDH-mediated apoptosis. The interaction between Akt2 and GAPDH may be important in ovarian cancer as immunohistochemical analysis of 10 normal and 30 cancerous ovarian tissues revealed that decreased nuclear expression of GAPDH correlated with activation (phosphorylation) of Akt2. In conclusion, our study suggests that activated Akt2 may increase ovarian cancer cell survival via inhibition of GAPDH-induced apoptosis. This effect of Akt2 is partly mediated by its phosphorylation of GAPDH at Thr-237, which results in the inhibition of GAPDH nuclear translocation.

Controllable synthesis of nickel hydroxide and porous nickel oxide nanostructures with different morphologies.

Alpha-Ni(OH)(2) nanobelts, nanowires, short nanowires, and beta-Ni(OH)(2) nanoplates have been successfully prepared in high yields and purities by a convenient hydrothermal method under mild conditions from very simple systems composed only of NaOH, NiSO(4), and water. It has been found that the ratio of NaOH to NiSO(4) not only affects the morphology of the Ni(OH)(2) nanostructures, but also determines whether the product is of the alpha- or beta-crystal phase. A notable finding is that porous NiO nanobelts were produced after exposure of the Ni(OH)(2) products to an electron beam for several minutes during transmission electron microscopy (TEM) observations. Another unusual feature is that rectangular nanoplates with many gaps were obtained. Furthermore, porous NiO nanobelts, nanowires, and nanoplates could also be obtained by annealing the as-prepared Ni(OH)(2) products. A sequence of dissolution, recrystallization, and oriented attachment-assisted self-assembly of nanowires into nanobelts is proposed as a plausible mechanistic interpretation for the formation of the observed structures. The method presented here possesses several advantages, including high yields, high purities, low cost, and environmental benignity. It might feasibly be scaled-up for industrial mass production.

Microarray analysis of gene-expression profile in hepatocellular carcinoma cell, BEL-7402, with stable suppression of hLRH-1 via a DNA vector-based RNA interference.

To establish a cell line with a permanent suppression of hLRH-1 in this study, a stable RNAi vector (pSineohLRH-1) targeting hLRH-1 was constructed and introduced into hepatocellular carcinoma cell, BEL-7402. By semiquantitative RT-PCR analysis, the expression of hLRH-1 in BEL-7402 cells carrying pSineohLRH-1 was shown to be significantly suppressed by up to approximately 60%. In addition, microarray analysis was carried out to assess the extent of altered gene expression in BEL-7402 cells with stable knockdown of hLRH-1. Direct comparison of gene-expression profiles of more than 18,000 genes showed that 405 of the expressed genes in hLRH-1-knockdown cells differed dramatically in expression levels from those in controls, which suggested the even extensive biological functions of hLRH-1. Interestingly, among those differentially expressed genes, some are cancer-associated such as Gadd45beta and PTEN, and their expressions were further validated. Although the identification of the exact relationship between these genes and hLRH-1 awaits intensive investigation, the findings of this study provide new insights into the mechanism by which hLRH-1 is involved in tumorigenesis.