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1.  The Protective Effect of MicroRNA-320 on Left Ventricular Remodeling after Myocardial Ischemia-Reperfusion Injury in the Rat Model 
The primary objective of this study investigated the role of microRNA-320 (miR-320) on left ventricular remodeling in the rat model of myocardial ischemia-reperfusion (I/R) injury, and we intended to explore the myocardial mechanism of miR-320-mediated myocardium protection. We collected 120 male Wistar rats (240–280 g) in this study and then randomly divided them into three groups: (1) sham surgery group (sham group: n = 40); (2) ischemia-reperfusion model group (I/R group: n = 40); and (3) I/R model with antagomir-320 group (I/R + antagomir-320 group: n = 40). Value changes of heart function in transesophageal echocardiography were recorded at various time points (day 1, day 3, day 7, day 15 and day 30) after surgery in each group. Myocardial sections were stained with hematoxylin and eosin (H&E) and examined with optical microscope. The degree of myocardial fibrosis was assessed by Sirius Red staining. Terminal dUTP nick end-labeling (TUNEL) and qRT-PCR methods were used to measure the apoptosis rate and to determine the miR-320 expression levels in myocardial tissues. Transesophageal echocardiography showed that the values of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular systolic pressure (LVSP) and ±dp/dtmax in the I/R group were obviously lower than those in the sham group, while the left ventricular end-diastolic pressure (LVEDP) value was higher than that in the sham group. The values of LVEF, LVFS, LVSP and ±dp/dtmax showed a gradual decrease in the I/R group, while the LVEDP value showed an up tendency along with the extension of reperfusion time. The H&E staining revealed that rat myocardial tissue in the I/R group presented extensive myocardial damage; for the I/R + antagomir-320 group, however, the degree of damage in myocardial cells was obviously better than that of the I/R group. The Sirius Red staining results showed that the degree of myocardial fibrosis in the I/R group was more severe along with the extension of the time of reperfusion. For the I/R + antagomir-320 group, the degree of myocardial fibrosis was less severe than that in the I/R group. Tissues samples in both the sham and I/R + antagomir-320 groups showed a lower apoptosis rate compared to I/R group. The qRT-PCR results indicated that miR-320 expression in the I/R group was significantly higher than that in both the sham and I/R + antagomir-320 groups. The expression level of miR-320 is significantly up-regulated in the rat model of myocardial I/R injury, and it may be implicated in the prevention of myocardial I/R injury-triggered left ventricular remodeling.
PMCID: PMC4227171  PMID: 25268616
microRNA-320; myocardial ischemia-reperfusion injury; left ventricular remodeling
2.  Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways 
To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells.
mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot.
Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression.
Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.
PMCID: PMC3269723  PMID: 22312289
Lewis(y) antigen; cell cycle; cyclin; cyclin-dependent kinases; cyclin-dependent kinase inhibitors
3.  Beclin 1 Expression in Ovarian Tissues and Its Effects on Ovarian Cancer Prognosis 
Beclin 1 is an autophagy-associated protein involved in apoptosis and drug resistance, as well as various malignancies. We investigated the expression of Beclin 1 protein in ovarian epithelial tissues and correlated it with the prognosis of ovarian cancer. Beclin 1 protein expression was determined using immunohistochemistry in 148 patients with ovarian epithelial cancer, 26 with ovarian borderline tumor, 25 with benign ovarian tumor, and 30 with normal ovarian tissue. The relationships between Beclin 1 protein expression and ovarian cancer pathological characteristics were analyzed. The risk factors for ovarian cancer prognosis were analyzed using Cox’s regression model. A survival curve was plotted from the follow-up data of 93 patients with ovarian cancer to analyze the effects of Beclin 1 expression on the prognosis of ovarian cancer. The positive rates of Beclin 1 were significantly higher in ovarian epithelial cancer (148) and borderline tumor (26) than in benign ovarian tumor (25) or normal ovarian tissue (30) (all p < 0.001). The surgical stage and Beclin 1 expression were both independent risk factors for ovarian cancer prognosis (both p < 0.05). Patients with high Beclin 1 levels showed better survival than those with low Beclin 1 levels (p = 0.009). Beclin 1 protein is upregulated in ovarian epithelial cancer and is a prognostic factor of ovarian cancer.
PMCID: PMC4013564  PMID: 24675697
autophagy; Beclin 1; ovarian cancer; prognosis; overall survival
4.  Cetuximab-Induced MET Activation Acts as a Novel Resistance Mechanism in Colon Cancer Cells 
Aberrant MET expression and hepatocyte growth factor (HGF) signaling are implicated in promoting resistance to targeted agents; however, the induced MET activation by epidermal growth factor receptor (EGFR) inhibitors mediating resistance to targeted therapy remains elusive. In this study, we identified that cetuximab-induced MET activation contributed to cetuximab resistance in Caco-2 colon cancer cells. MET inhibition or knockdown sensitized Caco-2 cells to cetuximab-mediated growth inhibition. Additionally, SRC activation promoted cetuximab resistance by interacting with MET. Pretreatment with SRC inhibitors abolished cetuximab-mediated MET activation and rendered Caco-2 cells sensitive to cetuximab. Notably, cetuximab induced MET/SRC/EGFR complex formation. MET inhibitor or SRC inhibitor suppressed phosphorylation of MET and SRC in the complex, and MET inhibitor singly led to disruption of complex formation. These results implicate alternative targeting of MET or SRC as rational strategies for reversing cetuximab resistance in colon cancer.
PMCID: PMC4013599  PMID: 24714091
cetuximab; MET; EGFR; SRC; colon cancer
5.  Progressive Changes in Inflammatory and Matrix Adherence of Bronchial Epithelial Cells with Persistent Respiratory Syncytial Virus (RSV) Infection (Progressive Changes in RSV Infection) 
In addition to the acute manifestations of respiratory syncytial virus (RSV), persistent infection may be associated with long-term complications in the development of chronic respiratory diseases. To understand the mechanisms underlying RSV-induced long-term consequences, we established an in vitro RSV (strain A2) infection model using human bronchial epithelial (16HBE) cells that persists over four generations and analyzed cell inflammation and matrix adherence. Cells infected with RSV at multiplicity of infection (MOI) 0.0067 experienced cytolytic or abortive infections in the second generation (G2) or G3 but mostly survived up to G4. Cell morphology, leukocyte and matrix adherence of the cells did not change in G1 or G2, but subsequently, leukocyte adherence and cytokine/chemokine secretion, partially mediated by intercellular adhesion molecule-1 (ICAM-1), increased drastically, and matrix adherence, partially mediated by E-cadherin, decreased until the cells died. Tumor necrosis factor-α (TNF-α) secretion was inhibited by ICAM-1 antibody in infected-16HBE cells, suggesting that positive feedback between TNF-α secretion and ICAM-1 expression may be significant in exacerbated inflammation. These data demonstrate the susceptibility of 16HBE cells to RSV and their capacity to produce long-term progressive RSV infection, which may contribute to inflammation mobilization and epithelial shedding.
PMCID: PMC3794767  PMID: 24005865
respiratory syncytial virus; human bronchial epithelial cells; adherence; adhesive molecule; cytokine; chemokine
6.  Detection of Quantitative Trait Loci (QTLs) for Resistances to Small Brown Planthopper and Rice Stripe Virus in Rice Using Recombinant Inbred Lines 
Small brown planthopper (SBPH) and rice stripe virus (RSV) disease transmitted by SBPH cause serious damage to rice (Oryza sativa L.) in China. In the present study, we screened 312 rice accessions for resistance to SBPH. The indica variety, N22, is highly resistant to SBPH. One hundred and eighty two recombinant inbred lines (RILs) derived from a cross of N22 and the highly susceptible variety, USSR5, were used for quantitative trait locus (QTL) analysis of resistances to SBPH and RSV. In a modified seedbox screening test, three QTLs for SBPH resistance, qSBPH2, qSBPH3 and qSBPH7.1, were mapped on chromosomes 2, 3 and 7, a total explaining 35.1% of the phenotypic variance. qSBPH7.2 and qSBPH11.2, conferring antibiosis against SBPH, were detected on chromosomes 7 and 11 and accounted for 20.7% of the total phenotypic variance. In addition, qSBPH5 and qSBPH7.3, expressing antixenosis to SBPH, were detected on chromosomes 5 and 7, explaining 23.9% of the phenotypic variance. qSBPH7.1, qSBPH7.2 and qSBPH7.3, located in the same region between RM234 and RM429 on chromosome 7, using three different phenotyping methods indicate that the locus or region plays a major role in conferring resistance to SBPH in N22. Moreover, three QTLs, qSTV4, qSTV11.1 and qSTV11.2, for RSV resistance were detected on chromosomes 4 and 11. qSTV11.1 and qSTV11.2 are located in the same region between RM287 and RM209 on chromosome 11. Molecular markers spanning these QTLs should be useful in the development of varieties with resistance to SBPH and RSV.
PMCID: PMC3645751  PMID: 23591851
RIL population; quantitative trait locus; Oryza sativa L
7.  Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine 
Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX). Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX) can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.
PMCID: PMC3317715  PMID: 22489154
aptamer; SELEX; molecular medicine
8.  Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703 
Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.
PMCID: PMC3382817  PMID: 22754333
Pokemon; anoikis; Bim; hepatoma
9.  Elevated Levels of Lewis Y and Integrin α5β1 Correlate with Chemotherapeutic Drug Resistance in Epithelial Ovarian Carcinoma 
To measure Lewis y and integrin α5β1 expression in epithelial ovarian carcinoma and to correlate the levels of these molecules with ovarian carcinoma chemotherapy and prognosis.
The study population included 34 ovarian carcinoma patients with chemotherapeutic drug-resistance, six partially drug-sensitive cases, and 52 drug-sensitive cases (92 total). Immunochemistry was used to determine expression of Lewis y antigen and integrin α5β1 in ovarian carcinoma tissues, and correlation of these molecules with chemotherapy resistance was further investigated, Multi-factor logistic regression analysis was applied to investigate: age, surgical stage, grade, subtype of patient cases, metastasis of lymph nodes, residual tumor size, expression levels of Lewis y antigen and integrin α5β1 correlation with ovarian carcinoma chemotherapy resistance.
The expression rates of Lewis y antigen and integrins α5 and β1 were significantly greater in the drug-resistant group (91.17%, 85.29%, 88.24%) than the partially sensitive (50.00%, 33.33%, 50.00%) or sensitive groups (61.54%, 57.69%, 55.77%). Binary logistic regression analysis revealed that surgical stage, residual tumor size, and expression of integrin α5 and Lewis y in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance.
Overexpression of Lewis y and integrin α5 are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.
PMCID: PMC3546651  PMID: 23443083
integrins; Lewis y antigen; ovarian caricinoma; chemoresistance
10.  The Stimulation of IGF-1R Expression by Lewis(y) Antigen Provides a Powerful Development Mechanism of Epithelial Ovarian Carcinoma 
This study aimed to measure and correlate the expression of insulin-like growth factor receptor-1 (IGF-1R) and the Lewis(y) antigen in ovarian cancer cell lines and tissue samples.
Reverse transcriptase PCR (RT-PCR), Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence double-labeling techniques were applied to detect and measure the expression of Lewis(y) and IGF-1R.
In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, IGF-1R expression was significantly upregulated compared with cells that do not overexpress α1,2-FT (P < 0.05). The amount of Lewis(y) expressed on IGF-1R increased 1.81-fold in α1,2-FT-overexpressing cells (P < 0.05), but the ratio of Lewis(y) expressed on IGF-1R to total IGF-1R was unaltered between two cells (P > 0.05). In malignant epithelial ovarian tumors, the positivity rates of Lewis(y) and IGF-1R detection were 88.3% and 93.33%, respectively, which is higher than the positivity rates in marginal (60.00% and 63.33%, all P < 0.05), benign (33.00% and 53.33%, all P < 0.01), and normal (0% and 40%, all P < 0.01) ovarian samples. No correlations were detected in positivity rates of Lewis(y) or IGF-1R expression with respect to clinicopathological parameters in ovarian cancers (all P > 0.05). Both IGF-1R and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated (P < 0.05).
Overexpression of Lewis(y) results in overexpression of IGF-1R. Both IGF-1R and Lewis(y) are associated with the occurrence and development of ovarian cancers.
PMCID: PMC3211010  PMID: 22072919
epithelial ovarian tumor; Insulin-like growth factor receptor-1; Lewis(y) antigen; immunohistochemistry; immunofluorescence double labeling method
11.  Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro 
Tanshinone I (Tan-I) is a diterpene quinone extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge. Recently, Tan-I has been reported to have anti-tumor effects. In this study, we investigated the growth inhibition and apoptosis inducing effects of Tan-I on three kinds of monocytic leukemia cells (U937, THP-1 and SHI 1). Cell viability was measured by MTT assay. Cell apoptosis was assessed by flow cytometry (FCM) and AnnexinV/PI staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR–enzyme-linked immunosorbent assay (ELISA) were used to detect human telomerase reverse transcriptase (hTERT) expression and telomerase activity before and after apoptosis. The activity of caspase-3 was determined by Caspase colorimetric assay kit and Western blot analysis. Expression of the anti-apoptotic gene Survivin was assayed by Western blot and Real-time RT-PCR using the ABI PRISM 7500 Sequence Detection System. The results revealed that Tan-I could inhibit the growth of these three kinds of leukemia cells and cause apoptosis in a time- and dose-dependent manner. After treatment by Tan-I for 48 h, Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17-kD subunit, and a 89-kD cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also found clearly. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently in a dose-dependent manner. Moreover, Real-time RT-PCR and Western blot revealed a significant down-regulation of Survivin. We therefore conclude that the induction of apoptosis by Tan-I in monocytic leukemia U937 THP-1 and SHI 1 cells is highly correlated with activation of caspase-3 and decreasing of hTERT mRNA expression and telomerase activity as well as down-regulation of Survivin expression. To our knowledge, this is the first report about the effects of Tan-I on monocytic leukemia cells.
PMCID: PMC2904915  PMID: 20640151
Tanshinone I (Tan-I); telomerase; survivin; leukemia
12.  Differential Expression of Copper-Zinc Superoxide Dismutase Gene of Polygonum sibiricum Leaves, Stems and Underground Stems, Subjected to High-Salt Stress 
In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5′ untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3′ untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1–16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO3. The different mRNA levels’ expression of PS-CuZnSOD show the gene’s different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.
PMCID: PMC3100833  PMID: 21614204
P. sibiricum Laxm.; PS-CuZnSOD; RACE; real-time PCR; gene expression
13.  Analysis of Intestinal Injuries Induced by Ricin in Vitro Using SPR Technology and MS Identification 
The present study found that ricin toxicity did not only manifest itself as inhibition of protein synthesis, but also induced apoptosis of immune cells and played an extremely significant role in intestinal injury. In this report, we describe a novel method to estimate binding events occurring on intestinal brush border membranes (BBM) based on SPR technology in an attempt to mimic the real intestinal surface capable of interacting physically and/or actively with certain biological molecules. Combined with HPCE-ESI-MS indentification, we obtained 28 kinds of proteins in BBM that interacted with ricin.
PMCID: PMC2695285  PMID: 19564957
14.  Cdc42-Interacting Protein 4 Represses E-Cadherin Expression by Promoting β-Catenin Translocation to the Nucleus in Murine Renal Tubular Epithelial Cells 
Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. β-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with β-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating β-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in β-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-β1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of β-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-β1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of β-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and β-catenin was detected. We also show that β-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting β-catenin translocation to the nucleus.
PMCID: PMC4581292  PMID: 26287173
TGF-β1; CIP4; β-catenin; E-cadherin; renal tubular epithelial cells
15.  Association between the NF-E2 Related Factor 2 Gene Polymorphism and Oxidative Stress, Anti-Oxidative Status, and Newly-Diagnosed Type 2 Diabetes Mellitus in a Chinese Population 
Oxidative stress is a major risk factor in the onset and progression of type 2 diabetes mellitus (T2DM). NF-E2 related factor 2 (NRF2) is a pivotal transcription factor in oxidative stress related illnesses. This study included 2174 subjects with 879 cases of newly-diagnosed T2DM and 1295 healthy controls. Compared to individuals with the CC genotype, those with the AA genotype had lower total anti-oxidative capacity, superoxide dismutase, catalase, glutathione, glutathione peroxidase activity; and lower homeostasis model assessment of β-cell function index. Those with the AA genotype also had a higher malondialdehyde concentration and homeostasis model assessment of insulin resistance index values. The frequency of allele A was significantly higher in T2DM subjects (29.4%), compared to control subjects (26.1%; p = 0.019). Individuals with the AA genotype had a significantly higher risk of developing T2DM (OR 1.56; 95% CI 1.11, 2.20; p = 0.011), relative to those with the CC genotype, even after adjusting for known T2DM risk factors. Our results suggest that the NRF2 rs6721961 polymorphism was significantly associated with oxidative stress, anti-oxidative status, and risk of newly-diagnosed T2DM. This polymorphism may also contribute to impaired insulin secretory capacity and increased insulin resistance in a Chinese population.
PMCID: PMC4519961  PMID: 26204833
NRF2; oxidative stress; polymorphism; type 2 diabetes
16.  14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling 
As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.
PMCID: PMC4519969  PMID: 26204835
14-3-3γ; inflammatory responses; dairy cow mammary epithelial cells; NF-κB; MAPKs; mTOR signaling pathway
17.  Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells 
Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.
PMCID: PMC4490421  PMID: 26016500
chromate reduction; yieF; HepG2; transfection; Cr(VI) resistance
18.  Effect of Chronic Pioglitazone Treatment on Hepatic Gene Expression Profile in Obese C57BL/6J Mice 
Pioglitazone, a selective ligand of peroxisome proliferator-activated receptor gamma (PPARγ), is an insulin sensitizer drug that is being used in a number of insulin-resistant conditions, including non-alcoholic fatty liver disease (NAFLD). However, there is a discrepancy between preclinical and clinical data in the literature and the benefits of pioglitazone treatment as well as the precise mechanism of action remain unclear. In the present study, we determined the effect of chronic pioglitazone treatment on hepatic gene expression profile in diet-induced obesity (DIO) C57BL/6J mice in order to understand the mechanisms of NAFLD induced by PPARγ agonists. DIO mice were treated with pioglitazone (25 mg/kg/day) for 38 days, the gene expression profile in liver was evaluated using Affymetrix Mouse GeneChip 1.0 ST array. Pioglitazone treatment resulted in exacerbated hepatic steatosis and increased hepatic triglyceride and free fatty acids concentrations, though significantly increased the glucose infusion rate in hyperinsulinemic-euglycemic clamp test. The differentially expressed genes in liver of pioglitazone treated vs. untreated mice include 260 upregulated and 86 downregulated genes. Gene Ontology based enrichment analysis suggests that inflammation response is transcriptionally downregulated, while lipid metabolism is transcriptionally upregulated. This may underlie the observed aggravating liver steatosis and ameliorated systemic insulin resistance in DIO mice.
PMCID: PMC4490440  PMID: 26035752
pioglitazone; non-alcoholic fatty liver disease; Affymetrix Mouse GeneChip; inflammation response; lipid metabolism
19.  A Comparative Proteomic Analysis of the Buds and the Young Expanding Leaves of the Tea Plant (Camellia sinensis L.) 
Tea (Camellia sinensis L.) is a perennial woody plant that is widely cultivated to produce a popular non-alcoholic beverage; this beverage has received much attention due to its pleasant flavor and bioactive ingredients, particularly several important secondary metabolites. Due to the significant changes in the metabolite contents of the buds and the young expanding leaves of tea plants, high-performance liquid chromatography (HPLC) analysis and isobaric tags for relative and absolute quantitation (iTRAQ) analysis were performed. A total of 233 differentially expressed proteins were identified. Among these, 116 proteins were up-regulated and 117 proteins were down-regulated in the young expanding leaves compared with the buds. A large array of diverse functions was revealed, including roles in energy and carbohydrate metabolism, secondary metabolite metabolism, nucleic acid and protein metabolism, and photosynthesis- and defense-related processes. These results suggest that polyphenol biosynthesis- and photosynthesis-related proteins regulate the secondary metabolite content of tea plants. The energy and antioxidant metabolism-related proteins may promote tea leaf development. However, reverse transcription quantitative real-time PCR (RT-qPCR) showed that the protein expression levels were not well correlated with the gene expression levels. These findings improve our understanding of the molecular mechanism of the changes in the metabolite content of the buds and the young expanding leaves of tea plants.
PMCID: PMC4490536  PMID: 26096006
Camellia sinensis L.; proteome; iTRAQ
20.  Expression Profiling Reveals Genes Involved in the Regulation of Wool Follicle Bulb Regression and Regeneration in Sheep 
Wool is an important material in textile manufacturing. In order to investigate the intrinsic factors that regulate wool follicle cycling and wool fiber properties, Illumina sequencing was performed on wool follicle bulb samples from the middle anagen, catagen and late telogen/early anagen phases. In total, 13,898 genes were identified. KRTs and KRTAPs are the most highly expressed gene families in wool follicle bulb. In addition, 438 and 203 genes were identified to be differentially expressed in wool follicle bulb samples from the middle anagen phase compared to the catagen phase and the samples from the catagen phase compared to the late telogen/early anagen phase, respectively. Finally, our data revealed that two groups of genes presenting distinct expression patterns during the phase transformation may have important roles for wool follicle bulb regression and regeneration. In conclusion, our results demonstrated the gene expression patterns in the wool follicle bulb and add new data towards an understanding of the mechanisms involved in wool fiber growth in sheep.
PMCID: PMC4463583  PMID: 25915029
hair follicle; wool; sheep; gene expression profiling; high-throughput RNA sequencing
21.  Identification and Expression Analysis of Candidate Genes Associated with Defense Responses to Phytophthora capsici in Pepper Line “PI 201234” 
Phytophthora capsici (Leonian), classified as an oomycete, seriously threatens the production of pepper (Capsicum annuum). Current understanding of the defense responses in pepper to P. capsici is limited. In this study, RNA-sequencing analysis was utilized to identify differentially expressed genes in the resistant line “PI 201234”, with 1220 differentially expressed genes detected. Of those genes, 480 were up-regulated and 740 were down-regulated, with 211 candidate genes found to be involved in defense responses based on the gene annotations. Furthermore, the expression patterns of 12 candidate genes were further validated via quantitative real-time PCR (qPCR). These genes were found to be significantly up-regulated at different time points post-inoculation (6 hpi, 24 hpi, and 5 dpi) in the resistant line “PI 201234” and susceptible line “Qiemen”. Seven genes were found to be involved in cell wall modification, phytoalexin biosynthesis, symptom development, and phytohormone signaling pathways, thus possibly playing important roles in combating exogenous pathogens. The genes identified herein will provide a basis for further gene cloning and functional verification studies and will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.
PMCID: PMC4463708  PMID: 25993303
RNA-seq; Capsicum annuum; Phytophthora capsici; defense response; gene expression
22.  Rapid Characterization of Fatty Acids in Oleaginous Microalgae by Near-Infrared Spectroscopy 
The key properties of microalgal biodiesel are largely determined by the composition of its fatty acid methyl esters (FAMEs). The gas chromatography (GC) based techniques for fatty acid analysis involve energy-intensive and time-consuming procedures and thus are less suitable for high-throughput screening applications. In the present study, a novel quantification method for microalgal fatty acids was established based on the near-infrared spectroscopy (NIRS) technique. The lyophilized cells of oleaginous Chlorella containing different contents of lipids were scanned by NIRS and their fatty acid profiles were determined by GC-MS. NIRS models were developed based on the chemometric correlation of the near-infrared spectra with fatty acid profiles in algal biomass. The optimized NIRS models showed excellent performances for predicting the contents of total fatty acids, C16:0, C18:0, C18:1 and C18:3, with the coefficient of determination (R2) being 0.998, 0.997, 0.989, 0.991 and 0.997, respectively. Taken together, the NIRS method established here bypasses the procedures of cell disruption, oil extraction and transesterification, is rapid, reliable, and of great potential for high-throughput applications, and will facilitate the screening of microalgal mutants and optimization of their growth conditions for biodiesel production.
PMCID: PMC4425003  PMID: 25826532
biodiesel; Chlorella; fatty acids; microalgae; near-infrared spectroscopy
23.  Characterization of the Recognition Specificity of BH2, a Monoclonal Antibody Prepared against the HLA-B27 Heavy Chain 
BH2, a monoclonal antibody prepared against the denatured human leukocytic antigen-B27 heavy chain (HLA-B27 HC), can immunoprecipitate the misfolded HLA-B27 HC complexed with Bip in the endoplasmic reticulum and recognize the homodimerized HLA-B27 HC that is often observed on the cell membrane of patients suffered from ankylosing spondylitis (AS). However, the recognition specificity of BH2 toward the other molecules of HLA-B type and toward the different types of HLA molecules remained uncharacterized. In this study, we carried out the HLA-typing by using the Luminex Technology to characterize the recognition specificity of BH2 and analyzed the binding domain of HLA-B27 HC by BH2. Our results indicated that BH2 preferably binds to molecules of HLA-B and -C rather than HLA-A and the binding site is located within the α2 domain of HLA-B27 HC.
PMCID: PMC4425072  PMID: 25872138
HLA-B27; ankylosing spondylitis; monoclonal antibody; HLA-typing
24.  Tetraspanin 8-Rictor-Integrin α3 Complex Is Required for Glioma Cell Migration 
The malignant glioma remains one of the most aggressive human malignancies with extremely poor prognosis. Glioma cell invasion and migration are the main causes of death. In the current study, we studied the expression and the potential functions of tetraspanin 8 (Tspan8) in malignant gliomas. We found that Tspan8 expression level is high in both malignant glioma tissues and in several human glioma cell lines, where it formed a complex integrin α3 and rictor, the latter is a key component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Disruption of this complex, through siRNA-mediated knockdown of anyone of these three proteins, inhibited U251MG glioma cell migration in vitro. We further showed that Tspan8-rictor association appeared required for mTORC2 activation. Knockdown of Tspan8 by the targeted siRNAs prevented mTOR-rictor (mTORC2) assembly as well as phosphorylation of AKT (Ser-473) and protein kinase C α (PKCα) in U251MG cells. Together, these results demonstrate that over-expressed Tspan8 in malignant glioma forms a complex with rictor and integrin α3 to mediate mTORC2 activation and glioma cell migration. Therefore, targeting Tspan8-rictor-integrin α3 complex may provide a potential therapeutic intervention for malignant glioma.
PMCID: PMC4394480  PMID: 25761241
malignant glioma; tetraspanin 8; integrin α3; rictor; mTORC2; AKT and cell migration
25.  MiR-25 Protects Cardiomyocytes against Oxidative Damage by Targeting the Mitochondrial Calcium Uniporter 
MicroRNAs (miRNAs) are a class of small non-coding RNAs, whose expression levels vary in different cell types and tissues. Emerging evidence indicates that tissue-specific and -enriched miRNAs are closely associated with cellular development and stress responses in their tissues. MiR-25 has been documented to be abundant in cardiomyocytes, but its function in the heart remains unknown. Here, we report that miR-25 can protect cardiomyocytes against oxidative damage by down-regulating mitochondrial calcium uniporter (MCU). MiR-25 was markedly elevated in response to oxidative stimulation in cardiomyocytes. Further overexpression of miR-25 protected cardiomyocytes against oxidative damage by inactivating the mitochondrial apoptosis pathway. MCU was identified as a potential target of miR-25 by bioinformatical analysis. MCU mRNA level was reversely correlated with miR-25 under the exposure of H2O2, and MCU protein level was largely decreased by miR-25 overexpression. The luciferase reporter assay confirmed that miR-25 bound directly to the 3' untranslated region (UTR) of MCU mRNA. MiR-25 significantly decreased H2O2-induced elevation of mitochondrial Ca2+ concentration, which is likely to be the result of decreased activity of MCU. We conclude that miR-25 targets MCU to protect cardiomyocytes against oxidative damages. This finding provides novel insights into the involvement of miRNAs in oxidative stress in cardiomyocytes.
PMCID: PMC4394484  PMID: 25764156
cardiomyocytes; miR-25; mitochondrial calcium uniporter; oxidative stress

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