Relative Luciferase Activity Research Articles

Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter.

HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at −1205~−838 bp and −146~+93 bp, with the −838~−649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at −709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases.

Sweet Regulation of Human Glucocorticoid Receptor Transcriptional Activity

This study is to understanding the transcription profile of human Glucocorticoid Receptor GR, gene of a neuroendocrine, stress response and obesity related receptor. For GR, relative luciferase activity RLA and the distribution of phosphorylation [P], glycosylation sites [G] on transcription factor for promoter model were plotted on the same chart. Within the 3.2 kb upstream of the methionine ATG, for GR, trend lines of RLA with that of [G] or ([P]-[G]), both tend to have negative reciprocal relationship. It brings up the question: for the neuendocrine glucocorticoid receptor, does the nutrition and obesity related glycosylation regulated the transcriptional activity in a negative reciprocal way? In conclusion, for GR, the reciprocal relation between trend line of [G], ([P] ± [G]) or [P] and that of RLA, give a specific digit evidence for the first time to the theory, which the structure related glycosylation and the signal sensing phosphorylation, exhibit either independently or Interactively on regulation of transcription activity. Abbreviations: RLA: Relative Luciferase Activity; Luciferase/β-Galactosidase Activity; DLA: Dual Luciferase Assay; GR: Human Glucocorticoid Receptor; TF: Transcription Factor; P1: Construct Plasmid 1; P7: Construct Plasmid 7. GR-P1: Construct Plasmid 1 for Human Glucocorticoid Receptor; GR-P7: Constructs Plasmid 7 for Human Glucocorticoid Receptor; [P]: Number of Phosphorylation Sites on TF for Promoter Models; [G]: Number of Glycosylation Sites on TF for Promoter Models; [P]-[G]: Number of Difference between Phosphorylation and Glycosylation Sites on TF for Promoter Models; [P]+[G]: Number for Sum of Phosphoylation and Glycosylation Sites on TF for Promoter Models

Sweet Regulation of Melanocortin Receptor (MC1R, Mc3r, and MC4R) Transcription Activity

Objective: This study is to understanding the transcription profile of nociception-related melanocortin receptor MC1R, obesity related neural melanocortin receptor Mc3r and MC4R. Methods: For MC1R, Mc3r and MC4R, relative luciferase activity RLA and the distribution of phosphorylation [P], glycosylation sites [G] on transcription factor for promoter model were plotted on the same chart. Results: Within the 3.2 kb upstream of the methionine ATG, for all three receptors, trend lines of RLA with that of [G] or ([P]-[G]), both tend to have negative reciprocal relationship. It brings up the question: for the neural receptors MC1R, Mc3r and MC4R, does the nutrition and obesity related glycosylation regulated the transcriptional activity in a negative reciprocal way? Conclusion: The negative reciprocal relation between trend line of [G] or ([P]-[G]) and that of RLA, give a specific digit evidence for the first time to the theory, which the structure related glycosylation and the signal sensing phosphorylation, exhibit either independently or Interactively on regulation of transcription activity.

MiR-29a Participated in Nacre Formation and Immune Response by Targeting Y2R in Pinctada martensii.

miR-29a is a conserved miRNA that participates in bone formation and immune response in vertebrates. miR-29a of Pinctada martensii (Pm-miR-29a) was identified in the previous research though deep sequencing. In this report, the precise sequence of mature Pm-miR-29a was validated using miRNA rapid amplification of cDNA ends (miR-RACE) technology. The precursor sequence of Pm-miR-29a was predicted to have 87 bp. Stem loop qRT-PCR analysis showed that Pm-miR-29a was easily detected in all the tissues, although expressions in the mantle and gill were low. The microstructure showed the disrupted growth of the nacre after Pm-miR-29a over-expression, which was induced by mimic injection into P. martensii. Results of the target analysis indicated that neuropeptide Y receptor type 2 (Y2R) was the potential target of Pm-miR-29a. Meanwhile, Pm-miR-29a mimics could obviously inhibit the relative luciferase activity of the reporter containing 3′ UTR (Untranslated Regions) of the Y2R gene. Furthermore, the expression of Y2R was downregulated whereas expressions of interleukin 17 (IL-17) and nuclear factor κB (NF-κB) were upregulated after Pm-miR-29a over-expression in the mantle and gill, thereby suggesting that Pm-miR-29a could activate the immune response of the pearl oyster. Results showed that Pm-miR-29a was involved in nacre formation and immune response by regulating Y2R in pearl oyster P. martensii.

IRGM gene polymorphisms and haplotypes associate with susceptibility of pulmonary tuberculosis in Chinese Hubei Han population

Human immunity-related GTPase M (IRGM) is found to play an important role in defense against intracellular pathogen Mycobacterium tuberculosis (M. tuberculosis) in vitro by regulating autophagy. The objective of the study was to determine the association between IRGM polymorphisms and susceptibility to pulmonary tuberculosis (PTB) in Chinese Hubei Han population. In this study, 237 PTB patients and 269 healthy controls were screened for IRGM promoter single nucleotide polymorphisms (SNPs) by gene sequencing, and an association study was performed. A luciferase assay was used to determine the transcriptional activity of the promoter polymorphism. The relative expression level of IRGM gene was measured by Real time Quantitative PCR (qRT-PCR). We identified 3 polymorphisms [-1208 (rs4958842), -1161 (rs4958843), and -947 (rs4958846)] in the IRGM promoter region. Our finding showed that the IRGM -947 CT genotype as well as CC genotype decreased the risk of PTB in comparison with TT genotype (OR = 0.216, 95% CI = 0.141-0.331,P < 0.001 and OR = 0.167, 95% CI = 0.088-0.318, P < 0.001,respectively). The -947C allele decreased the risk of PTB in comparison with T allele (OR = 0.266, 95% CI = 0.196-0.362, P < 0.001). There was linkage disequilibrium between these three IRGM SNPs and we further analyzed the haplotypes of these SNPs. Six haplotypes were identified and we found that the haplotype ACC played a protective role in the susceptibility to PTB. In contrast, the ACT haplotype was associated with an increased susceptibility to PTB. In addition, the ACT haplotype reduced the relative luciferase activity of IRGM promoter and decreased the expression of IRGM in PTB patients. Our findings indicated that IRGM functional polymorphisms and haplotypes in promoter were associated with the susceptibility to PTB in Chinese Hubei Han population.

Epigenetic Disruption of ZEB1 Binding Causes Constitutive Activation of IL-15 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin's lymphoma of skin-homing T-cells that represents 70-80% of all cutaneous lymphomas. Studies have shown that interleukin-15 (IL-15) plays a key role in T-cell oncogenesis by increasing T-cell survival, proliferation, migration and invasion in patient-derived CTCL cell lines. Previously, we have demonstrated that IL-15 is overexpressed in lesional biopsies and peripheral blood of CTCL patients (Mishra et al., Blood, 122:1826, 2013). However, the underlying mechanisms of IL-15 deregulation are largely unknown. Since epigenetic modifications such as DNA methylation can alter gene expression, we selected samples highly enriched in neoplastic T-cells from the blood of CTCL patients and performed a pyro-sequencing analysis to determine methylation of the IL-15 gene promoter. We observed significantly higher methylation in the IL-15 promoter of CD4+ T-cells from CTCL patients vs. normal donors (mean ± SEM of relative % methylation= 232.0 ± 49.18, n=9 vs 100.1 ± 7.619, n=6, P =0.03). Furthermore, methylation at the IL-15 promoter was higher in malignant CD4+ T-cells compared to non-malignant neutrophils from the same patient (mean ± SEM of relative % methylation = 232.0 ± 49.18 vs 52.31 ± 6.28%, N=9 each, paired t-test, P =0.0025). Using this approach, we also analyzed methylation of each 'CpG dinucleotide' in the IL-15 promoter. Once again, methylation was higher in CD4+ T-cells of patient versus normal donors and, within each patient, in CD4+ T-cells versus neutrophils. The CpG rich region of the human IL-15 promoter contains three putative binding sites for the known transcriptional repressor Zeb1, which has been reported to be mutated or deleted in CTCL. In order to examine the effect of CpG methylation at the Zeb1 binding region on IL-15 transcription, the CpG rich 5' regulatory region of the IL-15 promoter was amplified and cloned into PGL3 luciferase vector. As measured by relative luciferase activity, the region of the IL-15 promoter containing the 3 ZEB1-binding sites is transcriptionally active in its native form (mean ± SEM of relative luciferase activity of promoter-less vector vs. IL-15 promoter vector = 77.00 ± 3.29 vs. 626.8 ± 25.05 respectively, N=3 each, P<0.0001). Deletion of the putative Zeb1 binding sites (BS#1, BS#2, BS#3 and BS#1-3) or the entire Zeb-binding region (BR) in the IL-15 promoter led to a significant increase in IL-15 transcription as determined by relative luciferase activity (mean ± SEM of relative luciferase activity of pGL3 IL-15 vs. BS#1 vs. BS#2 vs. BS#3 vs. BS#1-3 vs. BR vectors = 99.56 ± 0.33 vs. 7135 ± 108.5 vs. 3940 ± 62.36 vs. 2372 ± 24.65 vs. 1099 ± 9.72 vs. 1525 ± 32.00, n=4 each; P <0.0001 each). Since CpG methylation of DNA can physically prevent transcription factor binding to regulatory regions of DNA, we hypothesized that CD4+ cells from CTCL patients might display reduced binding of Zeb1 to the IL-15 promoter thereby increasing IL-15 transcription. Indeed, using chromatin immunoprecipitation (ChIP)-PCR, we observed a substantial loss of Zeb1 binding at the IL-15 promoter in the CD4+ cells of CTCL patients compared to those of normal donors (mean ± SEM of relative Zeb1 binding to IL-15 promoter in normal donor vs. CTCL patient CD4+ T-cells = 101.3 ± 1.48 vs. 25.31 ± 3.78, n=3 each; P <0.0001). Similarly, silencing Zeb1 in normal donor CD4+ T-cells caused an increase in IL-15 transcription. Finally, constitutive IL-15 overexpression was sufficient to cause a high penetrance lymphoproliferative disease in transgenic mice, with typical dermatologic features of human CTCL, including alopecia, scaly erythematous plaques/patches, and ulcerations, caused by an epidermotropic T-cell infiltrate. Thus, we defined Zeb1 as a novel repressor for IL-15 transcription in normal T-cells and show that epigenetic interference with its repressive function at the IL-15 promoter is associated with T-cell lymphoma in vivo, highlighting the role of epigenetic processes in inflammation-induced cancers and further supporting the role of Zeb1 as a putative tumor suppressor gene in CTCL DisclosuresPorcu:Cell Medica: Research Funding; Celgene: Research Funding; Shape: Research Funding; Infinity: Research Funding; Seattle Genetics: Research Funding.

Histone Deacetylase Inhibitors Induce microRNAs Targeting BTK in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous disease primarily affecting the elderly. Despite considerable progress in understanding the biology of AML, the prognosis of the majority of patients still remains poor due to lack of progress in improving therapy over the past 3 decades, thus creating the need to identify new pharmacological strategies.Bruton's Tyrosine Kinase (BTK) has been demonstrated to be functionally important in a variety of hematological malignancies and several groups have recently shown that BTK is highly expressed and constitutively active in AML cell lines and in a subset of AML patients. Moreover, treatment with ibrutinib, a covalent irreversible inhibitor of BTK, can reduce proliferation, blast adhesion to bone marrow stromal cells and migration of both AML cell lines and primary AML blasts. Validation of this target also occurred with shRNA experiments confirming the specificity of BTK for growth inhibition of AML.Dysregulation of microRNA (miRNA) expression contributes to the pathogenesis of numerous human malignancies including AML, where multiple miRNAs have been associated with specific cytogenetic and molecular subsets of the disease.Using a target prediction algorithm we identified several miRNAs as potential regulators of BTK (miR-147b, miR-210-3p, miR-425-5p, miR-1253, miR-4269, miR-4667-3p). We performed 3′UTR luciferase reporter assays in the AML cell line OCI-AML3 and found that relative luciferase activities were significantly decreased (20-30%) in cells transfected with miR-147b, miR-425-5p, miR-4269 and miR-4667-3p mimics. Flow cytometry and western blotting analysis confirmed that BTK protein expression was significantly down-regulated by all the predicted miRs except miR-1253.Previous data from our lab have shown that histone deacetylase (HDAC) 1 and 2 become recruited to multiple microRNA promoters, including the ones targeting BTK. To determine the consequence of HDAC inhibition on BTK signaling, we exposed primary blasts from 14 AML patients to 10nM of the pan-deacetylase inhibitor panobinostat (LBH589) for 48h. Our results showed that panobinostat induced a 2-23 fold increase in the expression miR-147b, miR-210-3p, miR-425-5p, and miR-4667-3p in most of the patients and a time-dependent depletion of total BTK protein and downstream BTK signaling (phospho- and total ERK and phospho- and total AKT) in about half of them. BTK protein at 24h directly correlated with BTK and HDAC1 mRNA expression at 24h and miR-210 induction at 24h inversely correlated with BTK mRNA levels at 24h. Moreover, 2 days of incubation with 10nM panobinostat is able to induce robust cell death in AML primary samples (9.6-92% apoptosis).We hypothesized that HDAC inhibitors would cooperate with kinase inhibitors of BTK to synergistically abolish BTK signaling and induce death in AML cells. Therefore, we treated OCI-AML3 and MV4-11 cell lines with 0.40 µM abexinostat, 1 µM ibrutinib for 2h alone followed by washout, or a combination of 0.40 µM abexinostat with 1 µM ibrutinib for 2h, and viability was measured at 48h. The combination of abexinostat and ibrutinib induced greater than additive cytotoxicity compared to either abexinostat or ibrutinib alone. Exposure to ibrutinib alone for 2h followed by washout inhibited the kinase activity of BTK (measured by the auto-phosphorylation on Y223) but did not affect total BTK protein. However, the combination of abexinostat and ibrutinib showed both loss of BTK kinase and decrease in total BTK protein, in addition to attenuation of BTK signaling, thus confirming the dual targeting of BTK via independent mechanisms.Our results provide the rationale for the clinical evaluation of ibrutininb in combination with histone deacetylase inhibitors in AML patients. DisclosuresNo relevant conflicts of interest to declare.

The Chinese medicine Sini-San inhibits HBx-induced migration and invasiveness of human hepatocellular carcinoma cells.

BackgroundSini-San (SNS) is a formulation of four Traditional Chinese Drugs that exhibits beneficial therapeutic effects in liver injury and hepatitis. However, there are no reports describing its effects on the hepatitis B X-protein (HBx)-induced invasion and metastasis in hepatoma cells, and the detailed molecular mechanisms of its actions are still unclear.MethodsIn this study, we investigated the mechanisms underlying SNS-mediated inhibition of HBx-induced cell invasion and the inhibition of secreted and cytosolic MMP-9 production, using gelatin zymography and Western blot analysis in a human hepatoma cell line (HepG2). Relative luciferase activity was assessed for MMP-9, NF-κB, or AP-1 reporter plasmid-transfected cells.ResultsSNS suppressed MMP-9 transcription by inhibiting activator protein (AP)-1 and nuclear factor-κ B (NF-κB) activity. SNS suppressed HBx-induced AP-1 activity through inhibition of phosphorylation in the extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. SNS also suppressed HBx-induced inhibition of NF-κB nuclear translocation through IκB and suppressed HBx-induced activation of ERK/phosphatidylinositol 3-kinase/Akt upstream of NF-κB and AP-1.ConclusionsSNS suppresses the invasiveness and metastatic potential of hepatocellular carcinoma cells by inhibiting multiple signal transduction pathways.

Blimp-1/PRDM1 regulates the transcription of human CS1 (SLAMF7) gene in NK and B cells

CS1 (CRACC/CD319/SLAMF7) is a member of SLAM (Signaling Lymphocyte Activation Molecule) family receptors and is expressed on NK cells, a subset of CD8+ T lymphocytes, activated monocytes, mature dendritic cells and activated B cells. In NK cells, CS1 signaling induces cytolytic function of NK cells against targets whereas in B cells CS1 induces proliferation and autocrine cytokine production. CS1 is upregulated in multiple myeloma cells and contributes to clonogenic growth and tumorigenicity. However, the mechanism of CS1 upregulation is unknown. In this study, we analyzed the transcriptional regulation of human CS1 gene in NK and B cells. The promoter region of CS1 contains a Blimp-1/PRDM1 binding site and relative luciferase activities of successive deletion mutants of CS1 promoter were different between Blimp-1/PRDM1-positive and Blimp-1/PRDM1-negative cells. Proximal region of CS1 promoter contains a CAAT box and atypical TATA-box that might result in common transcription initiation at −29 nucleotides upstream of the ATG translation start codon. Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assays revealed Blimp-1/PRDM1 binds to the CS1 promoter region. Mutating the Blimp-1/PRDM1 site at −750 to −746 decreased the transcriptional activity of CS1 promoter implicating a trans-activating function of Blimp-1/PRDM1 in human CS1 gene regulation. The finding that Blimp-1/PRDM1 enhances transcription of CS1 gene in multiple myeloma cells may help in developing novel strategies for therapeutic intervention in multiple myeloma.

Retinoblastoma-binding protein 2 induces epithelial-mesenchymal transition in esophageal squamous cancer cells.

To investigate the effect of retinoblastoma-binding protein 2 (RBP2) on epithelial-mesenchymal transition (EMT) in esophageal squamous cancer cells and to compare the effect of RBP2 in lung squamous cancer cells and esophageal squamous cancer cells. When transfected with RBP2 siRNA, the migrated cells were 36.3±6.03 by transwell migration assay, compared to 107±6.7 cells in the control group. The mRNA level of epithelial cadherin (E-cadherin) was 1.54±0.14 times higher than in the control group, and that of neural cadherin (N-cadherin) fell to 0.76±0.03 times. The relative luciferase activity of E-cadherin promoter rose to 3.84±0.23 times. Correspondingly, the expression of E-cadherin protein increased and that of N-cadherin protein decreased. When SK-MES-1 cells were transfected with RBP2 siRNA, their relative mRNA level of E-cadherin was 8.6±0.37 times as high as that in control group, which was higher than that in Eca-109 cells. The E-cadherin protein was also greater in SK-MES-1 cells. RBP2 could induce EMT in esophageal cancer cells and exert a greater effect on the expression of E-cadherin in lung squamous cells than in esophageal squamous cells.

An IL-13 promoter polymorphism associated with liver fibrosis in patients with Schistosoma japonicum.

The aim of this study was to determine whether two polymorphisms in the gene encoding IL13 previously associated with Schistosoma hematobium (S. hematobium) and S. mansoni infection are associated with S. japonicum infection. Single nucleotide polymorphisms (SNPs) rs1800925 (IL13/-1112C>T) and rs20541 (IL13R130Q) were genotyped in 947 unrelated individuals (307 chronically infected, 339 late-stage with liver fibrosis, 301 uninfected controls) from a schistosomiasis-endemic area of Hubei province in China. Regression models were used to evaluate allelic and haplotypic associations with chronic and late-stage schistosomiasis adjusted for non-genetic covariates. Expression of IL-13 was measured in S. japonicun-infected liver fibrosis tissue and normal liver tissue from uninfected controls by immunohistochemistry (IHC). The role of rs1800925 in IL-13 transcription was further determined by Luciferase report assay using the recombinant PGL4.17-rs180092 plasmid. We found SNP rs1800925T was associated with late-stage schistosomiasis caused by S. japonicum but not chronic schistosomiasis (OR = 1.39, 95%CI = 1.02–1.91, p = 0.03) and uninfected controls (OR = 1.49, 95%CI = 1.03–2.13, p = 0.03). Moreover, the haplotype rs1800925T-rs20541C increased the risk of disease progression to late-stage schistosomiasis (OR = 1.46, p = 0.035), whereas haplotype rs1800925C-rs20541A showed a protective role against development of late-stage schistosomiasis (F = 0.188, OR = 0.61, p = 0.002). Furthermore, S. japonicum-induced fibrotic liver tissue had higher IL13 expression than normal liver tissue. Plasmid PGL4.17-rs1800925T showed a stronger relative luciferase activity than Plasmid PGL4.17-rs1800925C in 293FT, QSG-7701 and HL-7702 cell lines. In conclusion, the functional IL13 polymorphism, rs1800925T, previously associated with risk of schistosomiasis, also contributes to risk of late-stage schistosomiasis caused by S. japonicum.

MicroRNA-141-3p and miR-200a-3p regulate insulin-like growth factor 2 during mouse placental development

Insulin-like growth factor 2 (IGF2) plays a vital role in fetal and placental development throughout gestation. Placental expression of IGF2 decreases substantially in intra-uterine growth restriction (IUGR) and Igf2 null mice develop small placentas. In this report, we examined the role of microRNAs in regulating Igf2 gene expression during mouse placental development. Using bioinformatic analysis, we have identified microRNAs that have conserved binding sites in the 3′-UTR of Igf2. Using luciferase reporter assay, we demonstrated that miR141-3p and miR-200a-3p mimics substantially down regulated relative luciferase activity by binding to 3′-UTR of Igf2, which was reversed by using miR141-3p and miR-200a-3p inhibitors. Furthermore, in a similar assay, use of Igf2 3′-UTR that lacked the binding site for the microRNAs did not have any effect on luceiferase activity. Interestingly, the expression of miR141-3p and miR-200a-3p were inversely and temporally correlated to the expression of IGF2 during mouse placental development. Overexpression of miR141-3p and miR-200a-3p in mouse trophoblast stem cells suppressed endogenous expression of IGF2. Consequently, IGF2 silencing by miR141-3p and miR-200a-3p diminished Akt activation in mouse trophoblast stem cells. Our study provides evidence for regulation of Igf2 by microRNAs and further elucidates the role of miR141-3p and miR-200a-3p in the mouse placental development.

Myeloid zinc finger 1 (MZF1) is the most important transcriptional factor for porcine follistatin promoter

Follistatin (FS) is a secreted protein, which was originally isolated from porcine follicular fluid. Expression of follistatin is tightly regulated during porcine growth and development. To study the essential transcriptional regions of the porcine FS promoter, ten primer pairs were designed to amplify segments with different lengths of the FS promoter from −1 800 to +16 bp. The products were then inserted into the pGL3-basic vector to analyze the relative luciferase activity. The results showed that the most remarkable changes of promoter activity were observed between constructs (−302/+16 bp)-FS and (−180/+16 bp)-FS (P<0.01). Further research showed that the reconstructed reporter plasmid lacking myeloid zinc finger 1 (MZF1) binding sequence had significantly decreased luciferase activity (P<0.05). Furthermore, the FS protein expression was significantly increased in PK15 cells while the MZF1 was overexpressed, suggesting that the short sequence “TCCCCACC” (the recognition site of transcription factor MZF1) was the most important for FS transcription activation in the porcine.

MicroRNA-218 suppresses the proliferation, invasion and promotes apoptosis of pancreatic cancer cells by targeting HMGB1.

To detect the expression profiles of microRNA-218 (miR-218) in human pancreatic cancer tissue (PCT) and cells and their effects on the biological features of human pancreatic cancer cell line PANC-1 and observe the effect of miR-218 on the expression of the target gene high mobility group box 1 (HMGB1), with an attempt to provide new treatment methods and strategies for pancreatic cancer. The expressions of miR-218 in PCT and normal pancreas tissue as well as in various pancreatic cancer cell lines including AsPC-1, BxPC-3, and PANC-1 were determined with quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The change of miR-218 expression in PANC-1 cells was detected using qRT-PCT after the transfection of miR-218 mimic for 48 h. Cell Counting Kit-8 (CCK-8) was applied for detecting the effect of miR-218 on the activity of PANC-1 cells. The effects of miR-218 on the proliferation and apoptosis of PANC-1 cells were analyzed using the flow cytometry. The effect of miR-218 on the migration of PANC-1 cells was detected using the Trans-well migration assay. The HMGB1 was found to be a target gene of miR-218 by luciferase reporter assay, and the effect of miR-218 on the expression of HMGB1 protein in cells were determined using Western blotting. As shown by qRT-PCR, the expressions of miR-218 in PCT and in pancreatic cancer cell line significantly decreased when compared with the normal pancreatic tissue (NPT) (P<0.01). Compared with the control group, the miR-218 expression significantly increased in the PANC-1 group after the transfection of miR-218 mimic for 48 h (P<0.01). Growth curve showed that the cell viability significantly dropped after the overexpression of miR-218 in the PANC-1 cells for two days (P<0.05). Flow cytometry showed that the S-phase fraction significantly dropped after the overexpression of miR-218 (P<0.01) and the percentage of apoptotic cells significantly increased (P<0.01). As shown by the Trans-well migration assay, the enhanced miR-218 expression was associated with a significantly lower number of cells that passed through a Transwell chamber (P<0.01). Luciferase reporter assay showed that, compared with the control group, the relative luciferase activity significantly decreased in the miR-218 mimic group (P<0.01). As shown by the Western blotting, compared with the control group, the HMGB1 protein expression significantly decreased in the PANC-1 group after the transfection of miR-218 mimic for 48 h (P<0.01). The miR-218 expression decreases in human PCT and cell lines. miR-218 can negatively regulate the HMGB1 protein expression and inhibit the proliferation and invasion of pancreatic cancer cells. A treatment strategy by enhancing the miR-218 expression may benefit the patients with pancreatic cancer.

Targeted inhibition of microRNA-200c on expression of AP-2α to enhance the proliferation of colon cancer cells in vitro

Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism. Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database, while its eukaryotic expression plasmids and specific inhibitor were synthesized. Plasmids PEZX-miR-200c, PEZX-NC, pmir-GLO-AP-2α3'UTR, pmir-GLO and the specific inhibitors miR-67-inhibtor, miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000. The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Western blot and immunocytochemical staining. CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis. Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c. Results The proliferation activity was significantly decreased in anti-miR-200c /SW480 group, while in PEZX-miR-200c/HCT-116 group, it was higher than that in PEZX-NC/HCT-116 group. The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7)% vs (66±1.1)%, P<0.05]. The expression of AP-2α both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group, while the protein level was increased in Anti-miR-200c/SW480 group. The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3'UTR (0.51±0.09 vs 0.98±0.04, P<0.01). Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells. Key words: Transcription factor AP-2α; microRNA-200c; Colonic neoplasms

Activation of HSPA1A promoter by environmental pollutants: An early and rapid response to cellular damage

We established the HepG2-luciferase cells containing a luciferase reporter gene regulated by human HSPA1A promoter. The screening of heat shock and three typical environmental toxicants revealed differences in their capacities to activate HSPA1A promoter in HepG2-luciferase cells. After heat shock, a progressive time-dependent increase in relative luciferase activity was detected peaking at 8h of recovery. Benzo[a]pyrene, formaldehyde and sodium bisulfite induced significant time-dependent elevation of relative luciferase activity, which were positively correlated with MDA concentration, Olive tail moment and micronuclei frequency. The significant increase in relative luciferase activity was already evident after 4h of benzo[a]pyrene, 1h of formaldehyde and sodium bisulfite exposure, when no increases in cellular damage were detected by other toxicity tests. Therefore, the HepG2-luciferase cells are useful model for examining the overall cellular responses to oxidative stress and genotoxic damage, and provide a reporter system for rapid and sensitive screening of environmental pollutants.

MicroRNA-18b modulates insulin-like growth factor-1 expression in deer antler cell proliferation by directly targeting its 3' untranslated region.

Insulin-like growth factor-1 (IGF-1) is a multipromoter gene that has complex biological functions and plays an important role in Chinese sika deer antler cell differentiation and proliferation. microRNAs and their roles in deer antler growth have attracted much attention. In the present study, to investigate the effect of microRNAs on the regulation of IGF-1 during the rapid growth of antlers, miRNA GeneChip analysis and TargetScan Human software were used to screen microRNAs that bind to the 3' untranslated region (3'UTR) of IGF-1. The results indicated that a significantly differential expression of miR-18b was observed in cartilage and mesenchymal of antler tip tissue and the presence of miR-18b-binding sites within the IGF-1 3'UTR. A miR-18b mimic was then transfected into antler cartilage cells to overexpress miR-18b and the expression levels were quantified by real-time PCR. Real-time PCR showed that the expression level of miR-18b in transfected cells was significantly increased compared with the control group (p<0.01). Dual luciferase assays revealed that miR-18b decreased the fluorescence value of the luciferase reporter gene in the group transfected with the wild-type vector of IGF-1 3'UTR. In contrast, the relative luciferase activity in the group transfected with the mutant vector of IGF-1 3'UTR did not change obviously. MTT assays and cell cycle analyses confirmed that overexpression of the miR-18b mimic inhibited the proliferation of cartilage cells. In contrast, transfection of a miR-18b inhibitor increased the cell proliferation rate. Furthermore, Western blot analyses revealed that overexpression of miR-18b mimics downregulated the protein levels of IGF-1, while IGF-1 expression increased after transfection of miR-18b inhibitors. Taken together, our findings show that miR-18b is a potentially novel target in deer antler cell proliferation. miR-18b may modulate IGF-1 expression of sika deer antler.

Study of the mechanism underlying the effect of SOCS3 rs4969170 A/G polymorphism on the occurrence of insulin resistance in chronic hepatitis C patients

To study the mechanism underlying the effect of the SOCS3 rs4969170 A/G alleles on the occurrence of insulin resistance (IR) in patients with chronic hepatitis C. The promoter region of the SOCS3 gene was amplified by PCR,and luciferase expression vectors were constructed and transfected into HepG2,Huh7 cell lines.The relative luciferase activity of each expression vector was assessed by the dual luciferase reporter gene assay system.Western blotting was used to detect SOCS3 protein expression in PBMCs from groups of patients with the rs4969170 AA and AG genotypes.The state of IR in eight patients was evaluated by determining their HOMA-IR. The pGL3-A, PGL3-G and pGL3-control vectors showed significantly different luciferase expression in the HepG2 cells (0.121 00 ± 0.022 07,0.027 00+/-0.012 49 and 0.043 33 ± 0.005 51; F =48.068, P=0.001) and in the Huh7 cell lines (0.164 70 ± 0.007 10,0.027 33 ± 0.017 04 and 0.033 67 ± 0.014 98; F =115.137, P=0.001). The expression of SOCS3 protein was significantly higher in the rs4969170 AA genotype group than in the AG genotype group (1.22 ± 0.40 vs. 0.30 ± 0.19; t =4.149, P=0.006).The IR index of patients with the rs4969170 AA genotype and the AG genotype was 4.11 ± 2.62 and 1.47 ± 1.01 respectively.There were three patients with IR in the rs4969170 AA genotype group and one in the rs4969170 AG group. There was no statistically significant difference between the two genotype groups (t=1.881, P=0.109). The SOCS3 rs4969170 A haplotype may enhance transcriptional activity of the gene promoter to regulate gene expression, thereby increasing intracellular SOCS3 protein level and ultimately interfering with insulin signaling and causing IR in patients with chronic hepatitis C.

Foxp3 gene polymorphisms and haplotypes associate with susceptibility of Graves' disease in Chinese Han population.

Foxp3 plays important roles in the pathogenesis of autoimmune diseases. To investigate the association between Foxp3 gene polymorphisms and the susceptibility to Graves' disease (GD) in Chinese Han population, four single nucleotide polymorphisms (SNPs) including -2383, -3279, -3499 in the promoter and IVS9+459 in the intron were genotyped. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 308 GD patients and 306 healthy controls. The relative expression level of Foxp3 gene was measured by qRT-PCR. The frequencies of AA/CA genotype of -3279 and CC genotype of IVS9+459 were significantly higher in GD patients than healthy controls. The AA/CA genotype of -3279 in female GD patients was more frequent than the male. For -3279, GD patients with higher TSH level or/and lower TRAb level were more frequent to carry A allele. We further analyzed the haplotypes of three SNPs and found that the haplotype CCA played a protective role in the susceptibility to GD. In contrast, the CAA and TCA haplotypes were associated with an increased susceptibility to GD. In addition, the mutation from C to A at the position of -3279 reduced the relative luciferase activity of Foxp3 promoter and decreased the expression of Foxp3 in GD patients. Our findings indicated that Foxp3 functional polymorphisms and haplotypes in promoter were associated with the susceptibility to GD in Chinese Han population.

MicroRNA-130b regulates scleroderma fibrosis by targeting peroxisome proliferator-activated receptor γ

Objectives. To investigate the role of microRNA-130b (miR-130b) in systemic sclerosis (SSc) skin fibrosis and its regulatory effect on peroxisome proliferator-activated receptor γ (PPARγ).Methods. miR-130b was identified from microarray analyses in our previous studies. The expression of miR-130b, PPARγ, and fibrosis-related genes were determined by real-time PCR analysis. PPARγ protein levels were detected by immunohistochemistry and Western blot. Cells were transfected with microRNA mimics/inhibitor/scramble of miR-130b using Lipofectamine. Luciferase reporter gene assays were used to identify the direct target of miR-130b. Transforming growth factor β (TGF-β) was used for stimulation.Results. The expression of miR-130b was significantly upregulated and level of PPARγ was decreased in the dermis of the SSc skin biopsy samples and fibroblasts. Similar to human SSc, the same expression patterns of miR-130b, PPARγ, and fibrosis-related genes were observed in the bleomycin-induced skin fibrosis model; TGF-β induced the expression of miR-130b and fibrosis-related genes expression, but downregulated the expression of PPARγ. Overexpression of miR-130b in normal or SSc skin fibroblasts significantly decreased, and accordingly, knockdown of miR-130b increased the levels of PPARγ and fibrosis-related genes. In the reporter gene assay, cotransfection with miR-130b mimics significantly decreased the relative luciferase activity, which suggested a direct regulation of PPARγ by miR-130b.Conclusions. These studies demonstrated that miR-130b played important profibrotic roles in SSc fibrosis, and enhanced TGF-β signaling through negative regulation of PPARγ expression. MiR-130b may be a potential therapeutic target in SSc fibrosis.