Luciferase Reporter Gene Experiment Research Articles

LncRNA UNC5B-AS1 promotes malignant progression of prostate cancer by competitive binding to caspase-9.

This study aims to investigate the expression of LncRNA UNC5B-AS1 in prostate cancer (PCa) and to further investigate whether it can prompt malignant progression of PCa via regulating caspase-9. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was conducted to examine UNC5B-AS1 expression in 50 pairs of tumor tissue specimens and paracancerous ones collected from PCa patients, and the interplay between UNC5B-AS1 expression and clinical indicators of PCa was also analyzed. Meanwhile, UNC5B-AS1 levels in PCa cell lines were also further verified by qRT-PCR. In addition, UNC5B-AS1 knockdown model was constructed using lentivirus in PCa cell lines, and cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), transwell and flow cytometry assays were performed to figure out the impact of UNC5B-AS1 on the biological function of PCa cells. Finally, cell recovery experiment was conducted to explore the underlying mechanism and the association between UNC5B-AS1 and caspase-9. QRT-PCR results suggested that UNC5B-AS1 expression in PCa tissue samples was remarkably higher than in adjacent ones, with a statistically significant difference. Compared with patients with low expression of UNC5B-AS1, patients with highly-expressed UNC5B-AS1 had a higher incidence of distant metastasis and more advanced pathological stage. At the same time, proliferation and invasion, as well as migration ability of cells in sh-UNC5B-AS1 group, was conspicuously attenuated while cell apoptosis ability was conversely enhanced. Furthermore, qRT-PCR results revealed that caspase-9 and UNC5B-AS1 showed a negative correlation in gene expression level in PCa tissues. The results of the luciferase reporter gene experiment demonstrated that UNC5B-AS1 can be targeted by caspase-9 through their binding site. Additionally, cell recovery experiment indicated that UNC5B-AS1 and caspase-9 can be mutually regulated, which then together affect the malignant progression of PCa. UNC5B-AS1 expression was found remarkably increased in both PCa tissues and cell lines, which was remarkably associated with pathological stage and incidence of distant metastasis of PCa patients. In addition, UNC5B-AS1 was able to accelerate the malignant progression of PCa by modulating caspase-9 expression.

MiR-1323 Promotes Cell Migration in Lung Adenocarcinoma by Targeting Cbl-b and Is an Early Prognostic Biomarker.

Purpose: MicroRNAs are known to regulate cellular processes in non-small cell lung cancer (NSCLC) cells and predict prognosis. However, identification of specific microRNAs in NSCLC as potential therapeutic targets is controversial. We aim to determine the clinical significance of miR-1323 in the prognosis of patients with lung cancer and the potential mechanism.Patients and methods: A bioinformatics approach was used to screen the importance microRNA in NSCLC through the online GEO database (GSE42425). The relationship between expression level of miR-1323 and overall survival of lung cancer patients was analyzed. Additionally, an independent corhort including 53 NSCLC cases that underwent resection validated the connection between miR-1323 and LUAD patients' overall survival. Next, the function of miR-1323 was studied in vitro by transient transfection. A more in-depth mechanism was studied through luciferase reporter gene experiments.Results: High miR-1323 expression correlated with poor survival in NSCLC patients (P = 0.011), and in lung adenocarcinoma (LUAD) patients (P = 0.015) based on GEO database (GSE42425). In the independent cohort based on our hospital, high miR-1323 expression was associated with LUAD patients (P = 0.025). Moreover, transfection with mimics of miR-1323 showed an increased migratory capacity in LUAD A549 and HCC827 cells. In addition, E3 ubiquitin-protein ligase Casitas B-lineage Lymphoma-b (Cbl-b) was found to be the target genes of miR-1323 and significantly down regulated after mimics of miR-1323 transfection, and high Cbl-b expression predicted better prognosis in NSCLC and LUAD (P = 0.00072 and P = 0.02, respectively).Conclusion: The miR-1323 promoted LUAD migration through inhibiting Cbl-b expression. High miR-1323 expression predicted poor prognosis in LUAD patients.

Effect of CASC19 on proliferation, apoptosis and radiation sensitivity of cervical cancer cells by regulating miR-449b-5p expression

Objective: To investigate the effects of cancer susceptibility candidate gene 19 (CASC19) regulating the expression of microRNA-449b-5p (miR-449b-5p) on the proliferation, apoptosis and radiation sensitivity of cervical cancer cells. Methods: (1) HeLa cells of cervical cancer cell line were cultured. HeLa cells were irradiated with X-ray at different doses (0, 2, 4, 6, 8 Gy, respectively), then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR. (2) HeLa cell proliferation, apoptosis, radiation sensitivity (expressed as a survival fraction) and its related protein expression included cyclin D1, cleaved-caspase-3, and histone variant H2AX (γ-H2AX) were examined after different treatment including silencing CASC19 expression, over-expressing miR-449b-5p, down-regulating miR-449b-5p and silencing CASC19 expression. (3) The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p. Results: (1) With the increase of X-ray irradiation different dose (0, 2, 4, 6, 8 Gy), the expression level of CASC19 mRNA in HeLa cells gradually increased (F=502.681, P=0.000), and the expression level of miR-449b-5p gradually decreased (F=202.936, P=0.000).(2) After silencing CASC19 expression or over-expressing miR-449b-5p, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression level of cyclin D1 protein was significantly reduced (P<0.05), and the expression levels of cleaved-caspase-3 and γ-H2AX protein were significantly increased (P<0.05). After down-regulating miR-449b-5p and silencing CASC19 expression, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression levels of cyclin D1 and γ-H2AX protein were significantly increased (P<0.05), and the expression level of cleaved-caspase-3 was significantly decreased (P<0.05). (3) Over expression of miR-449b-5p could significantly reduce the luciferase activity of CASC19 wild type (1.00±0.09 versus 0.37±0.05, P<0.01), but there were no significant effect on the luciferase activity of CASC19 mutant type (0.92±0.07 versus 0.94±0.05, P>0.05). After the expression of CASC19 was silenced, the expression level of miR-449b-5p in HeLa cells increased significantly (1.00±0.12 versus 4.84±0.49, P<0.05). After overexpression of CASC19, the expression level of miR-449b-5p in HeLa cells was significantly reduced (1.00±0.09 versus 0.38±0.04, P<0.05). Conclusion: CASC19 in HeLa cells negatively regulates the expression of miR-449b-5p, and down-regulating the expression of miR-449b-5p could partially reverse the effects of silencing CASC19 on HeLa cell proliferation, apoptosis and radiation sensitivity.

Long-chain non-coding RNA GACAT1 promotes development and progression of breast cancer by targeting microRNA-875-3p.

Role of long-chain non-coding ribonucleic acid (lncRNA) GACAT1 in the development of breast cancer and its possible mechanism were investigated. The levels of GACAT1, microRNA-875-3p and Stonin2 (STON2) in breast cancer tissues and adjacent normal tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The level of GACAT1 in breast cancer cell lines was further explored. The effects of GACAT1 and microRNA-875-3p on cell proliferation and cycle were detected by cell counting kit-8 (CCK-8) and flow cytometry. The binding relationship between microRNA-875-3p and STON2, microRNA-875-3p and GACAT1 was examined by a dual luciferase reporting assay. It was confirmed by rescue experiments whether GACAT1 canregulate the level of STON2 by binding to microRNA-875-3p. GACAT1 level was clearly enhanced in breast cancer tissues compared to that of the adjacent tissues. Similar result was observed in breast cancer cell lines. Upregulation of GACAT1 promoted the proliferation and cycle of breast cancer cells including MCF-7 and BCap-37. The dual luciferase reporting assay results indicated that GACAT1 had a binding relationship with microRNA-875-3p. Further experiments confirmed that microRNA-875-3p was conspicuously downregulated in breast cancer tissues, and upregulation of microRNA-875-3p could inhibit the proliferation ability of MCF-7 and BCap-37 cells, and partially reversed the promoting effect of GACAT1 on cell cycle. Through bioinformatics prediction and dual luciferase reporter gene experiments, we found that STON2 might be a target gene of microRNA-875-3p. Overexpression of STON2 could partially abolish the effect of microRNA-875-3p on cell proliferation and cycle of MCF-7 and BCap-37 cells. GACAT1 can participate in the progression of breast cancer by promoting the proliferation and cycle of breast cancer cells. The mechanism may be through the regulation of the level of STON2 by adsorbing microRNA-875-3p.

LINC00657 promotes malignant progression of oral squamous cell carcinoma via regulating microRNA-150.

Previous studies have shown that LINC00657 is a cancer-promoting gene. However, the role of LINC00657 in oral squamous cell carcinoma (OSCC) has not been reported. This study was designed to investigate the role of LINC00657 in OSCC and its regulatory mechanism. Quantitative Real Time-Polymerase Chain Reaction (qPCR) was used to detect the levels of LINC00657 and microRNA-150 in 32 pairs of OSCC tissues and normal ones, and the correlation between LINC00657 and clinical indicators and OSCC patient's prognosis was analyzed. qRT-PCR further verified the levels of LINC00657 and microRNA-150 in OSCC cells. In addition, LINC00657 overexpression and knockdown models were constructed using lentivirus in OSCC cell lines Fadu and Tca8113, and Cell Counting Kit-8 (CCK-8), plate clone experiment, and 5-Ethynyl-2'-deoxyuridine (EdU) assay were carried out to evaluate the influence of LINC00657 on the biological functions of OSCC cells. Further, Luciferase reporter gene and recovery experiments were used to explore its potential mechanism. qRT-PCR showed that LINC00657 expression in OSCC tissue specimens was increased in comparison to normal ones. Patients with high LINC00657 expression had higher pathological staging and lower overall survival. Besides, the cell proliferation ability of the LINC00657 silencing group was remarkably decreased, while the opposite result was observed in LINC00657 overexpression group. Subsequently, qRT-PCR demonstrated a significant decrease in microRNA-150 expression in OSCC cell lines and tissues and a negative correlation with LINC00657. Luciferase assay demonstrated that LINC00657 could be targeted by microRNA-150 in certain binding sites. In addition, cell reverse experiment also confirmed that LINC00657 and microRNA-150 can be mutually regulated, thereby jointly modulating the malignant progression of OSCC. LINC00657, remarkably upregulated in OSCC tissues, showed a close association with the poor prognosis of OSCC patients. Additionally, it may accelerate the malignant progression of OSCC via regulating microRNA-150.

EPAS1 targeting by miR-152-3p in Paclitaxel-resistant Breast Cancer.

Background: Paclitaxel plays a pivotal role in the chemotherapy of breast cancer, but resistance to this drug is an important obstacle in the treatment. It is reported that microRNA-152-3p (miR-152-3p) is involved in tamoxifen resistance in breast cancer, but whether it is involved in paclitaxel resistance in breast cancer remains unknown.Materials and methods: We examined the expression of miR-152-3p in breast cancer tissues and cells by qRT-PCR. After transfecting paclitaxel-resistant MCF-7/TAX cells with miR-152-3p mimics, we analyzed the function of miR-152-3p in these cells by MTT assay and flow cytometry. We screened the target gene, endothelial PAS domain-containing protein 1 (EPAS1), using bioinformatics analysis and verified it with the dual luciferase reporter gene experiment. The relationship between EPAS1 and miR-152-3p and their roles in paclitaxel resistance of breast cancer were further investigated using RNA interference and transfection techniques.Results: The expression of miR-152-3p in normal breast tissues and cells was markedly higher than that in breast cancer. Overexpression of miR-152-3p decreased the survival rate and increased the apoptosis rate and sensitivity of MCF-7/TAX cells to paclitaxel. We confirmed that EPAS1 is the target of miR-152-3p and is negatively regulated by this miRNA. Moreover, transfection with EPAS1 siRNA enhanced the susceptibility and apoptosis rate of MCF-7/TAX cells to paclitaxel. Co-transfection of miR-152-3p mimics and EPAS1 increased paclitaxel sensitivity and apoptosis induced by the drug.Conclusion: miR-152-3p inhibits the survival of MCF-7/TAX cells and promotes their apoptosis by targeting the expression of EPAS1, thereby, enhancing the sensitivity of these breast cancer cells to paclitaxel.

MiR-124-5p Inhibits the Progression of Gastric Cancer by Targeting MIEN1.

Objective:To observe the effect of miR-124-5p on progression of gastric cancer (GC) and explore the targeting mechanism.Methods:After collecting the specimens, we used real-time fluorescence quantitative PCR to detect the miR-124-5p level of GC tissue and corresponding adjacent tissue. Then MTT test and scratch wound-healing assay were hired to evaluate the influence of miR-124-5p in GC cell (SGC-803 and SGC7901) migration and proliferation ability. The binding of miR-124-5p to migration and invasion enhancer 1 (MIEN1) was detected through dual luciferase reporter gene experiment and western blot was utilized to assay the protein level of MIEN1.Results:Compared with adjacent tissues, miR-124-5p level in GC tissues was lower significantly. MiR-124-5p mimic inhibited the metastasis and proliferation ability of SGC7901 cells and miR-124-5p inhibitor promoted the migration and proliferation ability of SGC803 cells. In addition, miR-124-5p targeted MIEN1 and negatively modulated the MIEN1 expression in SGC-803 and SGC7901 cells. Silencing MIEN1 negatively regulated the metastasis and proliferation ability of SGC7901 cells.Conclusion:MiR-124-5p inhibited the GC cell proliferation and metastasis phenotypes through MIEN1, which probably becomes a novel molecular target for clinical GC treatment.

Overexpression of miR-450 affects the biological behavior of HepG2 cells by targeting DNMT3a.

PurposeThis study was designed to explore the regulation mechanism of miR-450 in the development of hepatocarcinoma, and the effects of overexpression of miR-450 on biological behaviors such as proliferation, migration, and invasion of hepatoma cells.MethodsHepG2 cells were divided into miR-450 mimics group, miR-450 inhibitor group, miR-450 mimics NC group, miR-450 inhibitor NC group, and blank group. MTT assay was served to measure cell proliferation, and Transwell assay was used to test cell migration and invasion. Additionally, cell cycle was detected by flow cytometry and apoptosis was examined with AnnexinV-PI double staining. After the target gene of miR-450 was predicted by bioinformatics software, Western blot and dual luciferase reporter gene experiment were applied to verify the relationship between miR-450 and target gene.ResultsThe MTT and Transwell assay indicated that overexpression of miR-450 inhibited the proliferation, invasion, and migration of HepG2 cells. The flow cytometry analysis showed that overexpression of miR-450 arrested the cell cycle in the G1 phase. Meanwhile, Annexin V-PI double staining assay revealed that overexpression of miR-450 promoted apoptosis of HepG2 cells. However, silencing miR-450 in HepG2 cells promoted proliferation and invasion, and reduced apoptosis. Moreover, we found that DNMT3a was the target gene of miR-450.ConclusionsmiR-450 could inhibit proliferation, invasion, and migration via regulating DNMT3a in hepatocarcinoma cells, which provided a theoretical basis for the treatment of liver cancer.

Potential Regulatory Effects of miR-182-3p in Osteosarcoma via Targeting EBF2.

Osteosarcoma (OS) is one of the most common primary malignant bone tumors in adolescents with a high mortality rate. MicroRNA (miRNA) is a kind of noncoding RNAs and has been proved to participate in many physiological processes. Many miRNAs have been reported to act as function regulators in OS. In our study, the miRNA and gene expression profiles of OS were downloaded from GEO Datasets and the differential expression analysis was performed using GEO2R. 58 up- and 126 downregulated miRNAs were found. In the three OS gene profiles, 125 up- and 27 downregulated genes were found to be differentially expressed in at least two profiles. The miRNA-mRNA networks were constructed to predict the potential target genes of 10 most up- and downregulated miRNA. Venn analysis was used to detect the coexpressed differentially expressed genes (DEGs). EBF2, one of the upregulated DEGs, was also a potential target gene of miR-182-3p. Knockdown and overexpression of miR-182-3p resulted in overexpression and downexpression of EBF2 separately. Luciferase reporter gene experiment further verified the binding site of miR-182-3p and EBF2. CCK8 assay showed that miR-182-3p knockdown can further enhance the proliferation activity of OS cells, while overexpressing miR-182-3p can inhibit the proliferation activity of OS cells. Our research indicated that downexpression of miR-182-3p in OS cells results in overexpression of EBF2 and promotes the progression of OS.

ROCK2 Confers Acquired Gemcitabine Resistance in Pancreatic Cancer Cells by Upregulating Transcription Factor ZEB1

Background: Gemcitabine resistance is a major clinical challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC). The increasing attention to Rho-associated coil protein kinase 2 (ROCK2)-mediated chemotherapy resistance in a variety of cancers reminded us to explore the effect of ROCK2 signalnaling in the acquired gemcitabine-resistant pancreatic cancer cells (GR cells). Methods: The relative expression of related proteins and mRNA were detected by western blot and real-time PCR, respectively. Short hairpin RNA (shRNA) was used to silencing related genes. The double luciferase reporter gene experiment was used to explore the activation of related gene promoter. Chromatin immunoprecipitation technique (CHIP) was used to detect the ability of transcription factor binding to the promoter. Many other cell based assays were adopted to explore the mechanism of ROCK2 on resistance of GR cells to gemcitabine. Findings: Pharmacological inhibition or gene silencing of ROCK2 markedly sensitized GR cells to gemcitabine by suppressing the expression of zinc-finger-enhancer binding protein 1 (ZEB1). Mechanically, ROCK2-induced sp1 phosphorylation at Thr-453 enhanced the ability of sp1 binding to ZEB1 promoter regions in a p38-dependent manner. Moreover, transcriptional activation of ZEB1 facilitated GR cells to repair gemcitabine-mediated DNA damage via ATM/p-CHK1 signaling pathway. Interpretation: ROCK2 contributed to EMT-induced gemcitabine resistance in pancreatic cancer cells and might provided strong evidence for the clinical application of fasudil, a ROCK2 inhibitor, in gemcitabine-refractory PDAC. Funding Statement: This work was supported by the National Natural Science Foundation of China (No.81372268, No.81672816 and No.81872337), the Program for Jiangsu Province Innovative Research (KYLX16_1120), the Natural Science Foundation for Distinguished Young Scholars of Jiangsu Province (No. BK20130026), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. ZJ11173). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all animal experimental procedures were approved by Experimentation Ethics Review Committee of China Pharmaceutical.

Highly expressed miR-182-5p can promote preeclampsia progression by degrading RND3 and inhibiting HTR-8/SVneo cell invasion.

The role of miR-182-5p in preeclampsia was studied, and its mechanism was also explored. Fifty patients with preeclampsia were assigned to the study group and 50 normal pregnant women to the control group. The age, weight, blood pressure, urinary protein, and weight of newborns were compared between the two groups. The placental tissues of the above-mentioned subjects were collected, and quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) assay was used to detect the expression of miR-182-5p. MiR-182-5p was overexpressed or knocked down using a cell transfection assay in HTR-8/SVneov cell, which is a kind of human chorionic trophoblast cell. Changes in cell migration and invasiveness before and after transfection were determined by wound healing test and transwell assay, respectively. Western blot was performed to analyze the change of RND3 protein level before and after transfection. The biological prediction of the relationship between miR-182-5p and RND3 was performed and a dual luciferase reporter gene experiment was designed to verify the results. Finally, a rescue experiment was conducted to investigate whether RND3 could affect the role of miRNA-182-5p in the capacity of cell migration and invasion. Preeclampsia patients had higher systolic blood pressure, diastolic blood pressure, and urinary protein than normal pregnant women, while neonatal weight decreased compared with normal pregnant women. MiRNA-182-5p was highly expressed in placental tissues of patients with preeclampsia. After miRNA-182-5p was overexpressed, the migration and invasion of HTR-8/SVneo cells were significantly attenuated, and the mRNA and protein levels of RND3 were markedly downregulated, and vice versa. The dual luciferase reporting assay confirmed that miRNA-182-5p could bind to 3'UTR of RND3. In addition, the results of rescue experiment showed that overexpressing miRNA-182-5p could markedly inhibit the migration and invasion of HTR-8/SVneo cells; however, when RND3 was simultaneously overexpressed, the inhibitory effect of miRNA-182-5p was partially reversed. The highly expressed miRNA-182-5p in patients with preeclampsia promoted the development of preeclampsia, the possible mechanism of which might be that the increased miRNA-182-5p expression could inhibit the migratory and invasive ability of trophoblast cells through targeted degrading RND3 protein.

MiR-137 Inhibits Proliferation and Metastasis of Hypertrophic Scar Fibroblasts via Targeting Pleiotrophin

Background/Aims: There are few effective treatment options for hypertrophic scars. MircoRNAs are a class of small, noncoding RNAs involved in multiple biological functions. Methods: Gene chip screening was used to screen out the differential expression of miRNAs in hypertrophic scars and normal tissues. Western blot and real-time PCR were used to confirm the expression of pleiotrophin (PTN) in hypertrophic scars. After analyze the correlation between PTN and miR-137 using correlation analysis, we used miRDB software to analyze the binding sites of miR-137 and PTN. Luciferase reporter gene, western blot and real-time PCR experiments were used to detect the regulatory effect of miR-137 on PTN. MTT and transwell assay were used to detect the effect of miR-137 on proliferation and metastasis. Western blot and real-time PCR were used to detect the regulatory effects of miR-137 on cyclin B1, matrix metalloproteinase 9 (MMP9), α-smooth muscle actin (α-SMA), vimentin, and type-I collagen (COL1A). Finally, miR-137 inhibitor was transfected into fibroblasts which was silent PTN, and the proliferation and migration of cells were detected by MTT and transwell. Western blot and real-time PCR were used to detect the expression of related proteins. Results: Various miRNAs was abnormal expressed in hypertrophic scars. miR-137 was decreased in hypertrophic scar, however PTN was up regulated in hypertrophic scars. miR-137 induced proliferation and metastasis in fibroblasts. This effect was accompanied by decreased expression of cyclin B1, MMP9, α-SMA, vimentin, and COL1A mediated via the target of PTN. Conclusion: Modulation of miR-137 expression in fibroblasts could provide an important therapeutic strategy for hypertrophic scars.

MiR-92a promotes colorectal cancer cell proliferation by regulation of KLF4 gene expression

Objective To evaluate the effect of microRNA-92a (miR-92a) on regulating cell proliferation by targeting Kruppel-like factor 4 (KLF4) in colon cancer. Methods The miR-92a expressions in 21 colon cancer tissues and matched normal tumor-adjacent tissues and 4 colon cancer cells (HT29, SW480, SW620, HCT116) were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Models of over-expression and suppression of miR-92a were established by transient transfection of miR-92a-3p mimic to HCT116 and transient transfection of miR-92a-3p inhibitor to SW620, respectively. Cell proliferation activity was detected by the CCK-8 colorimetry method, cell cycles were detected by flow cytometry, KLF4 protein expression was detected by Western blotting, and cell luciferase activity was detected by the dual luciferase reporter gene experiment. Results The expression level of miR-92a in colon cancer tissues was (0.648±0.489) fmol/μg total RNA, significantly higher than that in matched normal tumor-adjacent tissues [(0.064±0.062) fmol/μg total RNA], with statistically significant difference (t=-5.420, P<0.001). In 4 colon cancer cell lines, the miR-92a expression level in HCT116 cells was the lowest, and highest in SW620 cell. When the expression of miR-92a was up-regulated, the cell proliferation activity of 72 h in HCT116 cells was higher than that in the negative control group (0.919±0.014 vs. 0.765±0.025), with statistically significant difference (t=-9.309, P=0.001), the proportion of S phase cells was also significantly increased [(41.670±0.461)% vs. (38.703±0.554)%, t=-7.127, P=0.002), and KLF4 protein expression was decreased (0.460±0.048 vs. 0.758±0.109, t=22.865, P=0.028). When the expression of miR-92a was down-regulated, the cell proliferation activity of 72 h in SW620 cells was lower than that in the negative control group (0.608±0.011 vs. 0.713±0.005), with statistically significant difference (t=15.920, P<0.001), while the proportion of S phase cells was decreased [(31.935±0.365)% vs. (34.955±0.465)%, t=8.849, P=0.001], and KLF4 protein expression was increased (0.694±0.121 vs. 0.479±0.044, t=-5.246, P=0.034). KLF4 3′UTR wild-type dual luciferase report plasmids were co-transfected with miR-92a-3p mimic to HCT116 cell, and dual luciferase assay showed that miR-92a slightly repressed firefly lucife-rase actively, but the difference was not statistically significant (t=0.878, P=0.429). There was a negative correlation between the expression of miR-92a and the expression of KLF4 protein in colon cancer tissues, but with no statistical significance (r=-0.163, P=0.699). Conclusion miR-92a is highly expressed in colon cancer tissues. It can promote colon cancer cells proliferation via enhancement of the cell cycle transition of G0-G1 phase to S phase. Up-expression of miR-92a may play a role in down-regulating the expression of KLF4 protein in colon cancer cells. However, KLF4 is not a direct target gene of miR-92a. Key words: Colonic neoplasms; Cell proliferation; MicroRNA-92a; Kruppel-like factor 4

20-HETE regulated PSMB5 expression via TGF-β/Smad signaling pathway

We previously found that 20-hydroxyeicosatetraeonic acid (20-HETE) showed an effect on proteasome activity in cytochrome P450 F2 (CYP4F2) transgenic mice. Proteasome subunit β5 (PSMB5) is a primary subunit of the proteasome. In the current study, we examine whether 20-HETE has any affect on PSMB5. We found that PSMB5 was upregulated in the liver, but downregulated in the kidney of transgenic mice, when compared with wild-type mice. Luciferase reporter gene experiments and electrophoretic mobility shift assays (EMSA) suggested that Smad3 directly associated with the putative Smad binding element (SBE) of the Psmb5 promoter. Furthermore, the binding affinity was different between the liver and kidney, and can be regulated by 20-HETE. Compared to wild mice, both TGF-β1 and Smad3 phosphorylation were increased in the liver but decreased in the kidney of transgenic mice. SB431542, an inhibitor of TGF-β receptor I kinase activity, can reverse the changes induced in PSMB5 by 20-HETE in vitro. Taken together, our data demonstrated that 20-HETE upregulated the expression of PSMB5 by activating the TGF-β/Smad signaling pathway in the liver, but downregulated the expression of PSMB5 by inhibiting the TGF-β/Smad signaling pathway in the kidney of transgenic mice.

Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway.

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor (TNF)-α. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of NF-κB and miR-503. We found that overexpression of NS5A inhibited TNF-αinduced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the TNF-α induced Hep-mock cells was significantly less than the viability of the TNF-α induced Hep-NS5A cells, which demonstrates that NS5A inhibited TNF-α-induced HepG2 cell apoptosis. Under TNF-α treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited TNF-α-induced NF-κB activation and NF-κB regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse TNF-α-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits NF-κB activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin.

Mechanistic insights into Lp(a)-induced IL-8 expression: a role for oxidized phospholipid modification of apo(a)

Elevated lipoprotein (a) [Lp(a)] levels are a causal risk factor for coronary heart disease. Accumulating evidence suggests that Lp(a) can stimulate cellular inflammatory responses through the kringle-containing apolipoprotein (a) [apo(a)] component. Here, we report that recombinant apo(a) containing 17 kringle (17K) IV domains elicits a dose-dependent increase in interleukin (IL)-8 mRNA and protein expression in THP-1 and U937 macrophages. This effect was blunted by mutation of the lysine binding site in apo(a) kringle IV type 10, which resulted in the loss of oxidized phospholipid (oxPL) on apo(a). Trypsin-digested 17K had the same stimulatory effect on IL-8 expression as intact apo(a), while enzymatic removal of oxPL from apo(a) significantly blunted this effect. Using siRNA to assess candidate receptors, we found that CD36 and TLR2 may play roles in apo(a)-mediated IL-8 stimulation. Downstream of these receptors, inhibitors of MAPKs, Jun N-terminal kinase and ERK1/2, abolished the effect of apo(a) on IL-8 gene expression. To assess the roles of downstream transcription factors, luciferase reporter gene experiments were conducted using an IL-8 promoter fragment. The apo(a)-induced expression of this reporter construct was eliminated by mutation of IL-8 promoter binding sites for either NF-κB or AP-1. Our results provide a mechanistic link between oxPL modification of apo(a) and stimulation of proinflammatory intracellular signaling pathways.

Prenatal hypoxia promotes atherosclerosis via vascular inflammation in the offspring rats

BackgroundHypoxia is a critical contributor to increased risks of cardiovascular diseases, including atherosclerosis, but the detailed mechanism that hypoxia leads to atherosclerosis remains unknown. MethodsPregnant rats were treated with hypoxia (10.5% oxygen) during pregnancy, and HUVEC cells treated with 1% of oxygen. Blood lipids were tested at fetal stage and adult stage of offspring rats; the level of pro-inflammatory cytokines of HUVEC and offspring rats were investigated, and HIF-1α and NFκB mRNA level were also measured by Q-PCR and Elisa. ResultsWe found that TC, LDL-C, ox-LDL-C, and the receptors of ox-LDL-C (lox-1) of the adult offspring were significantly higher than that of the control, while HDL-C was significantly reduced in hypoxia group. The internal elastic lamina was blocked by smooth muscle cells; and the migration of smooth muscle cells into the intima were observed in hypoxia offspring. Luciferase reporter gene experiment showed that HIF-1α activated NFκB transcription at four discrete binding sites of NFκBp65 promoter, although there was no obvious difference among the four discrete binding sites. Using transfection of pCDNA3.1-HIF-1α on HUVEC cells, HIF-1α significantly activated NFκB transcription at hypoxic conditions (1% O2), and concurrent with increased expression of IL-1β and TNF-α. ConclusionHypoxia during pregnancy activated NFκB transcription to induce pro-inflammatory cytokines, leading to the early stage of atherosclerosis.

Transcription factor CgMTF-1 regulates CgZnT1 and CgMT expression in Pacific oyster (Crassostrea gigas) under zinc stress

Oysters accumulate zinc at high tissue concentrations, and the metal response element (MRE)-binding transcription factor (MTF) functions as the cellular zinc sensor that coordinates the expression of genes involved in zinc efflux and storage, as well as those that protect against metal toxicity. In this study, we cloned MTF-1 in oysters and examined its regulation mechanism for its classic target genes, including MTs and ZnT1 under zinc exposure conditions. We cloned CgMTF-1 and determined the subcellular locations of its protein product in HEK293 cells. CgMTF-1 has a 2826bp open reading frame that encodes a predicted polypeptide with 707 amino acid residues, showing six well-conserved zinc finger domains that are required for metal binding. In HEK293 cell lines, CgMTF-1 primarily localizes in the cell nucleus under unstressed conditions and nuclear translocation was not critical for the activation of this gene. We searched for CgMTF-1-regulated genes in oysters using RNA interference. Decreased expression levels of CgMT1, CgMT4, and CgZnT1 were observed after CgMTF-1 interference (>70% inhibition) under zinc exposure, indicating the critical role of CgMTF-1 in the regulation of these genes. We searched for a direct regulation mechanism involving CgMTF-1 for CgMT1, CgMT4, and CgZnT1 in vitro. EMSA experiments indicated that CgMTF-1 can bind with the MREs found in the CgZnT1, CgMT1 and CgMT4 promoter regions. Additionally, luciferase reporter gene experiments indicated that CgMTF-1 could activate the CgMT1, CgMT4, and CgZnT1 promoters. Overall, our results suggest that CgMTF-1 directly coordinates the regulation of CgMTs and CgZnT1 expression and plays important roles in protecting oysters under zinc exposure conditions. To our knowledge, this is the first study to elucidate the function of MTF-1 in marine bivalves and provides new insights into the mechanisms of zinc accumulation and tolerance in mollusks.

Parathyroid hormone activates the orphan nuclear receptor Nurr1 to induce FGF23 transcription.

Parathyroid hormone (PTH) increases FGF23 mRNA and protein levels in vivo and in vitro. Here we tested whether the increased FGF23 expression by PTH is mediated by the orphan nuclear receptor Nurr1. PTH increased Nurr1 mRNA levels prior to elevation of FGF23 mRNA levels in UMR-106 rat osteoblast-like cells. Activation of PKA increased both FGF23 and Nurr1 mRNA levels. Modification of Nurr1 expression showed that Nurr1 is essential for the PTH-mediated increase in FGF23 and luciferase reporter gene experiments identified a functional promoter region containing several potential Nurr1 binding sites. Chromatin immunoprecipitation assays confirmed the binding of Nurr1 to these regions in the FGF23 promoter. In vivo, Nurr1 mRNA and protein levels were increased in calvaria from rats with experimental CKD together with high PTH and FGF23 expression. Calcimimetics decrease PTH and FGF23 levels in CKD patients. Importantly, in rats with experimental CKD, the calcimimetic R568 decreased PTH expression, calvaria Nurr1 mRNA and protein levels, and FGF23 mRNA. Immunohistochemistry for Nurr1 showed an increase in the number of Nurr1 expressing osteocytes in the femurs of rats with CKD and this was decreased by R568. Thus, the effect of PTH to increase FGF23 transcription is mediated by Nurr1 in vitro and in vivo. In CKD, calcimimetics decrease PTH, which in turn decreases Nurr1 and consequently FGF23.