Zhen Yue

Alterations of Serum Levels of BDNF-Related miRNAs in Patients with Depression

Depression is a serious and potentially life-threatening mental disorder with unknown etiology. Emerging evidence shows that brain-derived neurotrophic factor (BDNF) and microRNAs (miRNAs) play critical roles in the etiology of depression. Here this study was aimed to identify and characterize the roles of BDNF and its putative regulatory miRNAs in depression. First, we identified that miR-182 may be a putative miRNA that regulates BDNF levels by bioinformatic studies, and characterized the effects of miR-182 on the BDNF levels using cell-based studies, side by side with miR-132 (a known miRNA that regulates BDNF expression). We showed that treatment of miR-132 and miR-182 respectively decreased the BDNF protein levels in a human neuronal cell model, supporting the regulatory roles of miR-132 and miR-182 on the BDNF expression. Furthermore, we explored the roles of miR-132 and miR-182 on the BDNF levels in depression using human subjects by assessing their serum levels. Compared with the healthy controls, patients with depression showed lower serum BDNF levels (via the enzyme-linked immunosorbent assays) and higher serum miR-132 and miR-182 levels (via the real-time PCR). Finally, the Pearson’s (or Spearman’s) correlation coefficient was calculated to study whether there was a relationship among the Self-Rating Depression Scale score, the serum BDNF levels, and serum BDNF-related miRNA levels. Our results revealed that there was a significant negative correlation between the SDS scores and the serum BDNF levels, and a positive correlation between the SDS scores and miR-132 levels. In addition, we found a reverse relationship between the serum BDNF levels and the miR-132/miR-182 levels in depression. Collectively, we provided evidence supporting that miR-182 is a putative BDNF-regulatory miRNA, and suggested that the serum BDNF and its related miRNAs may be utilized as important biomarkers in the diagnosis or as therapeutic targets of depression.

miR‑99 inhibits cervical carcinoma cell proliferation by targeting TRIB2

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3′-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

TRIB2 knockdown as a regulator of chemotherapy resistance and proliferation via the ERK/STAT3 signaling pathway in human chronic myelogenous leukemia K562/ADM cells

Acquired resistance to chemotherapy plays a critical role in human drug treatment failure in many tumor types. Multidrug resistance (MDR) to Adriamycin (ADM) also limits the efficacy of therapy in human chronic myelogenous leukemia (CML). The overexpression of drug efflux transporters is one mechanism uderlying MDR. In particular, the consistent activation of MDR1 and MDR‑associated protein 1 (MRP1) is involved in drug resistance. In the present study, ADM‑resistant human CML K562/ADM cells were stably transfected with a Tribbles homolog 2 (TRIB2)‑targeted vector. A CCK‑8 assay showed that the half maximal inhibitory concentration (IC50) of ADM and the cell proliferation were lower in the transfected cells compared with that in the parental K562/ADM cells. The mRNA and protein expression levels of MDR1 and MRP1 were determined by reverse transcription‑polymerase chain reaction (RT‑PCR), RT‑quantitative PCR and western blot analysis. The results showed that the expression of MDR1 and MRP1 was significantly reduced in K562/ADM cells transfected with pGPU6/GFP/Neo‑TRIB2. Due to the downregulation of MDR1 and MRP1, the intracellular accumulation of ADM was increased in the transfected cells compared with that in the parental K562/ADM cells. Therefore, the sensitivity of the K562/ADM cells to ADM was enhanced and proliferation was inhibited. Our research revealed that protein expression of the ERK signaling pathway was inhibited by downregulating TRIB2, indicating that the ERK pathway was involved in cell drug resistance and proliferation. Furthermore, we used the ERK‑specific blocker U0126 to demonstrate this phenomenon. In summary, our research suggested that knockdown of TRIB2 could slow cell growth and reverse resistance, implying that TRIB2 is a potential therapy target for resistant human CML.

Identification and functional characterization of unfolded protein response transcription factor ATF6 gene in kuruma shrimp Marsupenaeus japonicus

ABSTRACT Activating transcription factor 6 (ATF6) pathway is the key branch of unfolded protein response (UPR). In this study, a homolog of ATF&agr; from Marsupenaeus japonicus (MjATF6) was identified using genome sequencing and characterized, so as to investigate the role of ATF6 pathway in anti‐viral immunity of M. japonicus. The cDNA of MjATF6 obtained was 1008 bp in length, with an open reading frame (ORF) of 849bp, which had encoded a putative of 283 amino acid proteins. Results of qRT‐PCR showed that MjATF6 was distributed in all the six tested tissues, with the higher expression level being seen in hemocytes and hepatopancreas. Furthermore, MjATF6 expression would be up‐regulated from 1 day to 7 day under white spot syndrome virus (WSSV) challenge. In comparison, RNA interference‐induced MjATF6 knockdown had resulted in a lower 7‐day cumulative mortality of M. japonicus in the presence of WSSV infection. Additionally, our results also revealed that less VP28 mRNA was extracted from hemocytes or hepatopancreas of MjATF6 knockdown shrimp than that from the control. Taken together, these results have confirmed that ATF6 pathway is vital for WSSV replication, and that UPR in M. japonicus may facilitate WSSV infection. HighlightsATF6 was identified in M. japonicus.MjATF6 was up‐regulated in shrimp in the presence of WSSV infection.MjATF6 knockdown resulted in a lower cumulative mortality of M. japonicus in the presence of WSSV infection.MjATF6 knockdown resulted in a lower VP28 expression level.MjATF6 was involved in WSSV replication.

Celastrol suppresses the proliferation of lung adenocarcinoma cells by regulating microRNA-24 and microRNA-181b

Cumulative evidence has indicated that celastrol may suppress cancer growth; however, the underlying mechanism requires further investigation. In the present study, A549 cells were treated with various concentrations of celastrol. Lung cancer cell proliferation was evaluated using an MTT assay and observed under a microscope. Cell apoptosis was detected by Annexin V fluorescein isothiocyanate/propidium iodide double-labeled flow cytometry. The results demonstrated that celastrol suppressed proliferation and induced apoptosis in a dose-independent manner. Celastrol may also decrease the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and the B cell lymphoma-2 (Bcl-2)/Bcl-2 associated C protein (Bax) ratio. As microRNA (miR-24 and miR-181b) were predicated to target STAT3, STAT3 activation was inhibited in miR-24-or miR-181b-treated A549 cells compared with the control treatment. The ratio of Bcl-2/Bax was further reduced in miR-24 or miR-181b-treated A549 cells. The results were further confirmed by detecting in another lung adenocarcinoma cell line, LTEP-a-2. In summary, the results of the present study demonstrated that celastrol treatment suppressed the proliferation and induced apoptosis by regulating the expression levels of miR-24 and miR-181b.