Zifan Lu

Glycyrrhizin treatment is associated with attenuation of lipopolysaccharide-induced acute lung injury by inhibiting cyclooxygenase-2 and inducible nitric oxide synthase expression.

Glycyrrhizin (GL), a major active constituent of licorice root, has been attributed numerous pharmacologic effects, including anti-inflammatory, anti-viral, anti-tumor, and hepatoprotective activities. In this study, we investigated the anti-inflammatory effect of GL on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. ALI was induced in Balb/c mice by intratracheal instillation of LPS (1 mg/kg). Before 1 h of LPS administration, the mice received intraperitoneal injection of GL at varied doses (10, 25, and 50 mg/kg). The severity of pulmonary injury was evaluated 12 h after LPS administration. GL pretreatment led to significant attenuation of LPS induced evident lung histopathologic changes, alveolar hemorrhage, and neutrophil infiltration with evidence of reduced myeloperoxidase (MPO) activity. The lung wet/dry weight ratios, as an index of lung edema, were markedly reduced by GL pretreatment. The concentrations of pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α were elevated in bronchoalveolar lavage fluid (BALF) after LPS administration, which were significantly inhibited by GL pretreatment. GL pretreatment also reduced the concentrations of nitric oxide (NO) in lung tissues. Furthermore, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was suppressed by GL pretreatment. In conclusion, GL potently protected against LPS-induced ALI, and the protective effects of GL may attribute partly to the suppression of COX-2 and iNOS expression.

Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

The selective RNA‐binding protein QKI is essential for myelination in the central nervous system (CNS). QKI belongs to the family of signal transduction activators of RNA (STARs), characteristic of binding RNA and signaling molecules, therefore is postulated to regulate RNA homeostasis in response to developmental signals. Here we report that QKI acts downstream of the Src family protein tyrosine kinases (Src‐PTKs) during CNS myelination. QKI selectively interacted with the mRNA encoding the myelin basic protein (MBP). Such interaction stabilized MBP mRNA and was required for the rapid accumulation of MBP mRNA during active myelinogenesis. We found that the interaction between QKI and MBP mRNA was negatively regulated by Src‐PTK‐dependent phosphorylation of QKI. During early myelin development, tyrosine phosphorylation of QKI in the developing myelin drastically declined, presumably leading to enhanced interactions between QKI and MBP mRNA, which was associated with the rapid accumulation of MBP mRNA and accelerated myelin production. Therefore, developmental regulation of Src‐PTK‐dependent tyrosine phosphorylation of QKI suggests a novel mechanism for accelerating CNS myelinogenesis via regulating mRNA metabolism.

E2F1 and RNA binding protein QKI comprise a negative feedback in the cell cycle regulation.

pRb/E2F1 activity is coordinately regulated during the cell cycle progression, while the molecular strategies safeguarding this pathway are not fully understood. We have previously shown that RNA binding protein QKI inhibits the cell proliferation and promotes the differentiation of gastrointestinal epithelium, suggesting a role of QKI in cell cycle regulation. Here we found that with the cell entry into S phase, QKI expression increased both at the mRNA and protein levels, which was reminiscent of cyclin E expression. Forced expression of E2F1 increased the endogenous level of QKI. Promoter luciferase assay and ChIP analysis identified that the -542~-538 E2F1 binding site was responsible for the upregulation. Increased QKI expression by E2F1, in turn, reduced the E2F1 activity and delayed S-phase entry, forming a negative feedback. As a gene expression regulator, QKI overexpression increased p27, while it decreased cyclin D1 and c-fos expression. Molecularly, p27 and c-fos were direct targets of QKI, while cyclin D1 reduction might be an indirect effect. Taken together, our results reveal that E2F1 directly transcribes QKI, which, in turn, negatively regulates the cell cycle by targeting multiple cell cycle regulators, forming an E2F1-QKI-pRb/E2F1 negative feedback loop.

The tumor suppressing effects of QKI-5 in prostate cancer: A novel diagnostic and prognostic protein

In recent years, the RNA-binding protein quaking 5 (QKI-5) has been recognized as a novel tumor suppressor in many cancers. To date, no studies have examined the role of QKI-5 in prostate cancer. The present study was designed to elucidate the correlation of QKI-5 expression with the clinical pathological features and prognosis of prostate cancer. In an overwhelming majority of the 184 cases of prostate cancer samples analyzed, the QKI-5 expression was significantly decreased, which was largely due to the high promoter methylation levels. Using lentiviral vectors, we established two stable prostate cancer cell lines with altered QKI-5 expression, including a QKI-5 overexpressing PC3 cell line and a DU145 cell line with knocked-down QKI-5 expression. The effects of the lentiviral-mediated QKI-5 knockdown on the PC3 cells and DU145 cells were assessed by cell growth curves, flow cytometry (FCM), and an invasion assay. The PC3 cells were transplanted into nude mice, and then, the tumor growth curves and TUNEL staining were determined. These results demonstrated that QKI-5 was highly expressed in benign prostatic hyperplasia (BPH) tissues but not in carcinomatous tissues and that QKI-5 effectively inhibited prostate cancer cell proliferation in vitro and in vivo. In addition, the decrease in QKI-5 expression was closely correlated with the prostate cancer Gleason score, poor differentiation, degree of invasion, lymph node metastasis, distant metastasis, TNM grading, and poor survival. These results indicate that the QKI-5 expression may be a novel, independent factor in the prognosis of prostate cancer patients.

NDRG2 is highly expressed in pancreatic β cells and involved in protection against lipotoxicity

The N-myc downstream-regulated gene 2 (NDRG2) is involved in cell differentiation and apoptosis, but its function in the pancreas remains to be established. Herein we examine the expression and function of NDRG2 in the endocrine pancreas. NDRG2 immunoreactivity was localized mainly in the cytoplasm of pancreatic β cells. When β-TC3 cells were exposed chronically to high levels of free fatty acid (FFA), cell viability was impaired, and Akt and NDRG2 phosphorylation were reduced. NDRG2 is a potential substrate of protein kinase Akt. Overexpression of constitutively active Akt enhanced NDRG2 phosphorylation and abolished the apoptosis induced by FFA in β-TC3 cells, whereas NDRG2 knock-down attenuated Akt-mediated protection of β cells against fatty acid-triggered apoptosis. Collectively, these data indicate that NDRG2 acts as a key molecule in pancreatic β cells and is involved in the Akt-mediated protection of β cells against lipotoxicity.

Gastrointestinal tract specific gene GDDR inhibits the progression of gastric cancer in a TFF1 dependent manner.

The novel gene GDDR, also named trefoil factor interactions 1, is abundantly expressed in human gastric epithelial cells but significantly down-regulated in gastric cancer. In this study, we first demonstrated that GDDR was specifically expressed in the gastrointestinal tract epithelium, while suppressed in gastric cancer cells. Forced expression of GDDR suppressed the tumor growth as shown by MTT and xenograft assay. siRNA mediated TFF1 inhibition nearly blocked the growth inhibitory role of GDDR. In summary, our study suggested that gastrointestinal tract specific gene GDDR might inhibit gastric cancer growth in a TFF1 dependent manner.

RNA binding protein QKI inhibits the ischemia/reperfusion-induced apoptosis in neonatal cardiomyocytes.

Backgrounds: RNA-binding protein QKI is abundantly expressed in the brain and heart. The role of QKI in the nervous system has been well characterized, but its function in cardiac muscle is still poorly understood. The present study was to investigate the role of QKI in ischemia/reperfusion-induced apoptosis in cardiomyocytes. Methods: A simulated ischemia/reperfusion model was established in neonatal cardiomyocytes and adult rat heart. After QKI5 or QKI6 was expressed by adenovirus and QKI was knocked down QKI by RNAi in the cardiomyocytes, RT-PCR, western blot and immunofluorescence staining were applied to detect gene expression alterations. Apoptosis was evaluated by PARP degradation, DNA fragmentation (DNA laddering) and flow cytometry. Results: Our study demonstrated that both QKI5 and QKI6 were present in cardiomyocytes, while QKI5 expression was greatly inhibited by simulated ischemia/reperfusion. Knocking down endogenous QKI by RNAi enhanced cell susceptibility to apoptosis, whereas overexpression of either QKI5 or QKI6 suppressed IR-induced apoptosis substantially. The pro-apoptotic transcription factor FoxO1, a potential QKI target, was induced by ischemia/reperfusion at both total amount and nuclear distribution. Accordingly, FOXO1 downstream target genes were negatively affected by the presence of QKI with IR treatment. Conclusion: In summary, our study supports that both QKI-5 and 6 are anti-apoptotic proteins in cardiomyocytes, favoring cardiac survival via antagonizing the elevation of some pro-apoptotic factors in cardiac injury.

High-Mobility Group Box 1 Induces Calcineurin-Mediated Cell Hypertrophy in Neonatal Rat Ventricular Myocytes

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. In recent years, evidences suggest that high-mobility group box 1 (HMGB1) protein, an inflammatory cytokine, participates in cardiac remodeling; however, the involvement of HMGB1 in the pathogenesis of cardiac hypertrophy remains unknown. The aim of this study was to investigate whether HMGB1 is sufficient to induce cardiomyocyte hypertrophy and to identify the possible mechanisms underlying the hypertrophic response. Cardiomyocytes isolated from 1-day-old Sprague-Dawley rats were treated with recombinant HMGB1, at concentrations ranging from 50 ng/mL to 200 ng/mL. After 24 hours, cardiomyocytes were processed for the evaluation of atrial natriuretic peptide (ANP) and calcineurin A expression. Western blot and real-time RT-PCR was used to detect protein and mRNA expression levels, respectively. The activity of calcineurin was also evaluated using a biochemical enzyme assay. HMGB1 induced cardiomyocyte hypertrophy, characterized by enhanced expression of ANP, and increased protein synthesis. Meanwhile, increased calcineurin activity and calcineurin A protein expression were observed in cardiomyocytes preconditioned with HMGB1. Furthermore, cyclosporin A pretreatment partially inhibited the HMGB1-induced cardiomyocyte hypertrophy. Our findings suggest that HMGB1 leads to cardiac hypertrophy, at least in part through activating calcineurin.

QKI impairs self-renewal and tumorigenicity of oral cancer cells via repression of SOX2

Cancer stem cells (CSCs) may contribute to tumor initiation, distant metastasis and chemo-resistance. One of RNA-binding proteins, Quaking (QKI), was reported to be a tumor suppressor. Here we showed that reduced QKI levels were observed in many human oral cancer samples. Moreover further reduction of QKI expression in CSCs was detected compared with non-CSCs in oral cancer cell lines. Overexpressing QKI in oral cancer cells significantly reduced CSC sphere formation and stem cell-associated genes. In tumor implanting nude mice model, QKI significantly impeded tumor initiation rates, tumor sizes and lung metastasis rates. As a contrast, knocking down QKI enhanced the above effects. Among the putative CSC target genes, SOX2 expression was negatively affected by QKI, mechanism study revealed that QKI may directly regulate SOX2 expression via specific binding with its 3′UTR in a cis element-dependent way. Loss of SOX2 even completely reversed the sphere forming ability in QKI knockdown cell line. Taken together, these data demonstrated that SOX2 is an important CSC regulator in oral cancer. QKI is a novel CSC inhibitor and impaired multiple oral CSC properties via partial repression of SOX2. Therefore, reduced expression of QKI may provide a novel diagnostic marker for oral cancer.

The RNA-binding protein QKI5 is a direct target of C/EBPα and delays macrophage differentiation.

During monocyte–macrophage differentiation, C/EBPα transcriptionally activates QKI, which in turn represses CSF1R and thus provides negative feedback to C/EBPα-induced macrophage differentiation. This feedback loop should be important in keeping the balance between cell proliferation and differentiation.