Mengying Wei

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.

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.

Maternal exosomes in diabetes contribute to the cardiac development deficiency.

Maternal diabetes mellitus induces an increased risk of congenital heart defects (CHD), however, the exact mechanisms are still not fully illustrated. In this study, diabetic pregnant C57BL/6 mice were induced by injection of streptozotocin before mating. Compared with the control normal mice, diabetic pregnant mice displayed significant changes of the exosomal miRNA contents in the blood, as revealed by RNA-seq analysis. Multiple of these miRNAs were found involved in cardiac development regulation. Moreover, fluorescence labeled exosomes and gold nanoparticles could cross the placenta barrier and infiltrated into the embryonic organs/tissues, including the heart, during embryonic development. Injection of diabetic maternal exosomes strikingly increased the risk of CHD in the normal recipient pregnant mice. Taken together, we could draw the conclusion that maternal exosomes in diabetes could cross the maternal-fetal barrier and contribute to the cardiac development deficiency possibly via miRNAs, providing new insights in CHD prevention and treatment.

The sense strand pre-cleaved RNA duplex mediates an efficient RNA interference with less off-target and immune response effects

RNA interference is an appealing and promising therapeutic approach in cancer and other diseases. Designing novel strategies aiming to increase the efficiency, duration, and reduce the off-target silencing by sense strand is of great significance for its future application clinically. Here, we report that RNA duplex with the sense strand pre-cleaved at the base between base 10 and 11 relative to the 5′ end of the antisense strand induced a target-specific RNA silencing effectively. Furthermore, different from the canonical RNA duplex, this novel RNA duplex rarely inhibits the luciferase activity in the reporter, bearing the target sequence corresponding to the sense strand, suggesting a less off-target effects of this novel strategy. Furthermore, the immune response of the novel RNA duplex induced a much milder immune response as seen from the NFkappaB activity. In addition, our newly designed RNA duplex should be easier for preservation than the asymmetric RNA duplex. Our results establish a novel method to design a new class of RNA duplex for improved RNA interference.

Rab27A mediated by NF-κB promotes the stemness of colon cancer cells via up-regulation of cytokine secretion

Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-β. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.

Production and Characterization of Polyclonal and Monoclonal Abs Against the RNA-Binding Protein QKI

RNA-binding protein QKI, a member of the Signal Transduction and Activation of RNA family, is found to be essential in the blood vessel development and postnatal myelination in central nervous system (Woo et al., Oncogene 28:1176–1186, 2009; Lu et al., Nucleic Acids Res 31(15):4616–4624, 2003; Bohnsack et al., Genesis 44(2):93–104, 2006). However, its wide expression pattern suggests other fundamental roles in vivo (Kondo et al., Mamm Genome 10(7):662–669, 1999). To facilitate the understanding of QKI function in various systems, we prepared the polyclonal and monoclonal antibodies against QKI. To obtain the antigen, recombinant His-tagged QKI was expressed in Escherichia coli and highly purified by Ni2+-chelated column combined with hydrophobic and ion exchange methods. Following three types of immunizations with different adjuvants, including Freund’s, PAGE gel, and nitrocellulose membrane, only the antiserum produced with Freund’s adjuvant is effective for Western blot detection. Several McAb clones are able to recognize both endogenous and over-expressed QKI with high affinity in Western blot and immunofluorescence. The specificity of Ab was validated as weakening, and no specific signals were observed in cells with QKI knocking down. Immunohistochemistry analysis further showed positive staining of QKI in kidney where QKI mRNA was abundantly expressed, ensuring the wide applications of the QKI Abs in the ongoing mechanistic studies.