Guihong Sun

MicroRNA-720 promotes in vitro cell migration by targeting Rab35 expression in cervical cancer cells.

BackgroundMicroRNA-720 (miR-720), a nonclassical miRNA, is involved in the initiation and progression of several tumors. In our previous studies, miR-720 was shown to be significantly upregulated in cervical cancer tissues compared with normal cervical tissues. However, the precise biological functions of miR-720, and its molecular mechanisms of action, are still unknown.ResultsMicroarray expression profiles, luciferase reporter assays, and western blot assays were used to validate Rab35 as a target gene of miR-720 in HEK293T and HeLa cells. The regulation of Rab35 expression by miR-720 was assessed using qRT-PCR and western blot assays, and the effects of exogenous miR-720 and Rab35 on cell migration were evaluated in vitro using Transwell® assay, wound healing assay, and real-time analyses in HeLa cells. The influences of exogenous miR-720 on cell proliferation were evaluated in vitro by the MTT assay in HeLa cells. In addition, expression of E-cadherin and vimentin associated with epithelial-mesenchymal transition were also assessed using western blot analyses after transfection of miR-720 mimics and Rab35 expression vectors. The results showed that the small GTPase, Rab35, is a direct functional target of miR-720 in cervical cancer HeLa cells. By targeting Rab35, overexpression of miR-720 resulted in a decrease in E-cadherin expression and an increase in vimentin expression and finally led to promotion of HeLa cell migration. Furthermore, reintroduction of Rab35 3′-UTR(−) markedly reversed the induction of cell migration in miR-720-expressing HeLa cells.ConclusionsThe miR-720 promotes cell migration of HeLa cells by downregulating Rab35. The results show that miR-720 is a novel cell migration-associated gene in cervical cancer cells.

Epigenetic regulation of thyroid hormone-induced adult intestinal stem cell development during anuran metamorphosis.

Epigenetic modifications of histones are emerging as key factors in gene regulation by diverse transcription factors. Their roles during vertebrate development and pathogenesis are less clear. The causative effect of thyroid hormone (T3) on amphibian metamorphosis and the ability to manipulate this process for molecular and genetic studies have led to the demonstration that T3 receptor (TR) is necessary and sufficient for Xenopus metamorphosis, a process that resembles the postembryonic development (around birth) in mammals. Importantly, analyses during metamorphosis have provided some of the first in vivo evidence for the involvement of histone modifications in gene regulation by TR during vertebrate development. Furthermore, expression and functional studies suggest that various histone modifying epigenetic enzymes likely participate in multiple steps during the formation of adult intestinal stem cells during metamorphosis. The similarity between intestinal remodeling and the maturation of the mammalian intestine around birth when T3 levels are high suggests conserved roles for the epigenetic enzymes in mammalian adult intestinal stem cell development and/or proliferation.

Differential regulation of two histidine ammonia-lyase genes during Xenopus development implicates distinct functions during thyroid hormone-induced formation of adult stem cells

BackgroundOrgan-specific, adult stem cells are essential for organ-homeostasis and tissue repair and regeneration. The formation of such stem cells during vertebrate development remains to be investigated. Frog metamorphosis offers an excellent opportunity to study the formation of adult stem cells as this process involves essentially the transformations of all larval tissues/organs into the adult form. Of particular interest is the remodeling of the intestine. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. A major advantage of this metamorphosis model is its total dependence on thyroid hormone (T3). In an effort to identify genes that are important for stem cell development, we have previously carried out tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis.ResultsWe report the detailed characterization of one of the genes thus identified, the histidine ammonia-lyase (HAL) gene, which encodes an enzyme known as histidase or histidinase. We show that there are two duplicated HAL genes, HAL1 and HAL2, in both Xenopus laevis and Xenopus tropicalis, a highly related but diploid species. Interestingly, only HAL2 is highly upregulated by T3 and appears to be specifically expressed in the adult intestinal progenitor/stem cells while HAL1 is not expressed in the intestine during metamorphosis. Furthermore, when analyzed in whole animals, HAL1 appears to be expressed only during embryogenesis but not metamorphosis while the opposite appears to be true for HAL2.ConclusionsOur results suggest that the duplicated HAL genes have distinct functions with HAL2 likely involved in the formation and/or proliferation of the adult stem cells during metamorphosis.

Activation of Sox3 Gene by Thyroid Hormone in the Developing Adult Intestinal Stem Cell During Xenopus Metamorphosis

The maturation of the intestine into the adult form involves the formation of adult stem cells in a thyroid hormone (T3)-dependent process in vertebrates. In mammals, this takes place during postembryonic development, a period around birth when the T3 level peaks. Due to the difficulty of manipulating late-stage, uterus-enclosed embryos, very little is known about the development of the adult intestinal stem cells. Interestingly, the remodeling of the intestine during the T3-dependent amphibian metamorphosis mimics the maturation of mammalian intestine. Our earlier microarray studies in Xenopus laevis revealed that the transcription factor SRY (sex-determining region Y)-box 3 (Sox3), well known for its involvement in neural development, was upregulated in the intestinal epithelium during metamorphosis. Here, we show that Sox3 is highly and specifically expressed in the developing adult intestinal progenitor/stem cells. We further show that its induction by T3 is independent of new protein synthesis, suggesting that Sox3 is directly activated by liganded T3 receptor. Thus, T3 activates Sox3 as one of the earliest changes in the epithelium, and Sox3 in turn may facilitate the dedifferentiation of the larval epithelial cells into adult stem cells.

miR-638 suppresses DNA damage repair by targeting SMC1A expression in terminally differentiated cells

The reduction of DNA damage repair capacity in terminally differentiated cells may be involved in sensitivity to cancer chemotherapy drugs; however, the underlying molecular mechanism is still not fully understood. Herein, we evaluated the role of miR-638 in the regulation of DNA damage repair in terminally differentiated cells. Our results show that miR-638 expression was up-regulated during cellular terminal differentiation and involved in mediating DNA damage repair processes. Results from a luciferase reporting experiment show that structural maintenance of chromosomes (SMC)1A was a potential target of miR-638; this was verified by western blot assays during cell differentiation and DNA damage induction. Overexpression of miR-638 enhanced the sensitivity of cancer cells to cisplatin, thus reducing cell viability in response to chemotherapy drug treatment. Furthermore, miR-638 overexpression affected DNA damage repair processes by interfering with the recruitment of the DNA damage repair-related protein, γH2AX, to DNA break sites. These findings indicate that miR-638 might act as a sensitizer in cancer chemotherapy and accompany chemotherapy drugs to enhance chemotherapeutic efficacy and to improve the chance of recovery from cancer.

Hepatitis B virus promotes cancer cell migration by downregulating miR-340-5p expression to induce STAT3 overexpression

BackgroundHepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, and infection with hepatitis B virus (HBV) is a leading cause of HCC. Previous studies have demonstrated that expression of the tumor inhibitor miR-340 is significantly downregulated in HCC tissues compared with normal liver tissues. However, the precise biological role of miR-340-5p in HBV–HCC and its molecular mechanism of action remain unknown.ResultsExpression of miR-340-5p was downregulated in HBV-associated HCC liver tissue and HBV-infected cells, facilitating migration of liver cancer cells. Signal transducer and activator of transcription (STAT)3 was found to be a direct functional target of miR-340-5p. The regulation of STAT3 expression by miR-340-5p was assessed using qRT-PCR and western blotting, and the effects of exogenous miR-340-5p and STAT3 on the migration of HBV-infected cells were evaluated in vitro using Transwell® and wound-healing assays. The expression of E-cadherin and vimentin, associated with epithelial–mesenchymal transition, was also assessed using Western blotting after transfection of miR-340-5p mimics and/or STAT3 expression vectors. Overexpression of STAT3 resulted in rescue of HBV effects, decreased E-cadherin expression, increased vimentin expression, and ultimately, enhanced cell migration. Re-introduction of the STAT3 CDS led to marked reversal of the inhibition of cell migration in HBV-infected cells mediated by miR-340-5p.ConclusionsHepatitis B virus promotes the migration of liver cancer cells by downregulating miR-340-5p expression to induce STAT3 overexpression. Our results show that STAT3 plays a key role in regulating cell migration in HBV–HCC involving miR-340-5p.

Tissue-Specific Upregulation of MDS/EVI Gene Transcripts in the Intestine by Thyroid Hormone during Xenopus Metamorphosis

Background Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells. Methodology/Principal Findings The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium. Conclusions/Significance Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells.

The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription

RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65.

Characterization and selective incorporation of small non-coding RNAs in non-small cell lung cancer extracellular vesicles

BackgroundExtracellular vesicles (EVs) play important roles in intercellular communication through the delivery of their cargoes, which include proteins, lipids, and RNAs. Increasingly, multiple studies have reported the association between EV small non-coding RNAs and cancer, due to their regulatory functions in gene expression. Hence, analysis of the features of small non-coding RNA expression and their incorporation into EVs is important for cancer research.ResultsWe performed deep sequencing to investigate the expression of small RNAs in plasma EVs from lung adenocarcinoma (ADC) patients, lung squamous cell carcinoma (SQCC) patients, and healthy controls. Then, eighteen differently expressed miRNAs in plasma EVs was validated by QRT-PCR. The small RNA expression profiles of plasma EVs were different among lung ADC, SQCC patients, and healthy controls. And many small RNAs, including 5′ YRNA hY4-derived fragments, miR-451a, miR-122-5p, miR-20a-5p, miR-20b-5p, miR-30b-5p, and miR-665, were significantly upregulated in non-small cell lung cancer (NSCLC) EVs. And the cell viability assays indicated that hY4-derived fragments inhibited the proliferation of lung cancer cell A549. By comparing the cellular and EV expression levels of six miRNAs in NSCLC cells, we found that miR-451a and miR-122-5p were significantly downregulated in NSCLC cell lysates, while significantly upregulated in NSCLC EVs.ConclusionsThe differently expressed EV small RNAs may serve as potential circulating biomarkers for the diagnosis of NSCLC. Particularly, YRNA hY4-derived fragments can serve as a novel class of biomarkers, which function as tumor suppressors in NSCLC. Additionally, miR-451a and miR-122-5p may be sorted into NSCLC EVs in a selective manner.

Bivalent Copper Ions Promote Fibrillar Aggregation of KCTD1 and Induce Cytotoxicity

Potassium channel tetramerization domain containing 1 (KCTD1) family members have a BTB/POZ domain, which can facilitate protein-protein interactions involved in the regulation of different signaling pathways. KCTD proteins have potential Zn2+/Cu2+ binding sites with currently unknown structural and functional roles. We investigated potential Cu2+-specific effects on KCTD1 using circular dichroism, turbidity measurement, fluorescent dye binding, proteinase K (PK) digestion, cell proliferation and apoptosis assays. These experiments indicate that the KCTD1 secondary structure assumes greater β-sheet content and the proteins aggregate into a PK-resistant form under 20 μM Cu2+, and this β-sheet-rich aggregation with Cu2+ promotes fibril formation, which results in increased cell toxicity by apoptosis. Our results reveal a novel role for Cu2+ in determining the structure and function of KCTD1.