ZFP36 loss-mediated BARX1 stabilization promotes malignant phenotypes by transactivating master oncogenes in NSCLC.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, with high morbidity and mortality worldwide. Although the dysregulation of BARX1 expression has been shown to be associated with malignant cancers, including NSCLC, the underlying mechanism remains elusive. In this study, we identified BARX1 as a common differentially expressed gene in lung squamous cell carcinoma and adenocarcinoma. Importantly, we uncovered a novel mechanism behind the regulation of BARX1, in which ZFP36 interacted with 3'UTR of BARX1 mRNA to mediate its destabilization. Loss of ZFP36 led to the upregulation of BARX1, which further promoted the proliferation, migration and invasion of NSCLC cells. In addition, the knockdown of BARX1 inhibited tumorigenicity in mouse xenograft. We demonstrated that BARX1 promoted the malignant phenotypes by transactivating a set of master oncogenes involved in the cell cycle, DNA synthesis and metastasis. Overall, our study provides insights into the mechanism of BARX1 actions in NSCLC and aids a better understanding of NSCLC pathogenesis.

The m6A methyltransferase METTL3 modifies PGC-1α mRNA promoting mitochondrial dysfunction and oxLDL-induced inflammation in monocytes.

Mitochondrial biogenesis and energy metabolism are essential for regulating the inflammatory state of monocytes. This state is partially controlled by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a coactivator that regulates mitochondrial biogenesis and energy metabolism. Disruption of these processes can also contribute to the initiation of chronic inflammatory diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis. Methyltransferase-like 3 (METTL3)-dependent N6-methyladenosine (m6A) methylation has recently been shown to regulate a variety of inflammatory processes. However, the role of m6A mRNA methylation in affecting mitochondrial metabolism in monocytes under inflammation is unclear, nor is there an established relationship between m6A methylation and PGC-1α. In this study, we identified a novel mechanism by which METTL3 acts during oxidized low-density lipoprotein (oxLDL)-induced monocyte inflammation, where METTL3 and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) cooperatively modify PGC-1α mRNA, mediating its degradation, decreasing PGC-1α protein levels, and thereby enhancing the inflammatory response. METTL3 coordinated with YTHDF2 to suppress the expression of PGC-1α, as well as that of cytochrome c (CYCS) and NADH:ubiquinone oxidoreductase subunit C2 (NDUFC2) and reduced ATP production and oxygen consumption rate (OCR). This subsequently increased the accumulation of cellular and mitochondrial reactive oxygen species (ROS) and the levels of proinflammatory cytokines in inflammatory monocytes. These data may provide new insights into the role of METTL3-dependent m6A modification of PGC-1α mRNA in the monocyte inflammation response. These data also contribute to a more comprehensive understanding of the pathogenesis of monocyte-macrophage inflammation-associated diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis.

Incorporation of classical scientific research stories into traditional lecture classes to promote the active learning of students.

Biochemistry is an important curriculum for all medical students but has long been considered obscure and of very little or indirect relevance to medical or clinical practice, which markedly diminishes the enthusiasm and motivation of medical students in learning. Biochemistry teachers always face a tremendous challenge to deliver an attractive and high-quality lecture class. Inspired by convincing studies that show numerous benefits of undergraduate research, we tried to modify our teaching method in the past 5 years by incorporating classical scientific research stories into our traditional lecture class, such as the discovery of the semi-conservative DNA replication, telomeric DNA and telomerase, the tricarboxylic acid cycle (TCA cycle), and so on. Through this story-based teaching, we not only helped them deeply understand the textbook content, but also introduced the process of real scientific research to the students in an interesting way. Our efforts aim to combine the delivery of knowledge with the inspiration of students' active learning. We found that most students involved in our classes responded positively. As described in the survey, they were strongly attracted by those research stories; rather than feeling bored about the Biochemistry textbook, they experienced curiosity which fostered their active learning. They also learned to appreciate the beauty of science. More importantly, their impression on how the authentic science research was done was instructive for their critical thinking.

A Systematic Analysis of Dysregulated Long Non-Coding RNAs/microRNAs/mRNAs in Lung Squamous Cell Carcinoma.

BACKGROUND: Lung squamous cell carcinoma (LUSC) accounts up for approximately 30% of all lung cancers with a high mortality. The study was aimed at finding genes critical in the diagnosis and prognosis of LUSC. MATERIALS AND METHODS: The differentially expressed (DE) genes (DEGs) and DE lncRNAs (DELs) from 501 LUSC and 49 normal lung tissues, and DE miRNAs (DEMs) from 478 LUSC and 45 normal lung tissues were respectively obtained via the TCGA database. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and co-expression network analyses were performed. Survival analysis and receiver operating characteristic curve of hub mRNAs were also analyzed. Competitive endogenous RNA networks of lncRNAs, miRNAs and mRNAs were constructed. RESULTS: A total of 5747 DEGs, 378 DEMs and 3141 DELs in LUSC were identified in LUSC. The DEGs including AUARK, CDK1, KIF11 and EXO1 were proven to be significant metastatic indicators in LUSC, and 2 DEGs were significantly associated with the survival in LUSC patients. Some genes might have connections with many other gene nodes through a co-expression network. Four lncRNAs, 2 mRNAs and 2 miRNAs were identified as the candidates for the competitive miRNA-mRNA-lncRNA network and might serve as prognostic markers in LUSC. CONCLUSIONS: We identified the differentially expressed lncRNAs, miRNAs and mRNAs in LUSC, providing further insights into the molecular mechanism of LUSC tumorigenesis and the potential prognostic biomarkers or therapeutic targets for LUSC.

Hepatic HuR modulates lipid homeostasis in response to high-fat diet.

Lipid transport and ATP synthesis are critical for the progression of non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) forms complexes with NAFLD-relevant transcripts. It associates with intron 24 of Apob pre-mRNA, with the 3'UTR of Uqcrb, and with the 5'UTR of Ndufb6 mRNA, thereby regulating the splicing of Apob mRNA and the translation of UQCRB and NDUFB6. Hepatocyte-specific HuR knockout reduces the expression of APOB, UQCRB, and NDUFB6 in mice, reducing liver lipid transport and ATP synthesis, and aggravating high-fat diet (HFD)-induced NAFLD. Adenovirus-mediated re-expression of HuR in hepatocytes rescues the effect of HuR knockout in HFD-induced NAFLD. Our findings highlight a critical role of HuR in regulating lipid transport and ATP synthesis.

MiR-509-3-5p-NONHSAT112228.2 Axis Regulates p21 and Suppresses Proliferation and Migration of Lung Cancer Cells.

BACKGROUND: Although the involvement of individual microRNA and lncRNA in the regulation of p21 expression has largely been evidenced, less is known about the roles of functional interactions between miRNAs and lncRNAs in p21 expression. Our previous work demonstrated that miR-509- 3-5p could block cancer cell growth. METHODS: To gain an insight into the role of miR-509-3-5p in the regulation of p21 expression, we performed in silico prediction and showed that miR-509-3-5p might target the NONHSAT112228.2, a sense-overlapping lncRNA transcribed by a non-code gene overlapping with p21 gene. Mutation and luciferase report analysis suggested that miR-509-3-5p could target NONHSAT112228.2, thereby blocking its expression. Consistently, NONHSAT112228.2 expression was inversely correlated with both miR-509-3-5p and p21 expression in cancer cells. Ectopic expression of miR-509-3-5p and knockdown of NONHSAT112228.2 both promoted proliferation and migration of cancer cells. RESULTS: Interestingly, high-expression of NONHSAT112228.2 accompanied by low-expression of p21 was observed in lung cancer tissues and associated with lower overall survival. CONCLUSION: Taken together, our study found a new regulatory pathway of p21, in which MiR-509-3-5p functionally interacts with NONHSAT112228.2 to release p21 expression. MiR-509-3-5p- NONHSAT112228.2 regulatory axis can inhibit the proliferation and migration of lung cancer cells.

Influence of critical thinking disposition on the learning efficiency of problem-based learning in undergraduate medical students.

BACKGROUND: Problem-based learning (PBL), a pedagogical approach, is widely accepted in medical education. Manipulated by many factors, the internal motivation of learner is the most crucial determinant that affects the nature of the outcome, in which the influences of critical thinking (CT) remained elusive. METHODS: One hundred two third-year undergraduate medical students at Peking University were involved in this study. A Chinese version of the Critical Thinking Disposition Inventory (CTDI-CV) was used to assess the CT disposition, and the performance scores of students in PBL tutorials were compiled. A parametric bivariate correlation analysis was performed between the students' CT scores and their PBL average scores. The PBL scores were compared between the strong and weak CT disposition groups using independent t-test. The analysis of numerical data was conducted using SPSS 16.0. RESULTS: CT disposition of third-year undergraduate medical students at Peking University was at a positive level, with an average score of 297.72. The total CT scores had a positive correlation with the scores of the PBL performance and its five dimensions significantly. In the majority, students with Strong-CT disposition obtained higher scores in PBL tutorials compared with students with Weak-CT disposition. The performance of these two groups was significantly different in the Late-Half but not in the Early-Half PBL tutorials. Furthermore, a significant improvement was observed in the students with strong CT but not weak CT dispositions. CONCLUSION: CT disposition positively correlates to a students' PBL performance. Students with stronger CT dispositions perform better in the PBL process and obtain higher scores. Our work suggested that the open-mindedness of the CT disposition is the primary factor that determines the improvement of the preparation dimensions in the PBL process.

DNA Damage-Response Pathway Heterogeneity of Human Lung Cancer A549 and H1299 Cells Determines Sensitivity to 8-Chloro-Adenosine.

Human lung cancer H1299 (p53-null) cells often display enhanced susceptibility to chemotherapeutics comparing to A549 (p53-wt) cells. However, little is known regarding to the association of DNA damage-response (DDR) pathway heterogeneity with drug sensitivity in these two cells. We investigated the DDR pathway differences between A549 and H1299 cells exposed to 8-chloro-adenosine (8-Cl-Ado), a potential anticancer drug that can induce DNA double-strand breaks (DSBs), and found that the hypersensitivity of H1299 cells to 8-Cl-Ado is associated with its DSB overaccumulation. The major causes of excessive DSBs in H1299 cells are as follows: First, defect of p53-p21 signal and phosphorylation of SMC1 increase S phase cells, where replication of DNA containing single-strand DNA break (SSB) produces more DSBs in H1299 cells. Second, p53 defect and no available induction of DNA repair protein p53R2 impair DNA repair activity in H1299 cells more severely than A549 cells. Third, cleavage of PARP-1 inhibits topoisomerase I and/or topoisomerase I-like activity of PARP-1, aggravates DNA DSBs and DNA repair mechanism impairment in H1299 cells. Together, DDR pathway heterogeneity of cancer cells is linked to cancer susceptibility to DNA damage-based chemotherapeutics, which may provide aid in design of chemotherapy strategy to improve treatment outcomes.

MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells.

MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3'-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53(-/-) cancer cells, in which PLK1 protein was suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage.

The experimental teaching reform in biochemistry and molecular biology for undergraduate students in Peking University Health Science Center.

Since 2010, second-year undergraduate students of an eight-year training program leading to a Doctor of Medicine degree or Doctor of Philosophy degree in Peking University Health Science Center (PKUHSC) have been required to enter the "Innovative talent training project." During that time, the students joined a research lab and participated in some original research work. There is a critical educational need to prepare these students for the increasing accessibility of research experience. The redesigned experimental curriculum of biochemistry and molecular biology was developed to fulfill such a requirement, which keeps two original biochemistry experiments (Gel filtration and Enzyme kinetics) and adds a new two-experiment component called "Analysis of anti-tumor drug induced apoptosis." The additional component, also known as the "project-oriented experiment" or the "comprehensive experiment," consists of Western blotting and a DNA laddering assay to assess the effects of etoposide (VP16) on the apoptosis signaling pathways. This reformed laboratory teaching system aims to enhance the participating students overall understanding of important biological research techniques and the instrumentation involved, and to foster a better understanding of the research process all within a classroom setting. Student feedback indicated that the updated curriculum helped them improve their operational and self-learning capability, and helped to increase their understanding of theoretical knowledge and actual research processes, which laid the groundwork for their future research work.

E2F1 regulates p53R2 gene expression in p53-deficient cells.

The p53R2 gene encoding a small subunit of the ribonucleotide reductase has been identified as a p53-inducible gene. Although this gene is discovered as a target for p53 family proteins, the mechanism underlying p53R2 induction by DNA damage in p53-defiencient cells remains to be elucidated. In this study, we demonstrate that transcription factor E2F1 regulates the p53R2 gene expression in p53-deficient cells. We found that p53R2 was a target for E2F1 in DNA damage response (DDR), because ectopic expression of E2F1 in HCT116-p53(-/-) cells resulted in the increase of p53R2 mRNA and protein expression, and silencing E2F1 diminished its basic expression. Combination of luciferase reporter assay with overexpression or knockdown of E2F1 revealed that E2F1 directly activates the p53R2 gene. Chromatin immunoprecipitation (ChIP) assay showed E2F1 directly bound to the site (TTTGGCGG) at position -684 to -677 of the promoter under E2F1 overexpression or adriamycin (ADR) exposure. Moreover, silencing p53R2 could enhance apoptotic cell death in both HCT116-p53(-/-) and HCT116-p53(+/+) compared to ADR exposure, indicating that p53R2 may protect cancer cell from ADR-induced apoptosis. Together, we have identified a new role of E2F1 in the regulation of p53R2 expression in DDR, and silencing p53R2 may sensitize cancer cells to ADR-induced apoptosis. Our data support the notion that p53R2 is a potential target for cancer therapy. The involvement of E2F1-dependent p53R2 activation in DDR will provide further insight into the induction of p53R2 in p53-deficient cells. These data also give us a deeper understanding of E2F1 role in DDR.

E2F1-regulated DROSHA promotes miR-630 biosynthesis in cisplatin-exposed cancer cells.

DNA damage may regulate microRNA (miRNA) biosynthesis at the levels of miRNA transcription, processing and maturation. Although involvement of E2F1 in the regulation of miRNA gene activation in response to DNA damage has been documented, little is known about the role of E2F1 in miRNA processing. In this study we demonstrate that E2F1 enhances miR-630 biosynthesis under cisplatin (CIS) exposure through promoting DROSHA-mediated pri-miR-630 processing. Northern blot and RT-qPCR revealed that CIS exposure caused not only an increase in pri-miR-630 but also much more increase in pre-miR-630 and mature miR-630. The increases in pri-miR-630 and pre-miR-630 expression in unmatched proportion indicated that primary transcript processing was involved in CIS-stimulated miR-630 biosynthesis. Furthermore, combination of reporter enzyme assay with mutation and over-expression of E2F1 showed that induction of DROSHA promoted miR-630 expression, in which CIS-induced E2F1 activated DROSHA gene expression by recognizing and binding two E2F1 sites at the positions -214/-207 and -167/-160 of the DROSHA promoter. The increased binding of E2F1 to the DROSHA promoter in CIS-exposed cells was further evidenced by chromatin immunoprecipitation assay. Together, E2F1-regulated DROSHA promotes pri-miR-630 processing, thereby, contributes to CIS-stimulated miR-630 expression. The involvement of E2F1-dependent DROSHA activation in pri-miRNA processing under DNA damage stress will provide further insight into the regulation of miRNA biosynthesis. These data also give us a deeper understanding of E2F1 role in response to DNA damage.

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

p53 Suppresses E2F1-dependent PLK1 expression upon DNA damage by forming p53-E2F1-DNA complex.

E2F1 is implicated in transcriptional activation of polo-like kinase-1 (PLK1), but yet the mechanism is not fully understood. PLK1 suppression plays an important checkpoint role in response to DNA damage. Suppression of the PLK1 gene by binding of p53 to upstream p53RE2 element in the promoter has been recently revealed. Here we report another mechanism, in which p53 interacts with E2F1 to form p53-E2F1-DNA complex repressing E2F1-dependent PLK1 expression. PLK1 was downregulated in cisplatin exposed HCT116p53(+/+) but not HCT116p53(-/-) cells, indicating p53-suppressed PLK1 upon DNA damage. Co-transfection and reporter enzyme assays revealed that p53 suppressed but E2F1 promoted PLK1 gene activation. 5'-Deletion and substitution mutations showed multiple positive cis-elements residing in the PLK1 promoter, of which at least two E2F1 sites at positions -75/-68 and -40/-32 were required for the full activity of the promoter. Combination of 5'-deletion and substitution mutations with over-expression of p53 showed that suppression of the PLK1 gene by p53 was E2F1-dependent: mutation of the E2F1 site at position -75/-68 partially abrogated suppression activity of p53; mutation of E2F1 site at position -40/-32 released from p53 suppression of PLK1 gene completely. Co-immunoprecipitation and electrophoretic mobility shift assay showed that DNA damage promoted p53-E2F1 interaction, thereby creating a p53-E2F1 complex assembly on the PLK1 promoter in vitro. The in vivo formation of p53-E2F1-PLK1 promoter complex upon DNA damage was further evidenced by chromatin immunoprecipitation (ChIP) and re-ChIP. In addition, we showed that suppression of PLK1 by p53 promoted apoptosis. Our data suggest that p53 may interact with E2F1 to form p53-E2F1-DNA complex suppressing E2F1-dependent PLK1 expression. The model of p53 action on E2F1-activated PLK1 gene may explain at least partly how p53 as a suppressor regulates the downstream effects of E2F1 in cellular stresses including DNA damage stress.

E2F1-mediated DNA damage is implicated in 8-Cl-adenosine-induced chromosome missegregation and apoptosis in human lung cancer H1299 cells.

Although E2F1-mediated DNA double-stranded breaks (DSBs) and tetraploid have been extensively studied, the role of E2F1 in mitotic catastrophe is still unknown. We have previously shown that 8-chloro-adenosine (8-Cl-Ado) induces DNA DSBs and aberrant mitosis in human lung cancer cells, followed by delayed apoptosis. Here, we demonstrate that E2F1-mediated DNA damage is implicated in 8-Cl-Ado-induced chromosome missegregation and apoptosis in lung cancer H1299 cells. We showed that E2F1 was accumulated upon 8-Cl-Ado-induced DNA DSBs. Induction of E2F1 by 8-Cl-Ado caused DNA damage in cycling cells including M cells. In contrast, silencing of E2F1 expression decreased 8-Cl-Ado-induced DNA DSBs, particularly eliminated E2F1-mediated mitotic DNA damage. Over-expression of E2F1 and/or 8-Cl-Ado exposure resulted in aberrant mitotic spindles and chromosome segregation errors. Furthermore, over-expression of E2F1 expression enhanced 8-Cl-Ado-induced apoptosis. Together, our data indicate that E2F1-mediated DNA damage, in particular mitotic DNA damage, is an important fraction of 8-Cl-Ado-induced DNA damage, which is implicated in 8-Cl-Ado-induced mitotic catastrophe and delayed apoptosis. Induction of E2F1 by 8-Cl-Ado may contribute at least partly to the drug-inhibited proliferation of cancer cells.

Teaching arrangements of carbohydrate metabolism in biochemistry curriculum in Peking University Health Science Center.

Biochemistry occupies a unique place in the medical school curricula, but the teaching of biochemistry presents certain challenges. One of these challenges is facilitating students' interest in and mastery of metabolism. The many pathways and modes of regulation can be overwhelming for students to learn and difficult for professors to teach in an engaging manner. The first chapter of the metabolism section in current Chinese biochemistry textbooks covers carbohydrate metabolism. Medical students usually complain about the difficulty of this subject. Here we discuss how to facilitate learning by rearranging the subjects in this introductory chapter of biochemical metabolism and to lay a solid foundation for future study. The strategy involves reorganizing the order in which subjects are taught from simple to complex and from short to long metabolic pathways. Most students taking the curriculum consider that the strategy engages their learning interests in biochemistry and enhances their learning outcomes.

Comparison of the biological response of osteoblasts after tension and compression.

The aim of this study was to investigate the difference in the biological response of osteoblasts when stretched and compressed. A cellular cyclic tension and compression apparatus (CCTCA) was designed to stretch and compress cells under the same conditions. After stretching or compressing MC3T3-E1 with continuously increased strain for 5 hours, cellular cytoskeletal modulation was detected by immunohistochemical assay with actin antibody. Real-time polymerase chain reaction was performed at 1, 3, and 5 hours to detect local factors related to bone remodelling. Statistical analysis was undertaken with analysis of variance and the Kruskal-Wallis. Following stretching or compression for 5 hours, MC3T3-E1 attached to the culture dishes grew well. Compared with the control, the microfilaments orientated parallel with each other and were clearly observed by laser scanning confocal microscope after 5 hours of stretching. The morphology of MC3T3-E1 cells was thinner and longer than the control. However, microfilaments presented a disordered arrangement after 5 hours of compression, and the MC3T3-E1 cells decreased in size. Gene expression of Wnt10b and Lrp5 increased during tension but more in the compression groups at 1, 3, and 5 hours. The ratio of osteoprotegerin to receptor activator for nuclear factor kappa B ligand increased in the tension group compared with the control but decreased in the compression group at 5 hours.

Effects of myogenin on expression of late muscle genes through MyoD-dependent chromatin remodeling ability of myogenin.

MyoD and myogenin (Myog) recognize sets of distinct but overlapping target genes and play different roles in skeletal muscle differentiation. MyoD is sufficient for near-full expression of early targets, while Myog can only partially enhance expression of MyoD-initiated late muscle genes. However, the way in which Myog enhances the expression of MyoD-initiated late muscle genes remains unclear. Here, we examine the effects of Myog on chromatin remodeling at late muscle gene promoters and their activation within chromatin environment. Chromatin immunoprecipitation (ChIP) assay showed that Myog selectively bound to the regulatory sequences of late muscle genes. Overexpression of Myog was found to overcome sodium butyrateinhibited chromatin at late muscle genes in differentiating C2C12 myoblasts, shifting the transcriptional activation of these genes to an earlier time period. Furthermore, overexpression of Myog led to increased hyperacetylation of core histone H4 in differentiating C2C12 myoblasts but not NIH3T3 fibroblasts, and hyperacetylated H4 was associated directly with the late muscle genes in differentiating C2C12, indicating that Myog can induce chromatin remodeling in the presence of MyoD. In addition, co-immunoprecipitation (CoIP) revealed that Myog was associated with the nuclear protein Brd4 in differentiating C2C12 myoblasts. Together, these results suggest that Myog enhances the expression of MyoD-initiated late muscle genes through MyoD-dependent ability of Myog to induce chromatin remodeling, in which Myog-Brd4 interaction may be involved.

E2F1 enhances 8-chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells.

The E2F1 transcription factor is a well known regulator of cell proliferation and apoptosis, but its role in response to DNA damage is less clear. 8-Chloro-adenosine (8-Cl-Ado), a nucleoside analog, can inhibit proliferation in a variety of human tumor cells. However, it is still elusive how the agent acts on tumors. Here we show that A549 and H1299 cells formed DNA double-strand breaks after 8-Cl-Ado exposure, accompanied by E2F1 upregulation at protein level. Overexpressed wild-type (E2F1-wt) colocalized with double-strand break marker γ-H2AX and promoted G2/M arrest in 8-Cl-Ado-exposed A549 and H1299, while expressed S31A mutant of E2F1 (E2F1-mu) significantly reduced ability to accumulate at sites of DNA damage and G2/M arrest, suggesting that E2F1 is required for activating G2/M checkpoint pathway upon DNA damage. Transfection of either E2F1-wt or E2F1-mu plasmid promoted apoptosis in 8-Cl-Ado-exposed cells, indicating that 8-Cl-Ado may induce apoptosis in E2F1-dependent and E2F1-independent ways. These findings demonstrate that E2F1 plays a crucial role in 8-Cl-Ado-induced G2/M arrest but is dispensable for 8-Cl-Ado-induced apoptosis. These data also suggest that the mechanism of 8-Cl-Ado action is complicated.

Local osteoprotegerin gene transfer inhibits relapse of orthodontic tooth movement.

INTRODUCTION: In orthodontic treatment, teeth can relapse after tooth movement without retention. The aim of this study was to evaluate the inhibition effects of local osteoprotegerin (OPG) gene transfer on orthodontic relapse. METHODS: Eighteen male Wistar rats were divided into 3 groups. The maxillary right first molars of all animals were subjected to orthodontic force and moved mesially. Three weeks later, the force was removed, and the teeth relapsed. During the 2-week relapse period, the 3 groups of rats received local OPG gene transfer (experimental group), mock vector transfer (mock group), and no injections (control group). Tooth movement and relapse were measured by using palatal superimpositions of 3-dimensional digital models. Histomorphometric analysis was used to quantify osteoclasts, and microcomputed tomography analysis was done to quantify the alveolar bone and the tibia. RESULTS: Relapse was significantly inhibited and the number of osteoclasts was reduced in the experimental group. On the other hand, bone mineral density and bone volume fraction of alveolar bone were significantly increased. Bone mineral density and bone volume fraction of the tibia showed no significant difference between the groups. CONCLUSIONS: Local OPG gene transfer to periodontal tissues could inhibit relapse after orthodontic tooth movement, through the inhibition of osteoclastogenesis.