Long non-coding RNAs mediate the association between short-term PM2.5 exposure and circulating biomarkers of systemic inflammation.

Although short-term fine particulate matter (PM2.5) exposure is associated with systemic inflammation, the effect of lncRNA on these association remains unknown. This study aims to investigate whether the plasma lncRNA mediate the effect of short-term PM2.5 exposure on systemic inflammation. In this cross-sectional study, plasma Clara cell protein 16 (CC16), interleukin 6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α) and lncRNA expression levels were measured in 161 adults between March and April in 2018 in Shijiazhuang, China. PM2.5 concentrations were estimated 0-3 days prior to the examination date and the moving averages were calculated. Multiple linear regressions were used to evaluate the associations between PM2.5, the four biomarkers and lncRNA expression levels. Mediation analyses were performed to explore the potential roles of lncRNA expression in these associations. The median concentration of PM2.5 ranged from 39.65 to 60.91 mg/m3 across different lag days. The most significant effects on IL-6 and TNF-α per interquartile range increase in PM2.5 were observed at lag 0-3 days, with increases of 0.70 pg/mL (95% CI: 0.33, 1.07) and 0.21 pg/mL (95% CI: 0.06, 0.36), respectively. While the associations between PM2.5 and IL-8 (0.68 pg/mL, 95% CI: 0.34, 1.02) and CC16 (3.86 ng/mL, 95% CI: 1.60, 6.13) were stronger at lag 0 day. Interestingly, a negative association between PM2.5 and the expression of four novel lncRNAs (lnc-ACAD11-1:1, lnc-PRICKLE1-4:1, lnc-GPR39-7:2, and lnc-MTRNR2L12-3:6) were observed at each lag days. Furthermore, these lncRNAs mediated the effects of PM2.5 on the four biomarkers, with proportions of mediation ranged from 2.27% (95% CI: 1.19%, 9.82%) for CC16 to 35.60% (95% CI: 17.16%, 175.45%) for IL-6. Our findings suggested that plasma lncRNA expression mediat the acute effects of PM2.5 exposure on systematic inflammation. These highlight a need to consider circulating lncRNA expression as biomarkers to reduce health risks associated with PM2.5.

circCIMT Silencing Promotes Cadmium-Induced Malignant Transformation of Lung Epithelial Cells Through the DNA Base Excision Repair Pathway.

Changes in gene expression in lung epithelial cells are detected in cancer tissues during exposure to pollutants, highlighting the importance of gene-environmental interactions in disease. Here, a Cd-induced malignant transformation model in mouse lungs and bronchial epithelial cell lines is constructed, and differences in the expression of non-coding circRNAs are analyzed. The migratory and invasive abilities of Cd-transformed cells are suppressed by circCIMT. A significant DNA damage response is observed after exposure to Cd, which increased further following circCIMT-interference. It is found that APEX1 is significantly down-regulated following Cd exposure. Furthermore, it is demonstrated that circCIMT bound to APEX1 during Cd exposure to mediate the DNA base excision repair (BER) pathway, thereby reducing DNA damage. In addition, simultaneous knockdown of both circCIMT and APEX1 promotes the expression of cancer-related genes and malignant transformation after long-term Cd exposure. Overall, these findings emphasis the importance of genetic-epigenetic interactions in chemical-induced cancer transformation.

Upregulation of Circ_0035266 Contributes to the Malignant Progression of Inflammation-Associated Malignant Transformed Cells Induced by Tobacco-Specific Carcinogen NNK.

Cigarette smoking-induced chronic inflammation has been considered a vital driver of lung tumorigenesis. The compounds 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen, and lipopolysaccharide (LPS), an inflammatory inducer, are important components of tobacco smoke which have been implicated in inflammation-driven carcinogenesis. However, the biological effects and underlying mechanisms of LPS-mediated inflammation on NNK-induced tumorigenesis are still unclear. In this study, BEAS-2B human bronchial epithelial cells were exposed to NNK, LPS or both, for short- or long-term periods. We found that acute LPS exposure promoted the secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-6 in NNK-treated BEAS-2B cells. In addition, chronic LPS exposure facilitated the NNK-induced malignant transformation process by promoting cell proliferation, cell cycle alteration, migration, and clonal formation. Previously, we determined that circular RNA circ_0035266 enhanced cellular inflammation in response to NNK + LPS by sponging miR-181d-5p and regulating expression of its downstream target DEAD-Box Helicase 3 X-Linked (DDX3X). Here, we found that knockdown of circ_0035266 or DDX3X led to a remarkable inhibition of the proliferation, cell cycle progression, and migration of NNK + LPS-transformed BEAS-2B cells, whereas overexpression of these genes produced the opposite effects, indicating the oncogenic roles of circ_0035266 and DDX3X in the malignant progression of chronic inflammation-driven malignant transformed cells. Moreover, the regulatory relationships among circ_0035266, miR-181d-5p, and DDX3X were further confirmed using a group of lung cancer tissues. Conclusively, our findings provide novel insights into our understanding of inflammation-driven tumorigenesis using a cellular malignant transformation model, and indicate a novel tumor-promoting role for circ_0035266 in chemical carcinogenesis.

Circular RNA circNIPBL promotes NNK-induced DNA damage in bronchial epithelial cells via the base excision repair pathway.

Environmental chemical exposure often causes DNA damage, which leads to cellular dysfunction and the development of diseases. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen that is known to cause DNA damage, while remains unknown about the underlying mechanism. In this study, simulated doses of NNK exposure in smokers, ranging from 50 to 300 µM, were used to detect the DNA damage effects of NNK in two human bronchial epithelial cells, 16HBE and BEAS-2B. The comet assay revealed increased DNA damage in response to NNK treatment, as measured by increased Olive tail moment (OTM). NNK treatment also led to elevated foci formation and protein expression of γ-H2AX, a DNA damage sensor. Dysregulation of proliferation, cell cycle arrest and apoptosis, was also observed in NNK-treated cells. Furthermore, the most effective dose of NNK (300 µM) was used in subsequent mechanistic studies. A circular RNA circNIPBL was identified to be significantly up-regulated in NNK-treated cells, circNIPBL knockdown successfully alleviated NNK-induced DNA damage and reversed the cellular dysregulation, while circNIPBL overexpression had the opposite effect. Mechanistically, we identified an interaction between circNIPBL and PARP1, a critical enzyme of the base excision repair (BER) pathway. CircNIPBL silencing successfully alleviated the NNK-induced inhibition of BER pathway proteins, including PARP1, XRCC1, PCNA and FEN1, while overexpression of circNIPBL had the opposite effect. In summary, our study shows for the first time that circNIPBL promotes NNK-induced DNA damage and cellular dysfunction through the BER pathway. In addition, our findings reveal the crucial role of epigenetic regulation in carcinogen-induced genetic lesions and further our understanding of environmental carcinogenesis.

Circular RNA circ_Cabin1 promotes DNA damage in multiple mouse organs via inhibition of non-homologous end-joining repair upon PM2.5 exposure.

Fine particulate matter (PM2.5) has been shown to induce DNA damage. Circular RNAs (circRNAs) have been implicated in various disease processes related to environmental chemical exposure. However, the role of circRNAs in the regulation of DNA damage response (DDR) after PM2.5 exposure remains unclear. In this study, male ICR mice were exposed to PM2.5 at a daily mean concentration of 382.18 µg/m3 for 3 months in an enriched-ambient PM2.5 exposure system in Shijiazhuang, China, and PM2.5 collected form Shijiazhuang was applied to RAW264.7 cells at 100 µg/mL for 48 h. The results indicated that exposure to PM2.5 induced histopathological changes and DNA damage in the lung, kidney and spleen of male ICR mice, and led to decreased cell viability, increased LDH activity and DNA damage in RAW264.7 cells. Furthermore, circ_Cabin1 expression was significantly upregulated in multiple mouse organs as well as in RAW264.7 cells upon exposure to PM2.5. PM2.5 exposure also resulted in impairment of non-homologous end joining (NHEJ) repair via the downregulation of Lig4 or Dclre1c expression in vivo and in vitro. Importantly, circ_Cabin1 promoted PM2.5-induced DNA damage via inhibiting of NHEJ repair. Moreover, the expression of circ_Cabin1 and Lig4 or Dclre1c was strongly correlated in multiple mouse organs, as well as in the blood. In summary, our study provides a new perspective on circRNAs in the regulation of DDR after environmental chemical exposure.

The Circ_CARM1 controls cell migration by regulating CTNNBIP1 in anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide-transformed 16HBE cells.

BACKGROUND: Circular RNAs (circRNAs) have an important role in the development and progression of human tumors, including lung cancer. Yet, their role in lung cancer induced by benzo(a)pyrene (B[a]P) remains unclear. In this study, circRNA chips and qRT-PCR were used to examine downregulated circRNAs in malignantly transformed 16HBE cells (16HBE-T) induced by B[a]P. Five down-regulated circRNAs were found, among which hsa_circ_0004552 (circ_CARM1) had the most significant downregulation. Consequently, the role of circ_CARM1 on 16HBE-T cells biological behavior was further examined using several in vitro experiments. MATERIALS AND METHODS: Detecting RNA expression via qRT-PCR. Fluorescence in situ hybridization (FISH) was used to identify the localization of circ_CARM1 in 16HBE-T. The effect of circ_CARM1 on cell behavior (cell migration, proliferation, and apoptosis) was explored by transfecting cells with a vector carrying an overexpression and then using wound healing, transwell migration assay, and flow cytometry. Also, the regulation mechanism for circ_CARM1, miR-1288-3p, and CTNNBIP1 was studied by Dual-Luciferase® Reporter (DLR™) Assay System and western blotting. RESULTS: Reduced expression of circ_CARM1 is observed in 16HBE-T. The overexpression of circ_CARM1 further inhibited the migration of 16HBE-T cells but did not affect cell proliferation and apoptosis. Furthermore, bioinformatic analysis and Dual-Luciferase® Reporter (DLR™) Assay System showed that the competitive binding of circ_CARM1 and miR-1288-3p enhanced the expression of CTNNBIP1, thereby inhibiting the migration of 16HBE-T cells. CONCLUSION: Downregulation of circ_CARM1 can stimulate the expression of miR-1288-3p, thereby reducing the expression of CTNNBIP1, spurring cell migration.

Circular RNA circBbs9 promotes PM2.5-induced lung inflammation in mice via NLRP3 inflammasome activation.

Fine particulate matter (PM2.5) is one of the most important components of environmental pollutants, and is associated with pulmonary injury. However, the biological mechanisms of pulmonary damage caused by PM2.5 are poorly defined, especially the molecular pathways related to inflammation. Following system exposure to PM2.5 for 3 months in normal mice and in chronic obstructive pulmonary disease (COPD) model mice, it was found that PM2.5 exposure increased the expression of IL-1ß and IL-18 in lung tissues via NLRP3 activation, and these effects were more intense in COPD model mice. Circular RNA (circRNA) sequencing showed that the expression profiles of circRNAs were changed after PM2.5 exposure, and the positive roles of circBbs9 in inflammation induced by PM2.5 were verified. The circBbs9 knockdown alleviated PM2.5-induced inflammation via NLRP3 inflammasome inactivation, as well as IL-1ß and IL-18 inhibition in RAW264.7 cells, while overexpression of circBbs9 had the opposite effect. Bioinformatics and luciferase reporter assays showed that circBbs9 bound to microRNA-30e-5p (miR-30e-5p) and co-regulated the expression of Adar, a downstream target gene of miR-30e-5p. Taken together, these results revealed that PM2.5 induced pulmonary inflammation through NLRP3 inflammasome activation regulated by the circBbs9-miR-30e-5p-Adar pathway. Our findings provide a new target, circBbs9, for the assessment of lung inflammation and COPD exacerbation induced by PM2.5 exposure.

Circular RNA 406961 interacts with ILF2 to regulate PM2.5-induced inflammatory responses in human bronchial epithelial cells via activation of STAT3/JNK pathways.

Fine particulate matter (PM2.5) has been verified to augmented the incidence of pneumonia, asthma, pulmonary fibrosis, and other pulmonary diseases. Airway inflammation is the pathological basis of the respiratory system, and understanding the molecular mechanisms responsible for airway inflammation may thus support the diagnosis and treatment of respiratory diseases. In our study, human bronchial epithelial cells (BEAS-2B) were exposed to various concentrations of PM2.5 for 48 h. PM2.5 entered the cells, resulting in increased production of interleukin 6 (IL-6) and interleukin 8 (IL-8) and decreased the expression of circular RNA 406961 (circ_406961). Further, PM2.5 with a concentration of 75 µg/mL was applied to mechanism study. Functional experiments further confirmed that circ_406961 inhibited PM2.5-induced BEAS-2B cell inflammation. RNA pull-down and mass spectrometry showed that circ_406961 interacted with interleukin enhancer-binding factor 2 (ILF2), which could regulate phosphorylation of signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase 8 (MAPK8, JNK). Our studies showed that circ_406961 inhibited activation of STAT3/JNK pathways via interacting with ILF2 protein, thereby inhibiting the PM2.5-induced inflammatory reaction.

LncRNA LOC101927514 regulates PM2.5-driven inflammation in human bronchial epithelial cells through binding p-STAT3 protein.

Long-term exposure to fine particulate matter (PM2.5) may cause or exacerbate many diseases, including respiratory inflammation. However, the full mechanism is not yet fully understood. The newly discovered long chain non-coding RNA, though unable to encode proteins, regulates multiple life activities and participates in the development of inflammation. In this study, we set up a cell inflammation model by using normal bronchial 16HBE cells exposed to PM2.5. High-throughput sequencing, as well as real-time fluorescent quantitative PCR detection and validation, was performed on the inflamed cells to evaluate the expression level of long chain noncoding RNA that helped us to identify the LncRNA LOC101927514. Inhibiting LncRNA LOC101927514 expression by RNAi, reflected in a reduction in inflammation, is driven by PM2.5. In addition, we identify LncRNA LOC101927514 to be located within the nucleus and binds to STAT3, altering the inflammatory state of the cells and IL6 and IL8 release. This study identifies that LncRNA LOC101927514 is a new potential target for future treatment of the inflammatory response activated by PM2.5 in the respiratory system.

CircRNA104250 and lncRNAuc001.dgp.1 promote the PM2.5-induced inflammatory response by co-targeting miR-3607-5p in BEAS-2B cells.

Long-term exposure to particulate matter 2.5 (PM2.5) is closely related to the occurrence and development of airway inflammation. Exploration of the role of PM2.5 in inflammation is the first step towards clarifying the harmful effects of particulate pollution. However, the molecular mechanisms underlying PM2.5-induced airway inflammation are yet to be fully established. In this study, we focused on the specific roles of non-coding RNAs (ncRNAs) in PM2.5-induced airway inflammation. In a human bronchial epithelial cell line, BEAS-2B, PM2.5 at a concentration of 75 µg/mL induced the inflammatory response. Microarray and quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed significant upregulation of circRNA104250 and lncRNAuc001.dgp.1 during the PM2.5-induced inflammatory response in this cell line. Data from functional analyses further showed that both molecules promote an inflammatory response. CircRNA104250 and lncRNAuc001.dgp.1 target miR-3607-5p and affect expression of interleukin 1 receptor 1 (IL1R1), which influences the nuclear factor κB (NF-κB) signaling pathway. In summary, we have uncovered an underlying mechanism of airway inflammation by PM2.5 involving regulation of ncRNA for the first time, which provides further insights into the toxicological effects of PM2.5.

Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase iota.

Among human four Y-family DNA polymerases, hPol ι is exceptionally error-prone in DNA synthesis. 6 mA plays significant roles in epigenetic regulation of numerous biological processes. Nonetheless, its effects on DNA replication by hPol ι is still unclear. In this work, we found that 6 mA and Hyp, the intermediate of 6 mA, inhibited the replication of DNA by hPol ι. 6 mA lost priority in extension beyond 6 mA:T pair, partially reducing dTTP incorporation efficiency and inhibiting next-base extension. Hyp was prone to dCTP incorporation and extension beyond Hyp:C instead of Hyp:T pair. Statistically, 6 mA primarily reduced the burst incorporation rate (kpol) and slightly increased the dissociation constant (Kd,dTTP). However, Hyp mainly increased the Kd,dCTP yet did not affect the kpol, both reducing the burst incorporation efficiency (kpol/Kd,dCTP). 6 mA together with Hyp weakened the binding affinity of hPol ι to DNA in binary or ternary complex. The misincorporation opposite 6 mA or Hyp further weakened this binding affinity. The methyl group in 6 mA doesn't almost affect the H-bond formation with dTTP, therefore mildly inhibiting dTTP incorporation. As an analogue of G, Hyp can form only two H-bonds with dCTP, thus reducing dCTP incorporation. This work provides a new insight in how the epigenetically modified 6 mA and its intermediate Hyp affect replication of DNA by human DNA polymerase ι.

Accuracy Evaluation of Circular RNA in Diagnosing Lung Cancer in a Chinese Population.

Circular RNA (circRNA) is a class of recently discovered noncoding RNA. circRNAs can be used as a potent noninvasive biological marker of cancer owing to their varying expression levels among different cancers. This meta-analysis was performed to assess the accuracy of circRNAs in diagnosing lung cancer. A total of eight studies identified through searching the PubMed, Web of Science, Cochrane Library, and Embase from inception to March 20, 2019 were eligible for this meta-analysis. The pooled sensitivity, specificity, positive likelihood ratios, negative likelihood ratios, and diagnostic odds ratio were 0.77 (95% confidence interval (CI): 0.73-0.80; I 2 = 8.98%), 0.76 (95% CI: 0.69-0.82; I 2 = 63.12%), 3.17 (95% CI: 2.43-4.14; I 2 = 33.18%), 0.31 (95% CI: 0.26-0.37; I 2 = 20.36%), and 10.26 (95% CI: 6.87-15.31; I 2 = 97.18%), respectively. The area under the receiver operating characteristic curve was 0.78 (95% CI: 0.74-0.81). The study confirmed the use of circRNAs in diagnosing lung cancer in a Chinese population.

Epigenetic DNA modification N6-methyladenine inhibits DNA replication by Sulfolobus solfataricus Y-family DNA polymerase Dpo4.

Dpo4 is a representative model of Y-family DNA polymerase and is therefore one of the most intensively studied DNA polymerase. 6 mA, an epigenetic marker, plays important roles in regulation of various biological processes. However, its effects on DNA replication by Dpo4 is completely unknown. Here, we found that 6 mA and its intermediate Hyp inhibits primer extension by Dpo4, showing an obvious blockage just one nucleotide before 6 mA or Hyp. 6 mA reduces dTTP incorporation efficiency, next-base extension efficiency, binding affinity of DNA to Dpo4, binding affinity of dTTP to Dpo4-DNA complex, the fraction of productive Dpo4 or productive ternary complex, and the burst incorporation rate, explaining the inhibition effects of 6 mA on DNA replication by Dpo4. Hyp is similar to G and dCTP is preferentially incorporated opposite Hyp by Dpo4, resulting in A:T to G:C mutation. Relative to dTTP incorporation opposite unmodified A, Hyp reduces dCTP incorporation efficiency, next-base extension efficiency, the priority in extension beyond correct pair, binding affinity of Dpo4 to DNA, binding of dCTP to Dpo4-DNA complex, and the burst incorporation efficiency, explaining the inhibition effects of Hyp on DNA replication by Dpo4. This work provides insight in the effects of epigenetically modified 6 mA and Hyp on DNA replication by a representative Y-family DNA polymerase Dpo4.

Ribonucleoside triphosphates promote T7 DNA replication and the lysis of T7-Infected Escherichia coli.

rNTPs are structurally similar to dNTPs, but their concentrations are much higher than those of dNTPs in cells. rNTPs in solutions or rNMP at the primer terminus or embedded in template always inhibit or block DNA replication, due to the reduced Mg2+ apparent concentration, competition of rNTPs with dNTPs, and the extra repulsive interaction of rNTP or rNMP with polymerase active site. In this work, unexpectedly, we found rNTPs can promote T7 DNA replication with the maximal promotion at rNTPs/dNTPs concentration ratio of 20. This promotion was not due to the optimized Mg2+ apparent concentration or the direct incorporation of extra rNMPs into DNA. This promotion was dependent on the concentrations and types of rNTPs. Kinetic analysis showed that this promotion was originated from the increased fraction of polymerase-DNA productive complex and the accelerated DNA polymerization. Further evidence showed that more polymerase-DNA complex was formed and their binding affinity was also enhanced in the presence of extra rNTPs. Moreover, this promotion in T7 DNA replication also accelerated the lysis of T7-infected host Escherichia coli. This work discovered that rNTPs could promote DNA replication, completely different from the traditional concept that rNTPs always inhibit DNA replication.

Protein interactions in T7 DNA replisome inhibit the bypass of abasic site by DNA polymerase.

Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme-DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome.

Epigenetic DNA Modification N6-Methyladenine Inhibits DNA Replication by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1.

N6-methyladenine (6mA), a newly identified epigenetic modification, plays important roles in regulation of various biological processes. However, the effect of 6mA on DNA replication has been little addressed. In this work, we investigated how 6mA affected DNA replication by DNA polymerase of Pseudomonas aeruginosa Phage PaP1 (gp90 exo-). The presence of 6mA, as well as its intermediate hypoxanthine (Hyp), inhibited DNA replication by gp90 exo-. The 6mA reduced dTTP incorporation efficiency by 10-fold and inhibited next-base extension efficiency by 100-fold. Differently, dCTP was preferentially incorporated opposite Hyp among four dNTPs. Gp90 exo- reduced the extension priority beyond the 6mA:T pair rather than the 6mA:C mispair and preferred to extend beyond Hyp:C rather than the Hyp:T pair. Incorporation of dTTP opposite 6mA and dCTP opposite Hyp showed fast burst phases. The burst rate and burst amplitude were both reduced for 6mA compared with unmodified A. Moreover, the total incorporation efficiency ( kpol/ Kd,dNTP) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate ( kpol), and Hyp increased the dissociation constant ( Kd,dNTP). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight into the inhibited effects of epigenetic modification of 6mA on DNA replication in PaP1.

Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase η.

N6-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication remain elusive. In this work, we used Y-family human DNA polymerase η as a model to investigate the kinetics of bypass of 6mA by hPol η. We found 6mA and its intermediate hypoxanthine (I) on template partially inhibited DNA replication by hPol η. dTMP incorporation opposite 6mA and dCMP incorporation opposite I can be considered as correct incorporation. However, both 6mA and I reduced correct incorporation efficiency, next-base extension efficiency, and the priority in extension beyond correct base pair. Both dTMP incorporation opposite 6mA and dCTP opposite I showed fast burst phases. However, 6mA and I reduced the burst incorporation rates (kpol) and increased the dissociation constant (Kd,dNTP), compared with that of dTMP incorporation opposite unmodified A. Biophysical binding assays revealed that both 6mA and I on template reduced the binding affinity of hPol η to DNA in binary or ternary complex compared with unmodified A. All the results explain the inhibition effects of 6mA and I on DNA replication by hPol η, providing new insight in the effects of epigenetically modified 6mA on human DNA replication.

Circular RNA circNOL10 Inhibits Lung Cancer Development by Promoting SCLM1-Mediated Transcriptional Regulation of the Humanin Polypeptide Family.

circNOL10 is a circular RNA expressed at low levels in lung cancer, though its functions in lung cancer remain unknown. Here, the function and molecular mechanism of circNOL10 in lung cancer development are investigated using in vitro and in vivo studies, and it is shown that circNOL10 significantly inhibits the development of lung cancer and that circNOL10 expression is co-regulated by methylation of its parental gene Pre-NOL10 and by splicing factor epithelial splicing regulatory protein 1 (ESRP1). circNOL10 promotes the expression of transcription factor sex comb on midleg-like 1 (SCML1) by inhibiting transcription factor ubiquitination and thus also affects regulation of the humanin (HN) polypeptide family by SCML1. circNOL10 also affects mitochondrial function through regulating the humanin polypeptide family and affecting multiple signaling pathways, ultimately inhibiting cell proliferation and cell cycle progression, and promoting the apoptosis of lung cancer cells, thereby inhibiting lung cancer development. This study investigates the functions and molecular mechanisms of circNOL10 in the development of lung cancer and reveals its involvement in the transcriptional regulation of the HN polypeptide family by SCML1. The results also demonstrate the inhibitory effect of HN on lung cancer cells growth. These findings may identify novel targets for the molecular therapy of lung cancer.

Circular RNA 100146 functions as an oncogene through direct binding to miR-361-3p and miR-615-5p in non-small cell lung cancer.

Circular RNAs are widely expressed in eukaryotic cells and associated with cancer. However, limited studies to date have focused on the potential role of circRNAs in progression of lung cancer. Data from the current investigation showed that circRNA 100146 is highly expressed in non-small cell lung cancer (NSCLC) cell lines and the chemically induced malignant transformed bronchial cell line, 16HBE-T, as well as 40 paired tissue samples of NSCLC. Suppression of circRNA 100146 inhibited the proliferation and invasion of cells and promoted apoptosis. Furthermore, circRNA 100146 could interact with splicing factors and bind miR-361-3p and miR-615-5p to regulate multiple downstream mRNAs. Our collective findings support a role of circRNA 100146 in the development of NSCLC and further demonstrate endogenous competition among circRNA 100146, SF3B3 and miRNAs, providing novel insights into the mechanisms underlying non-small cell lung cancer.

Benzo[a]Pyrene-7, 8-Diol-9, 10-Epoxide Suppresses the Migration and Invasion of Human Extravillous Trophoblast Swan 71 Cells Due to the Inhibited Filopodia Formation and Down-Regulated PI3K/AKT/CDC42/PAK1 Pathway Mediated by the Increased miR-194-3p.

Proper migration and invasion of trophoblast cells into endometrium is vital for successful embryo implantation during early pregnancy. Benzo[a]pyrene-7, 8-diol-9, 10-epoxide (BPDE) is an ultimate carcinogenic product of benzo[a]pyrene (BaP), which causes multiple trophoblast-related diseases. However, the mechanism of BPDE-inhibited migration/invasion of trophoblast cells is still unclear. In this work, we found that BPDE significantly inhibited the filopodia formation and migration/invasion of human trophoblast Swan 71 cells. BPDE up-regulated the level of miR-194-3p, which further inhibited the phosphoinositide 3-kinase (PI3K)/AKT/ cell division cycle 42/ p21 (RAC1) activated kinase 1 signaling pathway and depressed the filophdia formation of Swan71 cells. Addition of 740 Y-P, the activator of phosphoinositide 3-kinase, could stimulate cell migration/invasion, confirming the involvement of this pathway. Knock-down of miR-194-3p up-regulated this pathway and promoted filopodia formation and migration/invasion. Conversely, overexpression of miR-194-3p down-regulated this pathway and inhibited cell migration/invasion. Therefore, miR-194-3p takes important roles in the BPDE-inhibited filopodia formation and cell migration/invasion, providing valuable information in the BPDE-induced dysfunctions of human extravillous trophoblast cells.