Involvement of a AS3MT/c-Fos/p53 signaling axis in arsenic-induced tumor in human lung cells.

Arsenite methyltransferase (AS3MT) is an enzyme that catalyzes the dimethylation of arsenite (+3 oxidation state). At present, the studies on arsenic carcinogenicity mainly focus on studying the polymorphisms of AS3MT and measuring their catalytic activities. We recently showed that AS3MT was overexpressed in lung cancer patients who had not been exposed to arsenic. However, little is known about the molecular mechanisms of AS3MT in arsenite-induced tumorigenesis. In this study, we showed that AS3MT protein expression was higher in the arsenic-exposed population compared to the unexposed population. AS3MT was also overexpressed in human lung adenocarcinoma (A549) and human bronchial epithelial (16HBE) cells exposed to arsenic (A549: 20-60 µmol/L; 16HBE: 2-6 µmol/L) for 48 h. Furthermore, we investigated the effects of AS3MT on cell proliferation and apoptosis using siRNA. The downregulation of AS3MT inhibited the proliferation and promoted the apoptosis of cells. Mechanistically, AS3MT was found to specifically bind to c-Fos, thereby inhibiting the binding of c-Fos to c-Jun. Additionally, the siRNA-mediated knockdown of AS3MT enhanced the phosphorylation of Ser392 in p53 by upregulating p38 MAPK expression. This led to the activation of p53 signaling and the upregulated expression of downstream targets, such as p21, Fas, PUMA, and Bax. Together, these studies revealed that the inorganic arsenic-mediated upregulation of AS3MT expression directly affected the proliferation and apoptosis of cells, leading to arsenic-induced toxicity or carcinogenicity.

Up-regulation of PUMA caused the activation of p53 phosphorylation and acetylation, enhancing the interaction between PUMA and Bcl-X and mediating arsenic-induced apoptosis.

Arsenic is a toxic metalloid vastly dispersed all over the occupational environments, manifesting multiple adverse health issues related to apoptosis. PUMA (p53 up-regulated modulator of apoptosis) is a crucial member of the Bcl-2 protein family and plays a key role in pro-apoptosis. The purpose of this work was to determine whether inorganic arsenic (NaAsO2) and its metabolites influenced the expression of PUMA in vivo and vitro, followed by investigating the mechanisms. RNA was extracted from serum and used to determine the expression of PUMA in vivo. The urine samples performed arsenic speciation analysis. This trial tested three-dose proportions in two cell lines (A549: 20, 40, 60 µM/L; 16HBE: 1.5, 3.0, 4.5 µM/L), respectively. According to the results of qRT-PCR and western blotting, NaAsO2 caused the overexpression of PUMA, not its metabolites. Furthermore, NaAsO2 induced phosphorylation of p53 at Ser315, 376, 392, and Thr55, and acetylation of p53 at K370, 382 with a dose-response relationship, suggesting the contribution of PUMA up-regulation to p53 phosphorylation and acetylation. CCK-8, JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetramethylbenzimi-dazolylcarbocyanine iodide), Hoechst33342/PI and the caspase3 and PARP1 blots were utilized to reveal apoptosis responding to NaAsO2 exposure. The co-immunoprecipitation assay showed that the interaction between PUMA and Bcl-X enhanced in intensity responding to NaAsO2 exposure, disrupting the complexes of Bcl-X with other pro-survival Bcl-2-related proteins. To our knowledge, we first reported that NaAsO2 activated phosphorylation of p53 at Ser315, 376, and Thr55, as well as acetylation of p53 at K370.

Inorganic arsenic influences cell apoptosis by regulating the expression of MEG3 gene.

Arsenic is a wildly distributed carcinogen in the environment. Arsenic-induced apoptosis has been extensively studied in therapeutics and toxicology. LncRNA MEG3 has been extensively studied as apoptosis regulatory gene in recent years. However, it stays unclear regarding how the mechanism of MEG3 regulates arsenic-induced apoptosis. Our focus was to explore the effects of MEG3 on arsenic-induced apoptosis. MTS assay was used to test cell viability, and qRT-PCR was for the examination of gene expressions. The effect of the apoptosis and necrosis after knockdown MEG3 was detected with double staining. Our results demonstrated that MEG3 expression was positively correlated with the concentration of three arsenic species (inorganic arsenic (iAs), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) (p < 0.05). The ability of iAs to induce MEG3 expression was much higher compared with that induced by MMA and DMA. In addition, our experiments confirmed that MEG3 knockdown increased cell viability and arsenic-induced apoptosis, but cell viability decreased after iAs treatment. Moreover, LncRNA MEG3 regulated apoptosis via down-regulate API5 while up-regulate CASP7, CCND3 and APAF1. It is further proved that arsenic-induced apoptosis increased after the knockdown of MEG3, which regulates these genes. These findings provide experimental evidence and possible mechanisms for subsequent research on the effects of arsenic on health.

The transcript NR 134251.1 of lncRNA APTR with an opposite function to all transcripts inhibits proliferation and induces apoptosis by regulating proliferation and apoptosis-related genes.

Arsenic (As) exposure has been a global public health concern for hundreds of millions worldwide. LncRNA APTR (Alu-mediated p21 transcriptional regulator) plays an essential role in tumor growth and development. However, its function in arsenic-induced toxicological responses is still unknown. In this study, we found that the expressions of all transcripts and the transcript NR 134251.1 of APTR were increased in a dose-dependent manner in 16HBE cells treated with sodium arsenite (NaAsO2). Silencing the transcript NR 134251.1 of APTR inhibited cell proliferation and induced apoptosis. However, silencing all transcripts of APTR had the opposite function to the transcript NR 134251.1. Then we examined the protein level of the proliferation and apoptosis-related genes after silencing the transcript NR 134251.1 of APTR. The results showed that silencing the transcript NR 134251.1 of APTR up-regulated the expression of transcription factor E2F1 and regulated its downstream genes involved in proliferation and apoptosis, including p53, phospho-p53-S392, phospho-p53-T55, p21, Cyclin D1, PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c. In conclusion, arsenic induced APTR expression and the transcript NR 134251.1 of APTR have an opposite function to all transcripts, providing a theoretical basis for the prevention and treatment of arsenic exposure.

Molecular characterization of porcine NECD, SNRPN and UBE3A genes and imprinting status in the skeletal muscle of neonate pigs.

Imprinted genes are expressed monoallelically depending on their parental origin, and play important roles in embryo survival and postnatal growth regulation. In this study, we characterized the porcine NECD (necdin), SNRPN (small nuclear ribonucleoprotein polypeptide N) and UBE3A (UBE3A ubiquitin protein ligase E3A) genes, analyzed their expression in nine tissues including liver, lung, small intestine, skeletal muscle, heart, kidney, spleen, inguinal lymph nodes and fat, and also examined their imprinting status in the skeletal muscle of neonate pigs. Results indicated that these three genes were highly homologous between pigs and cattle, being 95.02 % in nucleotide and 99.17 % in amino acid with the cattle SNRPN gene, and 96.46 % in nucleotide and 98.63 % in amino acid with the cattle UBE3A gene, respectively. The three genes were expressed in all the tissues investigated. Three single nucleotide polymorphisms (SNPs) in the coding region of these genes, i.e. g.263G>C, g.402T>C and g.340A>G for porcine NECD, SNRPN and UBE3A genes, respectively, were revealed; and imprinting analysis with which indicated that, in the skeletal muscle of neonate pigs, both NECD and SNRPN were maternally imprinted, while UBE3A was not imprinted.

Sodium arsenite-mediated upregulation of circDHX34 promotes apoptosis in hormone-independent breast cancer cells by regulating apoptotic genes.

Arsenic and the compounds thereof can be carcinogens or therapeutic agents for different cancer types. However, for breast cancer (BC), studies have yielded conflicted results on the role of arsenic. A previous study by the present authors indicated a potential relationship between circDHX34 and sodium arsenite-treated BC cells. As such, the expression, function, and potential mechanism of circDHX34 in sodium arsenite-treated MDA-MB-231 cells were further detected. In the present study, findings were made that sodium arsenite upregulated circDHX34 expression in MDA-MB-231 cells in a dose-dependent manner, and knockdown of circDHX34 could promote cell proliferation and inhibit apoptosis. Further investigations revealed that knockdown of circDHX34 upregulated the expression levels of antiapoptotic genes BCL2 and BCL2L1 and downregulated the expression levels of proapoptotic genes CASP8 and CASP9. To conclude, by regulating apoptotic genes, sodium arsenite-mediated upregulation of circDHX34 promotes apoptosis in hormone-independent breast cancer cells.

Hsa_circ_0005050 interacts with ILF3 and affects cell apoptosis and proliferation by disrupting the balance between p53 and p65.

The regulatory network between arsenic, genes and signaling pathways has been reported in arsenic carcinogenesis. Studies on circRNA represent a growing field, but the extent to circRNA potential mechanisms remains poorly understood. So this study we explore the systematic function of hsa_circ_0005050 in mediating the cell apoptosis and proliferation. We demonstrated that hsa_circ_0005050 was highly expressed in subjects who are long-term exposed to arsenic, and could be induced by NaAs2O3 in A549 and 16HBE. Knockdown of hsa_circ_0005050 promotes A549 cell viability, whereas exerts the opposite effects in 16HBE. Mechanistically, hsa_circ_0005050 regulates the p53 and NF-κB signaling pathway involved in the apoptosis and proliferation. And we found that hsa_circ_0005050 could directly bind to the RNA binding protein ILF3 and mutually influence each other's formation. Upon si-hsa_circ_0005050, ILF3 export to the cytoplasm resulting the formation of a ternary complex ILF3-p65-IκBA, breaks the balance of p53 and NF-κB pathway and induces A549 apoptosis and leads to 16HBE proliferation. As a result of these investigations, suggestions were identified for future research.

CircP50 functions through the phosphorylation- and acetylation-activated p53 pathway to mediate inorganic arsenic-induced apoptosis in A549 cells.

As a class I carcinogen, arsenic has been reported to cause diseases accompanied by circRNAs regulating proliferation and apoptosis at the molecular level, but whether circP50 (circBase ID: hsa_circ_0008012) does the same has not been demonstrated. The aim of this study is to provide the basis for anti-lung cancer mechanism research, by studying the expression of circP50 under arsenic-induced conditions, and the effect and mechanism on the proliferation and apoptosis of A549 cells based on the circP50 knockdown models. To explore whether the circP50 is responsive to arsenic exposure, the qRT-PCR was applied to discover that the relative expression of circP50 in A549 cells increased only with increasing NaAsO2 dose and independent of its metabolites. We further determined the mechanism of circP50 by establishing circP50 knockdown models. The results of cell viability and EdU assays indicated the proliferation of A549 cells. According to the western blotting, phosphorylation of p53 at Ser15, Ser376, and Ser392 and acetylation of p53 at Lys370 and Lys382 were inhibited, resulting in the deficiency of p53 expression. Subsequently, the expression of genes downstream of p53 was reduced, including p21, PUMA, Caspase3, and Bcl-xS. Furthermore, the expressions of IKB-α, p65, and p50 decreased, but C-myc expression did not change significantly, referring to the NF-κB pathway was not dominant. The results suggest that circP50 mainly functions through the p53 pathway to mediate apoptosis in response to arsenic exposure.

Long non-coding RNA DICER1-AS1-low expression in arsenic-treated A549 cells inhibits cell proliferation by regulating the cell cycle pathway.

Arsenic, an environmental pollution with diverse toxicities, incurs public health problems. Arsenic trioxide could inhibit cell proliferation in vitro experiments, but the underlying mechanisms are not fully known. LncRNAs are also involved in the arsenic-induced toxicological responses. In our study, we found that the expression of lncRNA DICER1-AS1 was significantly inhibited by sodium arsenite in a dose-dependent manner. DICER1-AS1 silencing decreased the A549 cell proliferation and inhibited cell cycle progression. Importantly, DICER1-AS1 silencing induced upregulation of p21 and downregulation of Cyclin A2, Cyclin E2, CDK1 and PCNA. In conclusion, our study provided a new lncRNA-dictated regulatory mechanism participating in arsenic-induced inhibition of cell proliferation.

Arsenic exposure increased expression of HOTAIR and LincRNA-p21 in vivo and vitro.

Arsenic is an environmental contaminant, its multiple effects on human tend to increase the rate of disease, cancer and other health problems. Some of long non-coding RNAs (lncRNAs) can be induced in major cellular processes such as necrosis, proliferation, and mutation. While the toxicity of arsenic is well established, the association between arsenic exposure and long non-coding RNAs has not been studied enough. This study investigated the association between arsenic and the expression of HOTAIR and LincRNA-p21 in vivo and vitro. In epidemiological studies, the expression of HOTAIR and LincRNA-p21 was increased after long-term arsenic exposure. HOTAIR and LincRNA-p21 expression were positively linked to monomethylarsenic acid (MMA), dimethylarsenic acid (DMA), inorganic arsenic (iAs), total arsenic (tAs), and MMA% and negatively linked to secondary methylation index (SMI). In A549 cells, arsenic exposure resulted in enhanced HOTAIR and LincRNA-p21 expression dose-dependently. The expression of HOTAIR was considerably high in the presence of NaAsO2 and MMA but showed no difference in DMA compared with control group. And LincRNA-p21 expression was increased in the presence of NaAsO2, MMA, and DMA. The expression of HOTAIR and LincRNA-p21 induced by iAs was much higher than that induced by MMA and DMA. Compared with the control group, treatment of A549 cells with NaAsO2/S-adenosylmethionine (SAM) and NaAsO2/glutathione (GSH) combination increased HOTAIR and LincRNA-p21 expression. The expression of LincRNA-p21 in combination of NaAsO2/GSH was significantly decreased compared with NaAsO2 alone. Besides, in the presence of arsenic, both of HOTAIR and LincRNA-p21 were upregulated significantly when P53 was knocked down. We revealed that inorganic arsenic, its methylated metabolites, and arsenic metabolism efficiency affect the expression of HOTAIR and LincRNA-p21.

Air pollution and DNA methylation alterations in lung cancer: A systematic and comparative study.

The lung cancer incidence in the Xuanwei and neighboring region, Yunnan, China, is among the highest in China and is attributed to severe air pollution with high benzo(a)pyrene levels. We systematically and comparatively analyzed DNA methylation alterations at genome and gene levels in Xuanwei lung cancer tissues and cell lines, as well as benzo(a)pyrene-treated cells and mouse samples. We obtained a comprehensive dataset of genome-wide cytosine-phosphate-guanine island methylation in air pollution-related lung cancer samples. Benzo(a)pyrene exposure induced multiple alterations in DNA methylation and in mRNA expressions of DNA methyltransferases and ten-11 translocation proteins; these alterations partially occurred in Xuanwei lung cancer. Furthermore, benzo(a)pyrene-induced DKK2 and EN1 promoter hypermethylation and LPAR2 promoter hypomethylation led to down-regulation and up-regulation of the genes, respectively; the down-regulation of DKK2 and EN1 promoted the cellular proliferation. Thus, DNA methylation alterations induced by benzo(a)pyrene contribute partially to abnormal DNA methylation in air pollution-related lung cancer, and these DNA methylation alterations may affect the development and progression of lung cancer. Additionally, vitamin C and B6 can reduce benzo(a)pyrene-induced DNA methylation alterations and may be used as chemopreventive agents for air pollution-related lung cancer.

Genome-wide gene expression profiles in lung tissues of pig breeds differing in resistance to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an infectious disease characterized by severe reproductive deficiency in pregnant sows, typical respiratory symptoms in piglets, and high mortality rate of piglets. In this study, we employed an Affymetrix microarray chip to compare the gene expression profiles of lung tissue samples from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs after infection with PRRSV. During infection with PRRSV, the DLY pigs exhibited a range of clinical features that typify the disease, whereas the DPL pigs showed only mild signs of the disease. Overall, the DPL group had a lower percentage of CD4(+) cells and lower CD4(+)/CD8(+)ratios than the DLY group (p<0.05). For both IL-10 and TNF-α, the DLY pigs had significantly higher levels than the DPL pigs (p<0.01). The DLY pigs have lower serum IFN-γ levels than the DPL pigs (p<0.01). The serum IgG levels increased slightly from 0 dpi to 7 dpi, and peaked at 14 dpi (p<0.0001). Microarray data analysis revealed 16 differentially expressed (DE) genes in the lung tissue samples from the DLY and DPL pigs (q≤5%), of which LOC100516029 and LOC100523005 were up-regulated in the PRRSV-infected DPL pigs, while the other 14 genes were down-regulated in the PRRSV-infected DPL pigs compared with the PRRSV-infected DLY pigs. The mRNA expression levels of 10 out of the 16 DE genes were validated by real-time quantitative RT-PCR and their fold change was consistent with the result of microarray data analysis. We further analyzed the mRNA expression level of 8 differentially expressed genes between the DPL and DLY pigs for both uninfected and infected groups, and found that TF and USP18 genes were important in underlying porcine resistance or susceptibility to PRRSV.

Different expression patterns of PRRSV mediator genes in the lung tissues of PRRSV resistant and susceptible pigs.

Porcine reproductive and respiratory syndrome (PRRS) has caused severe economic loss in most swine-producing countries. The resistance to PRRS virus (PRRSV) infection varies among pig breeds and lines. In this study, we found that the Chinese Dapulian pigs (DPL) were more resistant to PRRSV than commercial Duroc×Landrace×Yorkshire (DLY) crossbred pigs in that lower rectal temperature and lower PRRSV copy number in the serum were detected in the former. Analysis of the mRNA expression of five PRRSV mediator genes (SIGLEC1, NMMHC-IIA, CD163, VIM and HSPG2) in the lung tissues indicated differences in expression between DLY and DPL pigs. In uninfected porcine lung tissues, the levels of SIGLEC1, NMMHC-IIA, CD163 and VIM genes were significantly higher in DLY than in DPL pigs (P<0.05); in PRRSV-infected pigs, the expression levels of NMMHC-IIA and CD163 mRNA were significantly higher in DPL pigs compared to uninfected ones (P<0.05), whereas these levels were not different in DLY pigs or between infected DPL and DLY pigs. Thus, the different expression of PRRSV mediator genes is likely related to pig resistance to PRRSV.