Genomic and epigenetic characterization of the arsenic-induced oncogenic microRNA-21.

As one of the first identified oncogenic microRNAs, the precise details concerning the transcriptional regulation and function of microRNA-21 (miR-21) are still not completely established. The miR-21 gene is situated on chromosome 17q23.2, positioned at the 3'-UTR of the gene that encodes vacuole membrane protein-1 (VMP1). In this current study, we presented evidence indicating that miR-21 possesses its own gene promoter, which can be found in the intron 10 of the VMP1 gene. Chromatin immunoprecipitation followed by global DNA sequencing (ChIP-seq) revealed the presence of a broad H3K4me3 peak spanning the entire gene body of the primary miR-21 and the existence of super-enhancer clusters in the close proximity to both the miR-21 gene promoter and the transcription termination site in arsenic (As3+)-induced cancer stem-like cells (CSCs) and human induced pluripotent stem cells (hiPSCs). In non-transformed human bronchial epithelial cells (BEAS-2B), As3+ treatment enhanced Nrf2 binding to both the host gene VMP1 of miR-21 and the miR-21 gene. Knockout of Nrf2 inhibited both the basal and As3+-induced expressions of miR-21. Furthermore, the As3+-enhanced Nrf2 peaks in ChIP-seq fully overlap with these super-enhancers enriched with H3K4me1 and H3K27ac in the miR-21 gene, suggesting that Nrf2 may coordinate with other transcription factors through the super-enhancers to regulate the expression of miR-21 in cellular response to As3+. These findings demonstrate the unique genetic and epigenetic characteristics of miR-21 and may provide insights into understanding the novel mechanisms linking environmental As3+ exposure and human cancers.

Tumor suppressive activity of AHR in environmental arsenic-induced carcinogenesis.

The aryl hydrocarbon receptor (AHR) is a highly conserved pleiotropic transcription factor that senses environmental pollutants, microbial products, and endogenous ligands. The transcriptional targets of AHR include phase I and phase II detoxification enzymes, as well as numerous signaling molecules that affect a wide spectrum of biological and biochemical processes in a manner of cellular context-dependent. In this review, we systematically assess the latest discoveries of AHR in carcinogenesis with an emphasis on its tumor suppressor-like property that represses the expression of genes in oncogenic signaling pathways. Additionally, we outline recent progress in our studies on the interaction among AHR, TGFb and NRF2 in cellular responses to arsenic and malignant transformation. Our findings indicate that AHR antagonized TGFb and NRF2, suggesting that AHR could serve as a potential tumor suppressor in arsenic-induced carcinogenesis. Notably, while AHR can exhibit both oncogenic and tumor-suppressive properties in cancer development and the generation of the cancer stem-like cells (CSCs), the tumor suppressor-like effect of AHR warrants further extensive exploration for the prevention and clinical treatment of cancers.

Disruption of the tumor suppressor-like activity of aryl hydrocarbon receptor by arsenic in epithelial cells and human lung cancer.

As the most classic and extensively studied transcription factor in response to environmental toxic chemicals, the human aryl hydrocarbon receptor (AHR) has been implicated in mediating some oncogenic responses also. Limited information is available, however, on whether arsenic, a widely presented environmental carcinogen, can regulate AHR to exert its carcinogenic activity. Through chromatin immunoprecipitation and sequencing (ChIP-seq), CRISPR-Cas9 gene editing, RNA-seq, and immunohistochemistry (IHC), in this report we provided evidence showing that arsenic enforces TGFß and other oncogenic signaling pathways in bronchial epithelial cells through disrupting the tumor suppressor-like activity of AHR. AHR is normally enriched on a number of oncogenic genes in addition to the known phase I/II enzymes, such as genes in TGFß and Nrf2 signaling pathways and several known oncogenes. Arsenic treatment substantially reduced the binding of AHR on these genes followed by an increased expression of these genes. CRISPR-Cas9-based knockout of AHR followed by RNA-seq further demonstrated increased expression of the TGFß signaling and some oncogenic signaling pathway genes in the AHR knockout cells. IHC studies on human tissue samples revealed that normal human lung tissues expressed high level of AHR. In contrast, the AHR expression was diminished in the lung cancer tissues. Accordingly, the data from this study suggest that AHR has tumor suppressor-like activity for human lung cancer, and one of the carcinogenic mechanisms of arsenic is likely mediated by the inhibition of arsenic on the tumor suppressor-like activity of AHR.

Epigenetics and environment in breast cancer: New paradigms for anti-cancer therapies.

Breast cancer remains the most frequently diagnosed cancer in women worldwide. Delayed presentation of the disease, late stage at diagnosis, limited therapeutic options, metastasis, and relapse are the major factors contributing to breast cancer mortality. The development and progression of breast cancer is a complex and multi-step process that incorporates an accumulation of several genetic and epigenetic alterations. External environmental factors and internal cellular microenvironmental cues influence the occurrence of these alterations that drives tumorigenesis. Here, we discuss state-of-the-art information on the epigenetics of breast cancer and how environmental risk factors orchestrate major epigenetic events, emphasizing the necessity for a multidisciplinary approach toward a better understanding of the gene-environment interactions implicated in breast cancer. Since epigenetic modifications are reversible and are susceptible to extrinsic and intrinsic stimuli, they offer potential avenues that can be targeted for designing robust breast cancer therapies.

Molecular Characterization of Two Genes Encoding Novel Ca2+-Independent Phospholipase A2s from the Silkworm, Bombyx mori.

Eicosanoids are crucial downstream signals in the insect immune responses. Phospholipase A2 (PLA2) catalyzes phospholipids, the initial step in eicosanoid biosynthesis. In mammals, the biological roles of Ca2+-independent Phospholipase A2 (iPLA2) have been extensively studied; however, only a few studies have attempted to explore iPLA2 functions in insects. In this study, we identified two iPLA2 genes (designated as BmiPLA2A and BmiPLA2B) in the silkworm, Bombyx mori. BmiPLA2A had a 2427 base pair (bp) open reading frame (ORF) that coded for a protein with 808 amino acids. In contrast, BmiPLA2B had a 1731 bp ORF that coded for a protein with 576 amino acids. Domain analysis revealed that BmiPLA2A had six ankyrin repeat domains, but BmiPLA2B lacks these domains. BmiPLA2A and BmiPLA2B were transcribed widely in various tissues and developmental stages with different expression patterns. The administration of 20-hydroxyecdysone increased their expression levels in the epidermis and hemocytes. Furthermore, challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria induced the expression of BmiPLA2A and BmiPLA2B with variable degrees along with different time points. Our findings imply that BmiPLA2A and BmiPLA2B may have important biological roles in the development and innate immunity of B. mori.

Deoxyelephantopin Induces Apoptosis and Enhances Chemosensitivity of Colon Cancer via miR-205/Bcl2 Axis.

Colon cancer (CC) is one of the major causes of cancer death in humans. Despite recent advances in the management of CC, the prognosis is still poor and a new strategy for effective therapy is imperative. Deoxyelephantopin (DET), extracted from an important medicinal plant, Elephantopus scaber L., has been reported to exhibit excellent anti-inflammatory and -cancer activities, while the detailed anti-cancer mechanism remains unclear. Herein, we found that DET showed a significant CC inhibiting effect in vitro and in vivo without obvious organ toxicity. Mechanistically, DET inhibited CC cells and tumor growth by inducing G2/M phase arrest and subsequent apoptosis. DET-mediated cell cycle arrest was caused by severe DNA damage, and DET decreased the Bcl2 expression level in a dose-dependent manner to promote CC cell apoptosis, whereas restoring Bcl2 expression reduced apoptosis to a certain extent. Moreover, we identified a microRNA complementary to the 3'-UTR of Bcl2, miR-205, that responded to the DET treatment. An inhibitor of miR-205 could recover Bcl2 expression and promoted the survival of CC cells upon DET treatment. To further examine the potential value of the drug, we evaluated the combinative effects of DET and 5-Fluorouracil (5FU) through Jin's formula and revealed that DET acted synergistically with 5FU, resulting in enhancing the chemotherapeutic sensitivity of CC to 5FU. Our results consolidate DET as a potent drug for the treatment of CC when it is used alone or combined with 5FU, and elucidate the importance of the miR-205-Bcl2 axis in DET treatment.

PHF14 knockdown causes apoptosis by inducing DNA damage and impairing the activity of the damage response complex in colorectal cancer.

The abnormal expression or mutation of the plant homeodomain finger protein 14 (PHF14), a recently discovered PHD finger protein, has been reported to link to a wide range of disorders, like the aetiology and pathophysiology of multiple malignancies. Its detailed biological functions, however, still remain unclear. Herein, we discovered that PHF14 expression is strongly associated with the gastrointestinal tumor grade and gastrointestinal disorders, especially colorectal cancer (CRC), with high PHF14 expressions indicating a poor prognosis. Additionally, the mutation rate of PHF14 in CRC patients accounts for a striking proportion of 18%. PHF14 is also implicated in the expression of several oncogenes. In vitro, PHF14 was significantly expressed in patient tissues and in various CRC cell lines, and its expression was closely associated with cell proliferation and growth. Knockdown of PHF14 mediated severe DNA damage and activation of the ATR-CHK1-H2A.X pathway, leading to apoptosis. Strikingly, PHF14 interacted with KIF4A and contributes to the formation of BRCA2/Rad51 foci, indicating that PHF14 is a newly discovered factor that may participate in the formation and recruitment of DNA damage response complexes. These impairments, however, could be alleviated by restoring PHF14 expression. Importantly, inhibiting PHF14 expression in CRC cells might reduce carcinogenesis in vivo. In conclusion, PHF14 is necessary for CRC cell proliferation and growth, and therefore, it might be used as a novel biomarker and therapeutic target for the disease.

Scavenger receptor C regulates antimicrobial peptide expression by activating toll signaling in silkworm, Bombyx mori.

Scavenger receptor is pattern-recognition receptor (PRR) that plays a crucial function in host defense against pathogens. Scavenger receptor C (SR-C) is present only in invertebrates and its function has not been studied in detail. In this study, an SR-C homologous gene from the silkworm, Bombyx mori, was identified and characterized. SR-C was largely expressed in hemocytes and Malpighian tubules, with continuous expression in hemocytes. The peak expression was observed in hemocytes during molting and wandering stages both at mRNA and protein levels. Furthermore, immunofluorescence demonstrated it to be mainly distributed in the cell membranes of hemocytes, including oenocytoids and granulocytes. The recombinant SR-C protein (rSR-C) could bind to different types of bacteria and pathogen-associated molecular patterns (PAMPs), with strong binding to gram-positive bacteria and Lys-type peptidoglycans. The overexpression of SR-C induced the expression of genes related to the Toll pathway and antibacterial peptides. While the knockdown of SR-C reduced the expression of AMPs and inhibited the Toll pathway, it impaired the bacterial clearance ability of silkworm larvae, thus decreasing silkworm larvae's survival rate. Altogether, SR-C is a PRR that protect silkworms against bacterial pathogens by enhancing the expression of AMPs expression via the Toll pathway in hemocytes.

Dehydrodiisoeugenol inhibits colorectal cancer growth by endoplasmic reticulum stress-induced autophagic pathways.

BACKGROUND: Dehydrodiisoeugenol (DEH), a novel lignan component extracted from nutmeg, which is the seed of Myristica fragrans Houtt, displays noticeable anti-inflammatory and anti-allergic effects in digestive system diseases. However, the mechanism of its anticancer activity in gastrointestinal cancer remains to be investigated. METHODS: In this study, the anticancer effect of DEH on human colorectal cancer and its underlying mechanism were evaluated. Assays including MTT, EdU, Plate clone formation, Soft agar, Flow cytometry, Electron microscopy, Immunofluorescence and Western blotting were used in vitro. The CDX and PDX tumor xenograft models were used in vivo. RESULTS: Our findings indicated that treatment with DEH arrested the cell cycle of colorectal cancer cells at the G1/S phase, leading to significant inhibition in cell growth. Moreover, DEH induced strong cellular autophagy, which could be inhibited through autophagic inhibitors, with a rction in the DEH-induced inhibition of cell growth in colorectal cancer cells. Further analysis indicated that DEH also induced endoplasmic reticulum (ER) stress and subsequently stimulated autophagy through the activation of PERK/eIF2α and IRE1α/XBP-1 s/CHOP pathways. Knockdown of PERK or IRE1α significantly decreased DEH-induced autophagy and retrieved cell viability in cells treated with DEH. Furthermore, DEH also exhibited significant anticancer activities in the CDX- and PDX-models. CONCLUSIONS: Collectively, our studies strongly suggest that DEH might be a potential anticancer agent against colorectal cancer by activating ER stress-induced inhibition of autophagy.

Scavenger receptor B8 improves survivability by mediating innate immunity in silkworm, Bombyx mori.

Scavenger receptor class B (SR-B) is an extracellular transmembrane glycoprotein that plays a vital role in innate immunity. Although SR-Bs have been widely studied in vertebrates, their functions remained to elucidate in insects. Here, we identified and characterized a scavenger receptor class B member from the silkworm, Bombyx mori (designated as BmSCRB8). BmSCRB8 is broadly expressed in various immune tissues/organs, including fat body, gut, and hemocyte. Its expression is dramatically enhanced after challenge with different types of bacteria or pathogen-associated molecular patterns (PAMPs). The recombinant BmSCRB8 protein can detect different types of bacteria by directly binding to PAMPs and significantly improve the bacterial clearance in vivo. After knockdown of BmSCRB8, the pathogenic bacterial clearance was strongly impaired, and several AMP genes were down-regulated following E. coli challenge. Moreover, pathogenic bacteria's treatment following the depletion of BmSCRB8 remarkably decreased silkworm larvae's survival rate. Taken together, these results demonstrate that BmSCRB8 acts as a pattern recognition protein and plays an essential role in silkworm innate immunity by enhancing bacterial clearance and contributing to the production of AMPs in vivo.

Lycorine hydrochloride inhibits cell proliferation and induces apoptosis through promoting FBXW7-MCL1 axis in gastric cancer.

BACKGROUND: Lycorine hydrochloride (LH), an alkaloid extracted from the bulb of the Lycoris radiata, is considered to have anti-viral, anti-malarial, and anti-tumorous effects. At present, the underlying mechanisms of LH in gastric cancer remain unclear. MCL1, an anti-apoptotic protein of BCL2 family, is closely related to drug resistance of tumor. Therefore, MCL1 is considered as a potential target for cancer treatment. METHODS: The effect of LH on gastric cancer was assessed in vitro (by MTT, BrdU, western blotting…) and in vivo (by immunohistochemistry). RESULTS: In this study, we showed that LH has an anti-tumorous effect by down-regulating MCL1 in gastric cancer. Besides, we unveiled that LH reduced the protein stability of MCL1 by up-regulating ubiquitin E3 ligase FBXW7, arrested cell cycle at S phase and triggered apoptosis of gastric cancer cells. Meanwhile, we also demonstrated that LH could induce apoptosis of the BCL2-drug-resistant-cell-lines. Moreover, PDX (Patient-Derived tumor xenograft) model experiment proved that LH combined with HA14-1 (inhibitor of BCL2), had a more significant therapeutic effect on gastric cancer. CONCLUSIONS: The efficacy showed in our data suggests that lycorine hydrochloride is a promising anti-tumor compound for gastric cancer.

Niemann-Pick type C1 regulates cholesterol transport and metamorphosis in silkworm, Bombyx mori (Dazao).

NPC1 is a large glycoprotein with 13 transmembrane-spanning domains, which plays a crucial biological role in cholesterol transport and metamorphosis of animals. However, the physiological functions of this gene have rarely been elucidated in insects. Here, we isolated the NPC1 gene from Bombyx mori (BmNPC1), sequenced and evaluated its physiological functions. BmNPC1 comprised of 3702 bp open reading frame, encoding a protein of 1233 amino acid residues. The recombinant protein was expressed, and anti-BmNPC1 antibodies were synthesized. Immunofluorescence assay revealed that BmNPC1 protein localized in the cytoplasm of the cells. The qRT-PCR analysis showed that BmNPC1 expression was most significant in the testis, followed by the malpighian tubules, hemocytes, and ovary. The knockdown of BmNPC1 by double-stranded RNA caused the accumulation of cholesterol in the cells. Furthermore, suppression of this gene influenced the expression of ecdysone-responsive genes and also prevented the molting in B. mori (Dazao) larvae. Overall, BmNPC1 may have different biological roles in the physiology of silkworm, B. mori (Dazao), since it regulates the cholesterol transport and molting process.