The m6 A reader YTHDC2 regulates UVB-induced DNA damage repair and histone modification.

Ultraviolet B (UVB) radiation represents a major carcinogen for the development of all skin cancer types. Mechanistically, UVB induces damage to DNA in the form of lesions, including cyclobutane pyrimidine dimers (CPDs). Disruption of the functional repair processes, such as nucleotide excision repair (NER), allows persistence of DNA damage and contributes to skin carcinogenesis. Recent work has implicated m6 A RNA methylation and its regulatory proteins as having critical roles in facilitating UVB-induced DNA damage repair. However, the biological functions of the m6 A reader YTHDC2 are unknown in this context. Here, we show that YTHDC2 inhibition enhances the repair of UVB-induced DNA damage. We discovered that YTHDC2 inhibition increased the expression of PTEN while it decreased the expression of the PRC2 component SUZ12 and the levels of the histone modification H3K27me3. However, none of these functions were causally linked to the improvements in DNA repair, suggesting that the mechanism utilized by YTHDC2 may be unconventional. Moreover, inhibition of the m6 A writer METTL14 reversed the effect of YTHDC2 inhibition on DNA repair while inhibition of the m6 A eraser FTO mimicked the effect of YTHDC2 inhibition, indicating that YTHDC2 may regulate DNA repair through the m6 A pathway. Finally, compared to normal human skin, YTHDC2 expression was upregulated in human cutaneous squamous cell carcinomas (cSCC), suggesting that it may function as a tumor-promoting factor in skin cancer. Taken together, our findings demonstrate that the m6 A reader YTHDC2 plays a role in regulating UVB-induced DNA damage repair and may serve as a potential biomarker in cSCC.

NAT10 regulates the repair of UVB-induced DNA damage and tumorigenicity.

Chemical modifications in messenger RNA (mRNA) regulate gene expression and play critical roles in stress responses and diseases. Recently we have shown that N6-methyladenosine (m6A), the most abundant mRNA modification, promotes the repair of UVB-induced DNA damage by regulating global genome nucleotide excision repair (GG-NER). However, the roles of other mRNA modifications in the UVB-induced damage response remain understudied. N4-acetylcytidine (ac4C) is deposited in mRNA by the RNA-binding acetyltransferase NAT10. This NAT10-mediated ac4C in mRNA has been reported to increase both mRNA stability and translation. However, the role of ac4C and NAT10 in the UVB-induced DNA damage response remains poorly understood. Here we show that NAT10 plays a critical role in the repair of UVB-induced DNA damage lesions through regulating the expression of the key GG-NER gene DDB2. We found that knockdown of NAT10 enhanced the repair of UVB-induced DNA damage lesions by promoting the mRNA stability of DDB2. Our findings are in contrast to the previously reported role of NAT10-mediated ac4C deposition in promoting mRNA stability and may represent a novel mechanism for ac4C in the UVB damage response. Furthermore, NAT10 knockdown in skin cancer cells decreased skin cancer cell proliferation in vitro and tumorigenicity in vivo. Chronic UVB irradiation increases NAT10 protein levels in mouse skin. Taken together, our findings demonstrate a novel role for NAT10 in the repair of UVB-induced DNA damage products by decreasing the mRNA stability of DDB2 and suggest that NAT10 is a potential novel target for preventing and treating skin cancer.

Versican secreted by the ovary links ovulation and migration in fallopian tube derived serous cancer.

High grade serous ovarian cancers (HGSOC) predominantly arise in the fallopian tube epithelium (FTE) and colonize the ovary first, before further metastasis to the peritoneum. Ovarian cancer risk is directly related to the number of ovulations, suggesting that the ovary may secrete specific factors that act as chemoattractants for fallopian tube derived tumor cells during ovulation. We found that 3D ovarian organ culture produced a secreted factor that enhanced the migration of FTE non-tumorigenic cells as well as cells harboring specific pathway modifications commonly found in high grade serous cancers. Through size fractionation and a small molecule inhibitors screen, the secreted protein was determined to be 50-100kDa in size and acted through the Epidermal Growth Factor Receptor (EGFR). To correlate the candidates with ovulation, the PREDICT organ-on-chip system was optimized to support ovulation in a perfused microfluidic platform. Versican was found in the correct molecular weight range, contained EGF-like domains, and correlated with ovulation in the PREDICT system. Exogenous versican increased migration, invasion, and enhanced adhesion of both murine and human FTE cells to the ovary in an EGFR-dependent manner. The identification of a protein secreted during ovulation that impacts the ability of FTE cells to colonize the ovary provides new insights into the development of strategies for limiting primary ovarian metastasis.

Cellular Immune Signal Exchange From Ischemic Stroke to Intestinal Lesions Through Brain-Gut Axis.

As a vital pivot for the human circulatory system, the brain-gut axis is now being considered as an important channel for many of the small immune molecules' transductions, including interleukins, interferons, neurotransmitters, peptides, and the chemokines penetrating the mesentery and blood brain barrier (BBB) during the development of an ischemic stroke (IS). Hypoxia-ischemia contributes to pituitary and neurofunctional disorders by interfering with the molecular signal release and communication then providing feedback to the gut. Suffering from such a disease on a long-term basis may cause the peripheral system's homeostasis to become imbalanced, and it can also lead to multiple intestinal complications such as gut microbiota dysbiosis (GMD), inflammatory bowel disease (IBD), necrotizing enterocolitis (NEC), and even the tumorigenesis of colorectal carcinoma (CRC). Correspondingly, these complications will deteriorate the cerebral infarctions and, in patients suffering with IS, it can even ruin the brain's immune system. This review summarized recent studies on abnormal immunological signal exchange mediated polarization subtype changes, in both macrophages and microglial cells as well as T-lymphocytes. How gut complications modulate the immune signal transduction from the brain are also elucidated and analyzed. The conclusions drawn in this review could provide guidance and novel strategies to benefit remedies for both IS and relative gut lesions from immune-prophylaxis and immunotherapy aspects.

The Pharmacological Rationales and Molecular Mechanisms of Ganoderma lucidum Polysaccharides for the Therapeutic Applications of Multiple Diseases.

As a versatile Chinese herb, Ganoderma lucidum (Leyss. ex Fr.) Karst (G. lucidum) has been applied to treat multiple diseases in clinics and improve the quality of life of patients. Among all of its extracts, the main bioactive components are G. lucidum polysaccharides (GLPs), which possess many therapeutic effects, such as antitumor, immunoregulatory, anti-oxidant, antidiabetic, antibacterial, and antifungal effects and neuroprotection activities. This review briefly summarized the recent studies of the pharmacological rationales of GLPs and their underlying molecular signaling transmission mechanisms in treating diseases. Until now, the clear mechanisms of GLPs for treating diseases have not been reported. In this review, we used the keywords of "Ganoderma lucidum polysaccharides" and "tumor" to search in PubMed (years of 1992-2020), then screened and obtained 160 targets of antitumor activities in the literatures. The network pharmacology and mechanism framework were employed in this study as powerful approaches to systematically analyze the complicated potential antitumor mechanisms and targets of GLPs in cancer. We then found that there are 69 targets and 21 network pathways in "Pathways in cancer". Besides, we summarized the effects of GLPs and the models and methods used in the research of GLPs. In conclusion, GLPs have been studied extensively, but more in-depth research is still needed to determine the exact mechanisms and pathways. Therefore, this review might provide new insights into the vital targets and pathways for researchers to study the pharmacological mechanisms of GLPs for the treatment of diseases.

[Therapeutic effect of Heirong Kidney-Tonifying Granule on busulfan-induced dyszoospermia in model mice].

OBJECTIVE: To explore the therapeutic effect of Heirong Kidney-Tonifying Granule (HKTG) on busulfan-induced dyszoospermia in mice, and its mechanism in regulating testicular spermatogenesis. METHODS: Forty-eight male mice were randomly divided into six groups of an equal number: blank control (BC), negative control (NC), HKTG-1, HKTG-2, HKTG-3 and HKTG-4. The model of dyszoospermia was established in the latter five groups by intraperitoneal injection of busulfan at 40 mg/kg and, 30 days after modeling, the mice in the BC and NC groups were given gavage of normal saline, and those in the latter four groups treated with HKTG + pilose antler at 400 mg/kg/d, HKTG + pilose antler at 800 mg/kg/d, HKTG + black ants at 400 mg/kg/d and HKTG + black ants at 800 mg/kg/d, respectively, all for 5 consecutive weeks. The mean body weight of the mice was recorded daily, and their testes weighed after treatment. The microstructure of the testis tissue was detected by HE staining, and the localization and expression of spermatogenesis markers in the testis were determined by immunofluorescence staining. RESULTS: The mice in the BC and NC groups showed no statistically significant difference from those in the HKTG groups in the body weight and daily body weight gain (P > 0.05). Compared with the NC mice, the animals in the HKTG-1 group exhibited significantly increased testis weight (P < 0.05), and those in the HKTG-1 and HKTG-1 groups presented a large number of germ cells in the seminiferous tubules, including deformed sperm cells in the lumen, and some seminomatogonia in the seminogenic tubules, but almost no deformed sperm cells. The expressions of the total germ cell marker gene Ddx4, spermatogonial cell marker gene Dazl, spermatic cell marker gene Sycp3 and sperm cell marker gene Tnp1 were significantly upregulated (P < 0.05) while that of the Sertoli cell marker gene Sox9 downregulated (P < 0.05) in the HKTG-1 group. The number of Sertoli cells in the HKTG-1 group was remarkably reduced (P<0.05), corresponding to the increased number of germ cells in the HKTG-1 group. There were no significant changes in the relative expressions of the DDX4, Dazl, Sycp3 and Tnp1 genes, nor in the number of Sertoli cells in the HKTG-3 and HKTG-4 groups. The expressions of meiosis-related genes Meioc, Stra8 and Spo11were markedly upreguated in the HKTG-1 group, indicating significantly improved spermatogenesis in the testis tissue of the mice. CONCLUSION: HKTG improves the function of spermatogenic cells and increases sperm production in the testis tissue of mice by promoting meiosis.

METTL14 facilitates global genome repair and suppresses skin tumorigenesis.

Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.

The regioselective glucuronidation of morphine by dimerized human UGT2B7, 1A1, 1A9 and their allelic variants.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGT1A1 and UGT1A9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B7*1 (wild type), UGT2B7*71S (A71S, 211G>T), UGT2B7*2 (H268Y, 802C>T), UGT2B7*5 (D398N, 1192G>A), and double-recombinant allozymes formed by the dimerization of UGT1A9*1 (wild type), UGT1A9*2 (C3Y, 8G>A), UGT1A9*3 (M33T, 98T>C), UGT1A9*5 (D256N, 766G>A), UGT1A1 (wild type) with its splice variant UGT1A1b were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B7*1, UGT2B7*2 singles or doubles have lower Km values for M3G and M6G, whereas UGT2B7*5 allozymes perform opposite effects. The double allozymes of UGT1A9*2 or UGT1A9*5 with UGT2B7 tend to produce M6G. Interestingly, the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGT1A9*2-UGT2B7*1 double enzyme has the lowest M3G:M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGT1A1 and UGT1A9 and their allelic variants can regioselectively affect the generation of two metabolites of morphine via altering the CLint ratios of M3G to M6G. These results may predict the effectiveness of morphine antinociception in individualized opioid treatment.

Brain-derived neurotrophic factor involved epigenetic repression of UGT2B7 in colorectal carcinoma: A mechanism to alter morphine glucuronidation in tumor.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7, as one of significant drug enzymes, is responsible on the glucuronidation of abundant endobiotics or xenobiotics. We here report that it is markedly repressed in the tumor tissues of colorectal carcinoma (CRC) patients. Accordingly, morphine in CRC cells will stimulate the expression of its main metabolic enzyme, UGT2B7 during tolerance generation by activating the positive signals in histone 3, especially for trimethylated lysine 27 (H3K4Me3) and acetylated lysine 4 (H3K27Ac). Further study reveals that brain-derived neutrophilic factor (BDNF), a secretory neurotrophin, enriched in CRC can interact and inhibit UGT2B7 by primarily blocking the positive signals of H3K4Me3 as well as activating H3K27Ac on the promoter region of UGT2B7. Meanwhile, BDNF repression attributes to the sensitizations of main core factors in poly-comb repressive complex (PRC) 1 rather than PRC2 as the reason of the depression of SUZ12 in the later complex. Besides that, the productions of two main morphine glucuronides are both increased in the BDNF deficient or TSA and BIX-01294 treated morphine tolerance-like HCT-116 cells. On the same condition, active metabolite, morphine-6-glucuronide (M6G) was accumulated more than inactive M3G. Our findings imply that enzymatic activity enhancement and substrate regioselective catalysis alteration of UGT2B7 may release morphine tolerance under the cure of tumor-induced pain.

Reverse of Acute and Chronic Morphine Tolerance by Lithocholic Acid via Down-Regulating UGT2B7.

Lithocholic acid (LCA) deposited in human livers always induces drastic pains which need analgesic drug, like morphine to release. Our research showed that LCA can effectively inhibit uridine 5'-diphospho-glucuronosyltransferase 2B7 (UGT2B7) in morphine tolerance-like human normal liver cells, HL-7702, then increase µ-opioid receptor (MOR) and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) expression. In vivo assay, UGT2B7 was significantly repressed in the livers of acute or chronic morphine tolerance mice pretreated with LCA (10, 50, and 100 mg/kg, p.o.). To investigate the connections between LCA function performance and change of UGT2B7 enzymatic activity in mice livers, two morphine metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) were quantified by solid phase extraction (SPE)-HPLC-MS/MS. The result indicated no matter in acute or chronic morphine tolerance, the concentrations of M3G and M6G were all decreased, the later one fell even more. Besides that, 50 mg/kg of LCA administration can prevent auto-phosphorylation of CaMKIIα at Thr286 in acute or chronic morphine tolerance mice prefrontal cortexes (mPFCs) due to synthesis increase of cyclic adenosine monophosphate. As a consequence, UGT2B7 depression mediated by LCA can affect its selective catalysis ability to morphine, that may be responsible to acute or chronic morphine tolerance alleviation. These findings might assist to modify antinociception of morphine in clinic.

siRNA capsulated brain-targeted nanoparticles specifically knock down OATP2B1 in mice: a mechanism for acute morphine tolerance suppression.

Regulating main brain-uptake transporter of morphine may restrict its tolerance generation, then modify its antinociception. In this study, more than 2 fold higher intracellular uptake concentrations for morphine and morphine-6-glucuronide (M6G) were observed in stable expression cells, HEK293-hOATP2B1 than HEK293-MOCK. Specifically, the Km value of morphine to OATP2B1 (57.58 ± 8.90 µM) is 1.4-time more than that of M6G (80.31 ± 21.75 µM); Cyclosporine A (CsA), an inhibitor of OATP2B1, can inhibit their intracellular accumulations with IC50 = 3.90 ± 0.50 µM for morphine and IC50 = 6.04 ± 0.86 µM for M6G, respectively. To further investigate the role of OATP2B1 in morphine brain transport and tolerance, the novel nanoparticles of DGL-PEG/dermorphin capsulated siRNA (OATP2B1) were applied to deliver siRNA into mouse brain. Along with OATP2B1 depressed, a main reduction was found for each of morphine or M6G in cerebrums or epencephalons of acute morphine tolerance mice. Furthermore, calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) in mouse prefrontal cortex (mPFC) underwent dephosphorylation at Thr286. In conclusion, OATP2B1 downregulation in mouse brain can suppress tolerance via blocking morphine and M6G brain transport. These findings might help to improve the pharmacological effects of morphine.

Simultaneous analysis of gemfibrozil, morphine, and its two active metabolites in different mouse brain structures using solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry with a deuterated internal standard.

A rapid and sensitive bioassay was established and validated to simultaneously determine gemfibrozil, morphine, morphine-3ß-glucuronide, and morphine-6ß-glucuronide in mouse cerebrum, epencephalon, and hippocampus based on ultra-high performance liquid chromatography and tandem mass spectrometry. The deuterated internal standard, M6G-d3, was mixed with the prepared samples at 10 ng/mL as the final concentration. The samples were transferred into the C18 solid-phase extraction columns with gradient elution for solid-phase extraction. The mobile phase consisted of methanol and 0.05% formic acid (pH 3.2). Multiple reaction monitoring has been applied to analyze gemfibrozil (m/z 249.0 → 121.0) in anion mode, and M6G-d3 (m/z 465.1 → 289.1), morphine (m/z 286.0 → 200.9), and M3G and M6G (m/z 462.1 → 286.1) in the positive ion mode. The method has a linear calibration range from 0.05 to 10 ng for gemfibrozil, morphine, and M3G and M6G with correlation coefficients >0.993. The lower limit of quantitation for all four analytes was 0.05 ng/mL, relative standard deviation of intra- and interday precision was less than 10.5%, and the relative error of accuracy was from -8.2 to 8.3% at low, medium, and high concentrations for all the analytes. In conclusion, gemfibrozil can influence the morphine antinociception after coronary heart disease induced chronic angina by the change in one of morphine metabolites', M3G, distribution in mouse brain.