bims-rimeca 2021-09-26 papers

Issue of 2021‒09‒26
six papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics

Biochem Pharmacol. 2021 Sep 15. pii: S0006-2952(21)00382-8. [Epub ahead of print] 114766

m6A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2.

Seiya Takemoto, Masataka Nakano, Tatsuki Fukami, Miki Nakajima.

Methylation of adenosine at the N6 position to form N6-methyladenosine (m6A) is the most prevalent epitranscriptomic modification of mammalian mRNA. This modification is catalyzed by a methyltransferase-like 3 (METTL3)-METTL14 complex and is erased by demethylases such as fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5). m6A modification regulates mRNA stability, nuclear export, splicing, and/or protein translation via recognition by reader proteins such as members of YT521-B homology (YTH) family. Carboxylesterase 2 (CES2) is a serine esterase responsible for the hydrolysis of drugs and endogenous substrates, such as triglycerides and diacylglycerides. Here, we examined the potential regulation of human CES2 expression by m6A modification. CES2 mRNA level was significantly increased by double knockdown of METTL3 and METTL14 but was decreased by knockdown of FTO or ALKBH5 in HepaRG and HepG2 cells, leading to changes in its protein level and hydrolase activity for 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), suggesting that m6A modification negatively regulates CES2 expression. Consistent with the changes in CES2 expression, lipid accumulation in the cells was decreased by double knockdown of METTL3 and METTL14 but was increased by knockdown of FTO or ALKBH5. RNA immunoprecipitation assays using an anti-m6A antibody showed that adenosines in the 5'-untranslated region (UTR) and the last exon of CES2 are methylated. Luciferase assays revealed that YTHDC2, which degrades m6A-containing mRNA, downregulates CES2 expression by recognition of m6A in the 5'-UTR of CES2. Collectively, we demonstrated that m6A modification has a great impact on the regulation of CES2, affecting pharmacokinetics, drug response and lipid metabolism.

Keywords: RNA degradation; RNA methylation; carboxylesterase; drug metabolism; lipid metabolism; post-transcriptional regulation

DOI: https://doi.org/10.1016/j.bcp.2021.114766

Mol Ther. 2021 Sep 18. pii: S1525-0016(21)00471-8. [Epub ahead of print]

METTL3-m6A-Rubicon axis inhibits autophagy in nonalcoholic fatty liver disease.

Zishan Peng, Yingying Gong, Xuejie Wang, Weiman He, Liting Wu, Luyao Zhang, Li Xiong, Yanrui Huang, Lei Su, Peijie Shi, Xiaopei Cao, Rengyun Liu, Yanbing Li, Haipeng Xiao.

N6-methyladenosine (m6A) mRNA modification plays critical roles in various biological events and involves in multiple complex diseases. However, the role of m6A modification in autophagy in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we report that m6A modification was increased in livers of NAFLD mouse models and in free fatty acids (FFAs)-treated hepatocytes, and the abnormal m6A modification was attributed to the upregulation of methyltransferase like 3 (METTL3) induced by lipotoxicity. Knockdown of METTL3 promoted hepatic autophagic flux and lipid drops (LDs) clearance, while overexpression METTL3 inhibited these process. Mechanistically, METTL3 directly bound to Rubicon mRNA and mediated the m6A modification. While YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), as a partner of METTL3, interacted with the m6A-marked Rubicon mRNA and promoted its stability. Subsequently, RUBICON inhibited autophagosome-lysosome fusion and further blocked LDs clearance. Together, our results showed a critical role of METTL3 and YTHDF1 in regulating lipid metabolism via autophagy pathway and provided a novel insight into m6A mRNA methylation in NAFLD.

Keywords: METTL3; NAFLD; YTHDF1; autophagy; m(6)A modification

DOI: https://doi.org/10.1016/j.ymthe.2021.09.016

J Exp Clin Cancer Res. 2021 Sep 20. 40(1): 294

RNA m6A demethylase FTO-mediated epigenetic up-regulation of LINC00022 promotes tumorigenesis in esophageal squamous cell carcinoma.

Yuanbo Cui, Chunyan Zhang, Shanshan Ma, Zhe Li, Wenjie Wang, Ya Li, Yingchao Ma, Jiarui Fang, Yaping Wang, Wei Cao, Fangxia Guan.

BACKGROUND: Long non-coding RNA (LncRNA) controls cell proliferation and plays a significant role in the initiation and progression of esophageal squamous cell carcinoma (ESCC). N6-methyladenosine (m6A) modification now is recognized as a master driver of RNA function to maintain homeostasis in cancer cells. However, how m6A regulates LncRNA function and its role in tumorigenesis of ESCC remain unclear.METHODS: Multiple ESCC datasets were used to analyze gene expression in tumor tissues and normal tissues. Kaplan-Meier method and the ROC curve were conducted to evaluate the prognostic value and diagnostic value of LINC00022 in ESCC, respectively. Both gain-of-function and loss-of-function experiments were employed to investigate the effects of LINC00022 on ESCC growth in vitro and in vivo. Bioinformatics analysis, colorimetric m6A assay, RIP, MeRIP and co-IP was performed to explore the epigenetic mechanism of LINC00022 up-regulation in ESCC.
RESULTS: Here we report that m6A demethylation of LncRNA LINC00022 by fat mass and obesity-associated protein (FTO) promotes tumor growth of ESCC in vivo. Clinically, we revealed that LINC00022 was up-regulated in primary ESCC samples and was predictive of poor clinical outcome for ESCC patients. Mechanistically, LINC00022 directly binds to p21 protein and promotes its ubiquitination-mediated degradation, thereby facilitating cell-cycle progression and proliferation. Further, the elevated FTO in ESCC decreased m6A methylation of LINC00022 transcript, leading to the inhibition of LINC00022 decay via the m6A reader YTHDF2. Over-expression of FTO was shown to drive LINC00022-dependent cell proliferation and tumor growth of ESCC.
CONCLUSIONS: Thus, this study demonstrated m6A-mediated epigenetic modification of LncRNA contributes to the tumorigenesis in ESCC and LINC00022, specific target of m6A, serves as a potential biomarker for this malignancy.

Keywords: Cell cycle; Esophageal squamous cell carcinoma; FTO; LINC00022; N6-methyladenosine; Tumorigenesis

DOI: https://doi.org/10.1186/s13046-021-02096-1

Toxicology. 2021 Sep 21. pii: S0300-483X(21)00283-3. [Epub ahead of print] 152961

METTL3-mediated M6A methylation modification is involved in colistin-induced nephrotoxicity through apoptosis mediated by Keap1/Nrf2 signaling pathway.

Chunli Xia, Jian Wang, Zhiyong Wu, Yusong Miao, Chunli Chen, Rui Li, Jichang Li, Hou Juan Xing.

Colistin is a cationic polypeptide antibiotic. Despite its nephrotoxicity, it is still widely used as a last-line antibiotic against infection worldwide with the emergence of multi-drug resistant Gram-negative bacilli. N-methyladenosine (m6A) methylation-mediated degradation of RNA is essential for kidney development. However, m6A methylation impacts not only RNA stability, but also other RNA metabolism processes. How RNA decay affects the nephrotoxicity of colistin is largely unknown. Therefore, in this study, we verified that colistin could induce mouse kidney apoptosis through some apoptotic indicators, and confirmed the relationship between methylation and apoptosis through the detection of m6A methylation, thus elucidating the potential mechanism of colistin nephrotoxicity. The results showed that the renal tubule dilation and tubular structure were observed in the colistin group, and the oxidative stress index and ATPase activities were significantly different from those in the control group. Under electron microscope, the kidney in colistin group showed typical apoptotic morphological changes such as nuclear pyknosis, chromatin edge aggregation, and intact nuclear membrane, accompanied by significant changes in apoptosis-related genes. The level of m6A in the colistin group was significantly decreased, accompanied by downregulation of METTL3 mRNA and protein levels, and METTL3 was significantly correlated with apoptotic gene proteins. Data from this study suggested that m6A methylation was involved in oxidative stress-mediated apoptosis in the mechanism of colistin nephrotoxicity.

Keywords: Apoptosis; Colistin; Nephrotoxicity; Oxidative stress; m6A methylation modification

DOI: https://doi.org/10.1016/j.tox.2021.152961

Aging (Albany NY). 2021 Sep 19. 13(undefined):

METTL3-mediated m6A modification of KIF3C-mRNA promotes prostate cancer progression and is negatively regulated by miR-320d.

Honggui Ma, Facai Zhang, Quliang Zhong, Jianquan Hou.

The occurrence of distant metastasis is one of the leading causes of death in patients with prostate cancer (PCa). It is confirmed that kinesin protein is associated with a variety of malignancies, and the KIF3 family is related to cancer, but the relationship between KIF3C and prostate cancer is not clear. Our experiments have confirmed that KIF3C is highly expressed in prostate cancer tissues and cell lines. Further, functional tests have proven that KIF3C can promote the growth migration and invasion of PCa. We used Starbase 3.0 to discover that methyltransferase like 3 (METTL3) interacts with KIF3C. Our hypothesis and experiments concluded that METTL3 induced m6A modification on KIF3C, promoting the stabilization of KIF3C-mRNA by IGF2 binding protein 1 (IGF2BP1). The prediction that miR-320d inhibits KIF3C expression by targeting METTL3 using the miRmap website, was later confirmed experimentally. Further, a recovery experiment was used to confirm that miR-320d inhibited the progression of prostate cancer. KIF3C was overexpressed in prostate cancer, promoting its growth migration and invasion was induced by miR-320d/METTL3 in an m6A dependent process.

Keywords: KIF3C; METTL3; m6A; miR-320d; prostate cancer

DOI: https://doi.org/10.18632/aging.203541

Chin Med. 2021 Sep 21. 16(1): 91

UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model.

Zhizhen Song, Zeyun Li, Xueqian Wen, Ruijuan Liu, Xin Tian.

BACKGROUND: Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA.METHODS: An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer.
RESULTS: In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40-20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2-100 ng/mL for guanosine, and 2-1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment.
CONCLUSION: The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.

Keywords: Epimedin C; Liver injury; Nucleosides; UPLC-MS/MS; mRNA methylation

DOI: https://doi.org/10.1186/s13020-021-00501-7