Inhibition of histone methyltransferase PRMT5 attenuates cisplatin-induced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway / 药物分析学报

This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5(PRMT5)in cisplatin-induced hearing loss.The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry,apoptosis assays,and auditory brainstem response.The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction(CUT&Tag-qPCR)analyses in the HEI-OC1 cell line.Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species.CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene,thus activating the expression of Pik3ca.These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatin-induced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.

Expression of PRMT5 protein in colorectal cancer tissues and its relationship with recurrence after radical resection / 中国医师进修杂志

Objective:To investigate the expression of PRMT5 protein in colorectal cancer tissues and its relationship with recurrence after curative resection.Methods:The clinical data of 154 patients with colorectal cancer who underwent radical resection in Zigong Fourth People′s Hospital from February 2016 to April 2018 were retrospectively reviewed, the colorectal cancer tissues and adjacent tissues samples of them were retained at the time of surgery. The PRMT5 protein expressions in each specimen were determined by immunohistochemistry. The recurrence status of patients during the 3-year postoperative follow-up period was counted, and they were divided into recurrence group and no recurrence group.The positive expression rates of PRMT5 protein in colorectal cancer tissues between the two group was compared, and Cox regression model analysis was used to analyze the influencing factors of recurrence after radical resection of colorectal cancer.Results:The positive expression rate of PRMT5 protein in the colorectal cancer tissues was higher than that in the adjacent tissues: 70.13%(108/154) vs. 16.23%(25/154), there was statistical difference ( χ2 = 91.16, P<0.01). Within 3 years after surgery, the recurrence rate was 20.13%(31/154), and the positive expression rate of PRMT5 protein in the recurrence group was higher than that in the no recurrence group: 93.55%(29/31) vs. 64.23%(79/123), there was statistical difference ( χ2 = 10.16, P<0.05). The results of Cox regression model analysis showed that the tumor stage, lymph node metastasis and positive expression of PRMT5 protein in colorectal cancer tissues were independent risk factors for recurrence after radical resection of colorectal cancer ( P<0.05). Conclusions:The PRMT5 protein shows high expression in colorectal cancer tissues, and it is closely related to postoperative recurrence, and its positive expression can increase the risk of postoperative recurrence in patients with colorectal cancer.

PRMT6 promotes tumorigenicity and cisplatin response of lung cancer through triggering 6PGD/ENO1 mediated cell metabolism

Metabolic reprogramming is a hallmark of cancer, including lung cancer. However, the exact underlying mechanism and therapeutic potential are largely unknown. Here we report that protein arginine methyltransferase 6 (PRMT6) is highly expressed in lung cancer and is required for cell metabolism, tumorigenicity, and cisplatin response of lung cancer. PRMT6 regulated the oxidative pentose phosphate pathway (PPP) flux and glycolysis pathway in human lung cancer by increasing the activity of 6-phospho-gluconate dehydrogenase (6PGD) and α-enolase (ENO1). Furthermore, PRMT6 methylated R324 of 6PGD to enhancing its activity; while methylation at R9 and R372 of ENO1 promotes formation of active ENO1 dimers and 2-phosphoglycerate (2-PG) binding to ENO1, respectively. Lastly, targeting PRMT6 blocked the oxidative PPP flux, glycolysis pathway, and tumor growth, as well as enhanced the anti-tumor effects of cisplatin in lung cancer. Together, this study demonstrates that PRMT6 acts as a post-translational modification (PTM) regulator of glucose metabolism, which leads to the pathogenesis of lung cancer. It was proven that the PRMT6-6PGD/ENO1 regulatory axis is an important determinant of carcinogenesis and may become a promising cancer therapeutic strategy.

Effect of PRMT5 Expression Knockdown on Proliferation, Invasion and Apoptosis of Gastric Cancer Cells and Its Mechanism / 肿瘤防治研究

Objective To observe the expression level of PRMT5 in gastric cancer cells, and explore the effect of knocking down the expression level of PRMT5 on the biological behavior of gastric cancer cells. Methods Western blot was used to detect the expression of PRMT5 protein in gastric cancer cell lines MGC803, SGC7901, MKN45 and human gastric epithelial cells GES-1. siRNA1 and siRNA2 plasmids were transfected to knock down the expression of PRMT5. Cell proliferation and apoptosis assay, transwell assay were used to detect cell proliferation, apoptosis and invasion abilities, respectively. The protein expression levels of β-catenin, cyclin D1 and Bax were detected. Results Compared with GES-1 cells, PRMT5 protein expression levels increased in MGC803, SGC7901 and MKN45 cells (P < 0.001). After knocking down the expression of PRMT5, the cell proliferation and invasion abilities were weakened, the apoptosis rate increased (P < 0.05), the expression of β-catenin and Cyclin D1 protein decreased, and the expression of Bax protein increased (P < 0.001). Conclusion The expression level of PRMT5 is increased in gastric cancer cells. Knockdown of PRMT5 expression level could inhibit cell proliferation, invasion and promote cell apoptosis via reducing Wnt/β-catenin signaling pathway.

Research progress of protein arginine methyltransferase 5 inhibitors / 中国药科大学学报

@#Protein arginine methyltransferases, which proceed the post-translational modification of both histones and non-histones, play an important role in many biological pathways. Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for symmetric di-methylation of arginine residues and has been suggested as a potential therapeutic target for tumors.In the past decade,the discovery and development of PRMT5 inhibitors have become one of the most important research fields.This article introduces the structure and biochemical function of PRMT5 and its correlation with cancer reviews, the binding modes and biological data of PRMT5 inhibitors under development, and discusses the clinical application potential of PRMT5 inhibitors in the treatment of cancer.

Mining histone methyltransferases and demethylases from whole genome sequence

Epigenetic regulation through post-translational modification of histones, especially methylation, is wellconserved in evolution. Although there are several insect genomes sequenced, an analysis with a focus on theirepigenetic repertoire is limited. We have utilized a novel work-flow to identify one or more domains as highpriority domain (HPD), if present in at least 50% of the genes of a given functional class in the referencegenome, namely, that of Drosophila melanogaster. Based on this approach, we have mined histone methyltransferases and demethylases from the whole genome sequence of Aedes aegypti (Diptera), the pea aphidAcyrthosiphon pisum, the triatomid bug Rhodnius prolixus (Hemiptera), the honeybee Apis mellifera (Hymenoptera), the silkworm Bombyx mori (Lepidoptera) and the red flour beetle Tribolium castaneum(Coleoptera). We identified 38 clusters consisting of arginine methyltransferases, lysine methyltransferases anddemethylases using OrthoFinder, and the presence of HPD was queried in these sequences using InterProScan.This approach led us to identify putative novel members and currently inaccurate ones. Other than the highpriority domains, these proteins contain shared and unique domains that can mediate protein–protein interaction. Phylogenetic analysis indicates that there is different extent of protein sequence similarity; averagesimilarity between histone lysine methyltransferases varies from 41% (for active mark) to 48% (for repressivemark), arginine methyltransferases is 51%, and demethylases is 52%. The method utilized here facilitatesreliable identification of desired functional class in newly sequenced genomes

Design, synthesis and activity evaluation of protein arginine methyltransferase 5 inhibitor / 药学学报

Protein arginine methyltransferase 5 (PRMT5) is an important type II human methyltransferase. It catalyzes the symmetrical double-methylation of many histones and non-histones, and it is highly expressed in many kinds of tumors. PRMT5 has been proven to be a potential new target for cancer treatment. Based on the reported crystal complex of EPZ015666 with PRMT5, a series of new compounds was designed using GSK3326595 (EPZ015938) as the lead compound and using the conformational restriction approach. We found that compounds B8 and the C series of derivatives displayed enzyme inhibitory activity comparable to that of GSK3326595. Compounds C3 and C4 showed poor permeability in Caco-2 cells, and that might be one of the reasons for their poor anti-proliferative activity against Z-138 cells. These data provide insights for further structural optimization.

Role and mechanism of PRMT4 in genesis and progression of gastric cancer / 上海交通大学学报（医学版）

Objective • To investigate the effect of protein arginine methyltransferase 4(PRMT4) on the development and progression of gastric carcinoma and the underlying molecular mechanisms. Methods • The expression levels of PRMT4 in 32 specimens of gastric cancer and adjacent tissues were measured by immunohistochemistry technology. Public databases were used to analyze the expression of PRMT4 in gastric cancer and its effect on the survival of patients with gastric cancer. PRMT4 expressions of gastric cancer MGC-803 and MKN-45 cells were down-regulated by short hairpin RNA-mediated knockdown, and PRMT4 expression of gastric cancer SGC-7901 cells was up-regulated by using plasmid-mediated overexpression. Cell biology methods, such as MTT assay, colony formation assay, flow cytometry, and Transwell assay were used to study the effect of PRMT4 on proliferation, apoptosis, migration and invasion of gastric cancer cells. Luciferase reporter assay was used to detect transcription activity of endogenous transcription factor TCF (T cell factor) after PRMT4 silencing. The mRNA level changes of Wnt/β-catenin pathway downstream target genes after PRMT4 silencing were detected by real-time PCR. The protein level changes of Wnt/β-catenin pathway-related proteins after PRMT4 silencing and overexpression were detected by Western blotting. Results • PRMT4 was highly expressed in gastric cancer tissues. Patients with higher expression of PRMT4 had shorter survival than those with lower expression of PRMT4. The proliferation, colony formation, migration and invasion ability of gastric cancer cells were inhibited; the cell apoptosis was induced by silencing of PRMT4. The Wnt/β-catenin signaling pathway was also inhibited by PRMT4 knockdown. The proliferation, colony formation, migration and invasion ability of gastric cancer cells were induced; the cell apoptosis was inhibited by overexpressed PRMT4. The Wnt/β-catenin signaling pathway was also activated by PRMT4 overexpression. Conclusion • PRMT4 is highly expressed in gastric cancer. It can promote the growth, migration and invasion of gastric cancer cells through Wnt/β-catenin signaling pathway.

Essential Role of Protein Arginine Methyltransferase 1 in Pancreas Development by Regulating Protein Stability of Neurogenin 3

BACKGROUND: Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells. Recent studies have revealed that PRMT1 plays important roles in the development of various tissues. However, its role in pancreas development has not yet been elucidated. METHODS: Pancreatic progenitor cell-specific Prmt1 knock-out (Prmt1 PKO) mice were generated and characterized for their metabolic and histological phenotypes and their levels of Neurog3 gene expression and neurogenin 3 (NGN3) protein expression. Protein degradation assays were performed in mPAC cells. RESULTS: Prmt1 PKO mice showed growth retardation and a severely diabetic phenotype. The pancreatic size and β-cell mass were significantly reduced in Prmt1 PKO mice. Proliferation of progenitor cells during the secondary transition was decreased and endocrine cell differentiation was impaired. These defects in pancreas development could be attributed to the sustained expression of NGN3 in progenitor cells. Protein degradation assays in mPAC cells revealed that PRMT1 was required for the rapid degradation of NGN3. CONCLUSION: PRMT1 critically contributes to pancreas development by destabilizing the NGN3 protein.

Roles of protein arginine methyltransferase 5 in cancerand its clinical application / 中国肿瘤生物治疗杂志

@#[摘 要] 蛋白质精氨酸甲基转移酶5（protein arginine methyltransferase 5，PRMT5）是一种Ⅱ型蛋白质精氨酸甲基转移酶，在胃 癌、肝癌、结直肠癌、肺癌和乳腺癌等多种肿瘤中异常表达。异常表达的PRMT5通过调节细胞周期蛋白、 PI3K细胞信号通路等途 径影响肿瘤细胞，通过对相关肿瘤抑制基因的调控进而影响肿瘤的发生和发展。本文围绕近年来肿瘤中PRMT5的最新研究进 展，包括对雄激素受体、免疫细胞、 E2F-1-Bcl-2途径、PTEN和P16等的作用及其机制，并结合目前相关抑制剂的研究，讨论如何在 未来临床治疗中靶向PRMT5达到治疗肿瘤的目的。

Effect of prmt3 knockout on A549 cell line using CRISPR/Cas9 / 军事医学

Objective To construct plasmids for knock-out of protein arginine methyltransferase 3 (prmt3) gene using CRISPR/Cas9 gene editing method and examine the effect of prmt3 knockout on the proliferation of human non-small cell lung cancer(NSCLC)A549 cells.Methods Synthesized sgRNA oligos targeting prmt3 gene were cloned into LentiCRISPR vector and positive constructs confirmed by sequencing later .After infection with the packaged virus , A549 cells were screened with puromycin , and then the single clones were isolated .The protein level of PRMT3 in individual cell clones was analyzed with Western blot . Biological assay of clone formation , wound healing , flow cytometry assay and mass spectrometry ( MS) analysis were used to compare cellular proliferation behavior changes between control cells and cells with prmt3 gene knockout .Results The LentiCRISPR plasmids targeting prmt3 gene were confirmed by sequencing , and the PRMT3 protein level was significantly decreased in PRMT 3 KO cells compared with control cells .Depletion of PRMT3 promoted cell proliferation and led to cell cycle arrest at G 2/M phase, but had no influence on cell migration .Besides, some PRMT3 substrate candidates were identified with mass spectrum assays .Conclusion A549 cells with prmt3 gene knockout based on CRISPR/Cas9 are successfully established .PRMT3 can regulate cell cycle and proliferation .

Research progress of effect and mechanism of protein arginine methyltransferase in tumors / 上海交通大学学报（医学版）

Protein arginine methyltransferase (PRMT) can methylate a variety of proteins,including histones and non-histones,affecting a variety of cellular processes such as transcription,RNA splicing,DNA repair,and cell cycle control.The activity of PRMT is affected by a variety of regulatory mechanisms,and its abnormal expression plays an important role in the development of the diseases,especially in tumors such as breast cancer and leukemia.PRMT has a unique value in the diagnosis or treatment of tumors.With the further exploration of PRMT methylation mechanism,PRMT selective inhibitors have made some progress.PRMT-specific inhibitors are expected to be targeted drugs for the treatment of tumors.

Protein Methylation and Interaction with the Antiproliferative Gene, BTG2(/TIS21/Pc3)

The last one and half a decade witnessed an outstanding re-emergence of attention and remarkable progress in the field of protein methylation. In the present article, we describe the early discoveries in research and review the role protein methylation played in the biological function of the antiproliferative gene, BTG2(/TIS21/PC3).

Study on Biological Activity of Various Truncations of Human PRMT5 in E.coli / 生物化学与生物物理进展

Protein arginine methyltransferase 5 (PRMT5) has been implicated as an important regulator of many cellular processes and signaling pathways,including chromatin remodeling,RNA splicing,DNA transcription,and cell proliferation.Therefore,structural and functional studies on PRMT5 are quite important.The full length ofPRMT5 gene was cloned into vector pGEX-4T-1,resulting in only low expression levels in Escherichia coli (E.colO.Here,it was showed that the several N-terminal amino acids deletions could result in a significant increase in the amount of soluble ft"action,while one of them did not affect the protein-arginine methyltransferase activity.And it was also found that the N-terminal 15 amino acids region of PRMT5 may be important for the catalytic activity.

Protein arginine methyltransferase 1 methylates SF2/ASF at arginine / 中国肿瘤生物治疗杂志

Objective:To investigate the arginine (Arg) sites in splicing factor 2/alternative splicing factor (SF2/ASF) methylated by protein arginine methyltransferase 1 (PRMT1). Methods:Wild-type and Arg93,Arg97,Arg109 mutant SF2/ASF plasmids were constructed,and GST-PRMT1,GST-SF2/ASF and arginine mutant GST-SF2/ASF fusion proteins were induced and purified. Methylation activity of PRMT1 on wild-type or mutant SF2/ASF protein and methylated sites of SF2/ASF were examined by methylation assay. The effect of SF2/ASF methylation on its subcellular localization was analyzed by immunofluorescence assay.Results:PRMT1 induced methylation of SF2/ASF at arginine,and PRMT1 did not methylate SF2/ASF when SF2/ASF was mutant at Arg93,Arg97 or Arg109,with Arg97 mutation showing the most profound inhibitory effect. Methylation of SF2/ASF did not affect its subcellular localization.Conclusion:SF2/ASF is a newly identified substrate of PRMT1; Arg93,Arg97 and Arg 109 are the three methylation sites in SF2/ASF,and Arg97 is the main methylation site. Methylation of SF2/ASF does not affect its subcellular localization.