AGAP2-AS1 affects TNM staging and prognosis of lung cancer patients by acting on SLC7A11 mRNA stability and ferroptosis

Objective The initiation and progression of lung carcinomas are critically regulated by long non-coding RNAs(lncRNAs).However,the role of lncRNAs in the pathways causing lung cancer remains unknown.Methods Cell morphology was regularly observed using an inverted phase-contrast microscope.Cell viability was assessed using CCK-8 according to the manufacturer’s instructions.Total RNA was retrotranscribed from each specimen using the RNAiso Plus Kit.The RT-PCR data were calculated using the Ct approach for comparison.Flow cytometric analyses were prepared by Click-iT™Plus TUNEL Assay for In Situ apoptosis detection,with Alexa Fluor^(TM)594 dye,as instructed.RNA immunoprecipitation assays were used to determine RNA concentration.Results Activated natural killer cells repeat and PH domain-containing protein 2 antisense RNA 1(AGAP2-AS1)levels in cancerous tissues were significantly correlated with cancerous tumor node metastasis(TNM)stage,with cancerous AGAP2-AS1 levels being higher in cancerous tissues than healthy tissues.Patients withelevated AGAP2-AS1 levels had considerably worse outcomes than those with reduced AGAP2-AS1 levels,regardless of the progression-free or overall survival.Functionally,AGAP2-AS1 downregulation represseslung cancer cell growth.AGAP2-AS1 elimination induces erastin-mediated ferroptosis in lung cancer cells.However,the ferritin inhibitor FERSINT-1 negated this result,whereas ERASTIN induced lung cancer cellmortality.After AGAP2-AS1 silencing,erastin-treated lung cancer cells showed a remarkable decrease inGSH levels.These results indicated that AGAP2-AS1 enhanced the stabilization of SLC7A11 mRNA via Recombinant Insulin Like Growth Factor Binding Protein 2(IGF BP2).Patients with elevated AGAP2-AS1 had considerably worse outcomes.Down-regulating AGAP2-AS1 was able to repress lung cancer cell growth and induce greater Erastin-mediated ferroptosis.Lungcancer cells treated with Erastin exhibited a remarkable decrease inglutathione(GSH)levels.The mechanical findingsindicated that AGAP2-AS1 enhanced the stabilization of SLC7A11 mRNA via the IGF2BP2.Conclusion We identified a novel effect of AGAP2-AS1 on TNM staging and the prognosis of patientswith lungcancer by modulating SLC7A11 mRNA stability and ferroptosis.

Post-transcriptional regulation of vascular endothelial growth factor: Implications for tumor angiogenesis

Vascular endothelial growth factor （VEGF） is a potent secreted mitogen critical for physiologic and tumor angiogenesis. Regulation of VEGF occurs at several levels, including transcription, mRNA stabilization, translation, and differential cellular localization of various isoforms. Recent advances in our understanding of post-transcriptional regulation of VEGF include identification of the stabilizing mRNA binding protein, HuR, and the discovery of internal ribosomal entry sites in the 5＇UTR of the VEGF mRNA. Monoclonal anti-VEGF antibody was recently approved for use in humans, but suffers from the need for high systemic doses. RNA interference （RNAi） technology is being used in vitro and in animal models with promising results. Here, we review the literature on post-transcriptional regulation of VEGF and describe recent progress in targeting these mechanisms for therapeutic benefit.

The m^(6)A reader SiYTH1 enhances drought tolerance by affecting the messenger RNA stability of genes related to stomatal closure and reactive oxygen species scavenging in Setaria italica

Foxtail millet(Setaria italica),a vital drought-resistant crop,plays a significant role in ensuring food and nutritional security.However,its drought resistance mechanism is not fully understood.N6-methyladenosine(m^(6)A)modification of RNA,a prevalent epi-transcriptomic modification in eukaryotes,provides a binding site for m^(6)A readers and affects plant growth and stress responses by regulating RNA metabolism.In this study,we unveiled that the YT521-B homology(YTH)family gene SiYTH1 positively regulated the drought tolerance of foxtail millet.Notably,the siyth1 mutant exhibited reduced stomatal closure and augmented accumulation of excessive H_(2)O_(2)under drought stress.Further investigations demonstrated that SiYTH1 positively regulated the transcripts harboring m^(6)A modification related to stomatal closure and reactive oxygen species(ROS)scavenging under drought stress.SiYTH1 was uniformly distributed in the cytoplasm of SiYTH1-GFP transgenic foxtail millet.It formed dynamic liquid-like SiYTH1 cytosol condensates in response to drought stress.Moreover,the cytoplasmic protein SiYTH1 was identified as a distinct m^(6)A reader,facilitating the stabilization of its directly bound SiARDP and ROS scavenging-related transcripts under drought stress.Furthermore,natural variation analysis revealed SiYTH1AGTG as the dominant allele responsible for drought tolerance in foxtail millet.Collectively,this study provides novel insights into the intricate mechanism of m^(6)A reader-mediated drought tolerance and presents a valuable genetic resource for improving drought tolerance in foxtail millet breeding.

mRNA stability in the nucleus

Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mRNA stability during RNA processing and translation. RNAs, especially mRNAs, are relatively vulnerable molecules in living cells for ribonucleases （RNases）. The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mRNA stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase Ⅱ in eukaryotes.

Long Non-coding RNAs in the Cytoplasm

An enormous amount of long non-coding RNAs（lnc RNAs） transcribed from eukaryotic genome are important regulators in different aspects of cellular events. Cytoplasm is the residence and the site of action for many lncRNAs. The cytoplasmic lncRNAs play indispensable roles with multiple molecular mechanisms in animal and human cells. In this review, we mainly talk about functions and the underlying mechanisms of lncRNAs in the cytoplasm. We highlight relatively well-studied examples of cytoplasmic lncRNAs for their roles in modulating mRNA stability,regulating m RNA translation, serving as competing endogenous RNAs, functioning as precursors of microRNAs, and mediating protein modifications. We also elaborate the perspectives of cytoplasmic lncRNA studies.

N-acetyltransferase 10 promotes colon cancer progression by inhibiting ferroptosis through N4-acetylation and stabilization of ferroptosis suppressor protein 1 (FSP1) mRNA

Background:N-acetyltransferase 10(NAT10)is the only enzyme known tomediate the N4-acetylcytidine(ac4C)modification of mRNA and is crucial formRNA stability and translation efficiency.However,its role in cancer development and prognosis has not yet been explored.This study aimed to examine the possible role of NAT10 in colon cancer.Methods:The expression levels ofNAT10were evaluated by immunohistochemical analyses with a colon cancer tissue microarray,and its prognostic value in patients was further analyzed.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blotting were performed to analyze NAT10 expression in harvested colon cancer tissues and cell lines.Stable NAT10-knockdown and NAT10-overexpressing colon cancer cell lines were constructed using lentivirus.The biological functions of NAT10 in colon cancer cell lines were analyzed in vitro by Cell Counting Kit-8(CCK-8),wound healing,Transwell,cell cycle,and ferroptosis assays.Xenograft models were used to analyze the effect of NAT10 on the tumorigenesis and metastasis of colon cancer cells in vivo.Dot blotting,acetylated RNA immunoprecipitation-qPCR,and RNA stability analyses were performed to explore the mechanism by which NAT10 functions in colon cancer progression.Results:NAT10 was upregulated in colon cancer tissues and various colon cancer cell lines.This increased NAT10 expression was associated with shorter patient survival.Knockdown of NAT10 in two colon cancer cell lines(HT-29 and LoVo)impaired the proliferation,migration,invasion,tumor formation and metastasis of these cells,whereas overexpression of NAT10 promoted these abilities.Further analysis revealed that NAT10 exerted a strong effect on the mRNA stability and expression of ferroptosis suppressor protein 1(FSP1)in HT-29 and LoVo cells.In these cells,FSP1 mRNA was found to be modified by ac4C acetylation,and this epigenetic modification was associated with the inhibition of ferroptosis.Conclusions:Our study revealed that NAT10 plays a critical role in colon cancer development by affecting FSP1 mRNA stability and ferroptosis,suggesting that NAT10 could be a novel prognostic and therapeutic target in colon cancer.

Loss of lncRNA SNHG8 promotes epithelial-mesenchymal transition by destabilizing CDH1 mRNA

Long non-coding RNAs(lnc RNAs) are widely involved in a variety of biological processes, including epithelial-mesenchymal transition(EMT). In the current study, we found that lnc RNA small nucleolar RNA host gene 8(SNHG8) was tightly correlated with EMT-associated gene signatures, and was down-regulated by Zinc finger E-box-binding homeobox 1(ZEB1) during EMT progress. Functionally, knockdown of SNHG8 induced EMT in epithelial cells, through destabilizing the CDH1 m RNA dependent on a 17-nucleotide sequence shared by SNHG8 and CDH1. In addition, analysis with public database showed that SNHG8 tended to be down-regulated in different cancer types and the lower expression of SNHG8 predicted poorer prognosis.Taken together, our study reports a ZEB1-repressed lnc RNA SNHG8 which is important for stabilizing CDH1 m RNA, thereby maintaining the epithelial status of epithelial cells.

Gene amplification-driven RNA methyltransferase KIAA1429 promotes tumorigenesis by regulating BTG2 via m6A-YTHDF2-dependent in lung adenocarcinoma

Background:Epigenetic alterations have been shown to contribute immensely to human carcinogenesis.Dynamic and reversible N6-methyladenosine(m6A)RNA modification regulates gene expression and cell fate.However,the reasons for activation of KIAA1429(also known as VIRMA,an RNA methyltransferase)and its underlying mechanism in lung adenocarcinoma(LUAD)remain largely unexplored.In this study,we aimed to clarify the oncogenic role of KIAA1429 in the tumorigenesis of LUAD.Methods:Whole-genome sequencing and transcriptome sequencing of LUAD data were used to analyze the gene amplification of RNA methyltransferase.The in vitro and in vivo functions of KIAA1429 were investigated.Transcriptome sequencing,methylated RNA immunoprecipitation sequencing(MeRIP-seq),m6A dot blot assays and RNA immunoprecipitation(RIP)were performed to confirm the modified gene mediated by KIAA1429.RNA stability assays were used to detect the half-life of the target gene.Results:Copy number amplification drove higher expression of KIAA1429 in LUAD,whichwas correlatedwith poor overall survival.Manipulating the expression of KIAA1429 could regulate the proliferation and metastasis of LUAD.Mechanistically,the target genes of KIAA1429-mediated m6A modification were confirmed by transcriptome sequencing and MeRIP-seq assays.We also revealed that KIAA1429 could regulate BTG2 expression in an m6A-dependent manner.Knockdown of KIAA1429 significantly decreased the m6A levels of BTG2 mRNA,leading to enhanced YTH m6A RNA binding protein 2(YTHDF2,the m6A“reader”)-dependent BTG2 mRNA stability and promoted the expression of BTG2;thus,participating in the tumorigenesis of LUAD.Conclusions:Our data revealed the activation mechanism and important role of KIAA1429 in LUAD tumorigenesis,which may provide a novel view on the targeted molecular therapy of LUAD.

Post-transcriptional regulation of erythropoiesis

Erythropoiesis is a complex,precise,and lifelong process that is essential for maintaining normal body functions.Its strict regulation is necessary to prevent a variety of blood diseases.Normal erythropoiesis is precisely regulated by an intricate network that involves transcription levels,signal transduction,and various epigenetic modifications.In recent years,research on posttranscriptional levels in erythropoiesis has expanded significantly.The dynamic regulation of splicing transitions is responsible for changes in protein isoform expression that add new functions beneficial for erythropoiesis.RNA-binding proteins adapt the translation of transcripts to the protein requirements of the cell,yielding mRNA with dynamic translation efficiency.Noncoding RNAs,such as microRNAs and lncRNAs,are indispensable for changing the translational efficiency and/or stability of targeted mRNAs to maintain the normal expression of genes related to erythropoiesis.N6-methyladenosine-dependent regulation of mRNA translation plays an important role in maintaining the expression programs of erythroid-related genes and promoting erythroid lineage determination.This review aims to describe our current understanding of the role of post-transcriptional regulation in erythropoiesis and erythroid-associated diseases,and to shed light on the physiological and pathological implications of the post-transcriptional regulation machinery in erythropoiesis.These may help to further enrich our understanding of the regulatory network of erythropoiesis and provide new strategies for the diagnosis and treatment of erythroid-related diseases.

Mevalonate improves anti-PD-1/PD-L1 efficacy by stabilizing CD274 mRNA

Mevalonate metabolism plays an important role in regulating tumor growth and progression;however,its role in immune evasion and immune checkpoint modulation remains unclear.Here,we found that non-small cell lung cancer(NSCLC)patients with higher plasma mevalonate response better to antiPD-(L)1 therapy,as indicated by prolonged progression-free survival and overall survival.Plasma mevalonate levels were positively correlated with programmed death ligand-1(PD-L1)expression in tumor tissues.In NSCLC cell lines and patient-derived cells,supplementation of mevalonate significantly upregulated the expression of PD-L1,whereas deprivation of mevalonate reduced PD-L1 expression.Mevalonate increased CD274 mRNA level but did not affect CD274 transcription.Further,we confirmed that mevalonate improved CD274 mRNA stability.Mevalonate promoted the affinity of the AU-rich elementbinding protein HuR to the 3'-UTR regions of CD274 mRNA and thereby stabilized CD274 mRNA.By in vivo study,we further confirmed that mevalonate addition enhanced the anti-tumor effect of anti-PD-L1,increased the infiltration of CD8^(+)T cells,and improved cytotoxic function of T cells.Collectively,our findings discovered plasma mevalonate levels positively correlated with the therapeutic efficacy of anti-PD-(L)1 antibody,and provided the evidence that mevalonate supplementation could be an immunosensitizer in NSCLC.

Ribonucleotide reductase small subunit M2 promotes the proliferation of esophageal squamous cell carcinoma cells via HuR-mediated mRNA stabilization

Esophageal squamous cell carcinoma(ESCC),a malignancy of the digestive system,is highly prevalent and the primary cause of cancer-related deaths worldwide due to the lack of early diagnostic biomarkers and effective therapeutic targets.Dysregulated ribonucleotide reductase(RNR)expression has been confirmed to be causally linked to tumorigenesis.This study demonstrated that ribonucleotide reductase small subunit M2(RRM2)is significantly upregulated in ESCC tissue and that its expression is negatively correlated with clinical outcomes.Mechanistically,HuR promotes RRM2 mRNA stabilization by binding to the adenine/uridine(AU)-rich elements(AREs)within the 3′UTR,resulting in persistent overexpression of RRM2.Furthermore,bifonazole is identified as an inhibitor of HuR via computational screening and molecular docking analysis.Bifonazole disrupts HuR-ARE interactions by competitively binding to HuR at F65,R97,I103,and R153 residues,resulting in reduced RRM2 expression.Furthermore,bifonazole exhibited antitumor effects on ESCC patient-derived xenograft(PDX)models by decreasing RRM2 expression and the dNTP pool.In summary,this study reveals the interaction network among HuR,RRM2,and bifonazole and demonstrated that bifonazole is a potential therapeutic compound for ESCC through inhibition of the HuR/RRM2 axis.