R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling

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FTO regulates adipogenesis by controlling cell cycle progression via m6A-YTHDF2 dependent mechanism

N⁶-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes. Loss of m⁶A demethylase FTO increases m⁶A levels and inhibits adipogenesis of preadipocytes. However, its underlying mechanism remains elusive. Here, we demonstrated that silencing FTO inhibited adipogenesis of preadipocytes through impairing cell cycle progression at the early stage of adipogenesis. FTO knockdown markedly decreased the expression of CCNA2 and CDK2, crucial cell cycle regulators, leading to delayed entry of MDI-induced cells into G2 phase. Furthermore, the m⁶A levels of CCNA2 and CDK2 mRNA were significantly upregulated following FTO knockdown. m⁶A-binding protein YTHDF2 recognized and decayed methylated mRNAs of CCNA2 and CDK2, leading to decreased protein expression, thereby prolonging cell cycle progression and suppressing adipogenesis. Our work unravels that FTO regulates adipogenesis by controlling cell cycle progression in an m⁶A-YTHDF2 dependent manner, which provides insights into critical roles of m⁶A methylation in adipogenesis.     

HPV E6/E7 promotes aerobic glycolysis in cervical cancer by regulating IGF2BP2 to stabilize m6A-MYC expression

Enhanced aerobic glycolysis constitutes an additional source of energy for tumor proliferation and metastasis. Human papillomavirus (HPV) infection is the main cause of cervical cancer (CC); however, the associated molecular mechanisms remain poorly defined, as does the relationship between CC and aerobic glycolysis. To investigate whether HPV 16/18 E6/E7 can enhance aerobic glycolysis in CC, E6/E7 expression was knocked down in SiHa and HeLa cells using small interfering RNA (siRNA). Then, glucose uptake, lactate production, ATP levels, reactive oxygen species (ROS) content, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were evaluated. RNA-seq was used to probe the molecular mechanism involved in E6/E7-driven aerobic glycolysis, and identified IGF2BP2 as a target of E6/E7. The regulatory effect of IGF2BP2 was confirmed by qRT-PCR, western blot, and RIP assay. The biological roles and mechanisms underlying how HPV E6/E7 and IGF2BP2 promote CC progression were confirmed in vitro and in vivo. Human CC tissue microarrays were used to analyze IGF2BP2 expression in CC. The knockdown of E6/E7 and IGF2BP2 attenuated the aerobic glycolytic capacity and growth of CC cells, while IGF2BP2 overexpression rescued this effect in vitro and in vivo. IGF2BP2 expression was higher in CC tissues than in adjacent tissues and was positively correlated with tumor stage. Mechanistically, E6/E7 proteins promoted aerobic glycolysis, proliferation, and metastasis in CC cells by regulating MYC mRNA m⁶A modifications through IGF2BP2. We found that E6/E7 promote CC by regulating MYC methylation sites via activating IGF2BP2 and established a link between E6/E7 and the promotion of aerobic glycolysis and CC progression. Blocking the HPV E6/E7-related metabolic pathway represents a potential strategy for the treatment of CC.     

Circ_0137287 suppresses cell tumroigenesis and aerobic glycolysis in papillary thyroid carcinoma through miR-183-5p/PPP2R2A axis

Cytotechnology - Circ_0137287 was found to be decreased in papillary thyroid cancer (PTC) tissues and related to aggressive clinicopathologic characteristics. However, the role and mechanism of...     

m6A甲基化修饰识别蛋白YTHDF2在肝癌组织中的表达及临床意义

摘要 目的：探讨m6A甲基化修饰结合蛋白YTHDF2(YTH domain-containing family protein 2)在肝癌（Hepatocellular carcinoma, HCC）组织中的表达及临床意义。方法：1）采用Western blot和实时荧光定量PCR（Quantitative Real Time PCR, qRT-PCR）检测20对肝癌和癌旁组织中YTHDF2在蛋白和mRNA水平的表达情况。2）通过免疫组织化学（Immunochemistry, IHC）检测40对肝癌和癌旁组织芯片YTHDF2的表达情况,并以H-score评分法进行半定量分析。3）通过基因表达谱数据动态分析数据库GEPIA（http://gepia.cancer-pku.cn/）分析YTHDF2在肝癌组织中的表达及对患者生存预后的影响;通过肿瘤免疫评估资源数据库TIMER(https://cistrome.shinyapps.io/timer/)分析YTHDF2与肝癌免疫微环境的相关性。结果：相比于癌旁肝组织,YTHDF2在肝癌组织中蛋白和mRNA水平均显著高表达（P＜0.05）;GEPIA数据库分析验证：YTHDF2肝癌组织表达水平高于正常肝组织,且YTHDF2在肝癌不同分期中表达水平均高于正常肝组织（P=0.0206）,Stage III期最为明显;YTHDF2高表达患者的总体生存率（Overall Survival,OS）和无复发生存率（Disease Free Survival, DFS）均较差（P=0.00027和P=0.013）;TIMER数据库分析表明：YTHDF2在肝癌免疫微环境中与各类免疫细胞呈正相关（P＜0.05）,但肝癌患者的累积存活率不受免疫细胞的影响（P＞0.05）,YTHDF2高表达对患者的生存预后具有不良影响（P=0.007）。结论：YTHDF2在肝癌组织中显著高表达,且YTHDF2高表达水平对肝癌患者生存预后具有不良影响,YTHDF2可作为肝癌临床预后判断的分子标志物,为今后肝癌治疗靶点提供新的策略。     

Hexokinase2 controls angiogenesis in melanoma by promoting aerobic glycolysis and activating the p38-MAPK signaling

In this study, we used endostatin (ES)-induced apoptosis of endothelial cells to study the role of Hexokinase2 (HK2) in the control of angiogenesis in melanoma. Real-time polymerase chain reaction and Western blot analysis were performed to explore the effect of HK2, lactate, and ES on the levels of caspase-9/3, ATP, and p38/MAPK activation. ES increased the levels of caspase-9/3 while decreasing the level of ATP, whereas ES + HK2 and lactate both restored the normal levels of caspase-9/3 and ATP. In addition, cells transfected with HK2 short hairpin RNA1 (HK2shRNA1) and HK2shRNA2 showed an evident decrease in the levels of caspase-9/3 along with an obvious increase in the level of ATP. Knockdown of HK2 also increased O₂ consumption while decreasing the extracellular level of lactate and the phosphorylation of p38-mitogen-activated protein kinase (MAPK). On the other hand, the lactate treatment elevated the phosphorylation of p38-MAPK under time- and concentration-dependent manner. In the study, we clarified the role of HK2 in the control of apoptosis of ECs, which plays an important role in the angiogenesis of melanoma by promoting aerobic glycolysis and activating the p38-MAPK signaling.     

Circular RNA circMDM2 accelerates the glycolysis of oral squamous cell carcinoma by targeting miR‐532‐3p/HK2

Circular RNAs (circRNAs) function as an essential regulator in the progression of oral squamous cell carcinoma (OSCC). However, the potential roles and mechanism of circRNAs in OSCC are still elusive. Here, this research investigates the roles and molecular mechanism of novel circRNA (circMDM2) in OSCC progression. Clinically, circMDM2 was overexpressed in OSCC tissue and cells, and the overexpression served as a poor prognostic factor for OSCC patients. Functionally, cellular experiments confirmed that circMDM2 accelerated OSCC cell proliferation and glycolysis in vitro and circMDM2 knockdown repressed the tumour growth in vivo. Mechanistically, circMDM2 sponged miR‐532‐3p to promote the hexokinase 2 (HK2), forming the circMDM2/miR‐532‐3p/HK2 axis. In conclusion, these findings demonstrated that circMDM2/miR‐532‐3p/HK2 axis promotes the proliferation and glycolysis of OSCC, rendering a potential diagnostic biomarker and prospective therapeutic target for OSCC.     

METTL3介导EZH2 m6A修饰在结直肠癌5-氟尿嘧啶耐药中的作用机制研究

摘要 目的：探讨甲基转移酶样蛋白3（METTL3）在5-氟尿嘧啶耐药中的作用机制。方法：大剂量间歇诱导法建立5-FU耐药细胞株。在耐药组细胞中分别敲减METTL3及EZH2抑制GSK343处理。qPCR及Western blot检测METTL3和EZH2表达。CCK-8检测各组细胞增殖情况。m6A甲基化RNA免疫沉淀技术分析EZH2 mRNA m6A修饰修饰情况。结果：各药物浓度处理下的耐药组细胞增殖活性与未处理细胞无显著差异（P>0.05）而原代细胞组肠癌细胞较未处理细胞在0.5-10 μg/mL处理下细胞增殖活性显著减低（P<0.01）。耐药组细胞与原代细胞组相比,METTL3及EZH2表达水平显著升高（P<0.01）。耐药组细胞METTL3敲减后或GSK343处理后,在0.5 μg/mL-10 μg/mL浓度间下的细胞增殖活性与0 μg/mL处理细胞增殖活性相比显著减低（P<0.05）。耐药组细胞METTL3敲减细胞的EZH2表达与对照细胞比,显著下调（P<0.01）。m6A甲基化RNA免疫沉淀实验显示耐药组细胞METTL3敲减细胞的EZH2 mRNA m6A修饰水平（m6A富集度为6361.95±67.47%）,较未敲减细胞修饰水平（396.30±57.74）显著减低（P<0.01）。结论：METTL3在肠癌细胞5-FU耐药抵抗中起到关键作用,靶向抑制METTL3有望成为缓解肠癌耐药的重要分子靶点。     

circ-BIRC6, a circular RNA,promotes hepatocellular carcinoma progression by targeting the miR-3918/Bcl2 axis

Circular (circ)RNA is a special type of endogenous RNA consisting of a covalently closed loop structure without 5‘ to 3‘ polarity and a polyadenylated tail. Accumulating evidence suggests that circRNAs play important roles in the development and progression of human cancers. However, the role of circRNAs in the progression of hepatocellular carcinoma (HCC) is largely unknown. This was addressed in the present study using high-throughput sequencing to identify aberrantly expressed circRNAs in HCC patient tissue and cell lines. We found that circ-baculoviral IAP repeat-containing (BIRC)6 was upregulated in HCC tissue samples and cells; this was associated with the overall survival of HCC patients. circ-BIRC6 knockdown reduced HCC cell proliferation, migration, and invasion and enhanced their apoptosis. Additionally, circ-BIRC6 overexpression negatively regulated the expression of microRNA miR-3918, which was identified as an inhibitor of B cell lymphoma (Bcl)2. The tumor-suppressive effect of circ-BIRC6 deletion was abrogated by inhibiting miR-3918. These results indicate that circ-BIRC6 functions as a competing endogenous RNA that regulates Bcl2 expression by sponging miR-3918, and may serve as a prognostic biomarker and therapeutic target for the treatment of HCC.