The skeletal muscle is the largest organ in mammals and is the primary motor function organ of the body. Our previous research has shown that long non-coding RNAs (lncRNAs) are significant in the epigenetic control of skeletal muscle development. Here, we observed progressive upregulation of lncRNA 4930581F22Rik expression during skeletal muscle differentiation. Knockdown of lncRNA 4930581F22Rik hindered skeletal muscle differentiation and resulted in the inhibition of the myogenic markers MyHC and MEF2C. Furthermore, we found that lncRNA 4930581F22Rik regulates myogenesis via the ERK/MAPK signaling pathway, and this effect could be attenuated by the ERK-specific inhibitor PD0325901. Additionally, in vivo mice injury model results revealed that lncRNA 4930581F22Rik is involved in skeletal muscle regeneration. These results establish a theoretical basis for understanding the contribution of lncRNAs in skeletal muscle development and regeneration.

目的 探讨体外扩增的外周血NK细胞对不同肿瘤细胞系的细胞毒作用.方法 人肝癌细胞株BEL-7402、HepG2、SMCC-7721,人肺癌细胞株A549和人宫颈癌细胞株Hela均为中山大学附属第三医院岭南医院生物治疗中心实验室保存并传代.10例健康志愿者外周血均采集自中山大学附属第三医院岭南医院,其中男5例,女5例;平均年龄(52±13)岁.所有受试者均签署知情同意书,符合医学伦理学规定.分离人外周血单核细胞,培养NK细胞,并采用流式细胞术鉴定.采用CCK-8法检测体外扩增的人外周血CD56+CD3-NK细胞对肿瘤细胞毒作用;观察不同波长、培养时间、靶细胞数和不同效靶比等条件下吸光度(A)值.结果 人外周血培养14 d后,CD56+CD3-NK细胞占(61±24)％.靶细胞最佳吸收波长为450 nm,最佳测定时间1～4 h,最佳细胞数量(1～4)x 104个/ml.NK细胞对不同肿瘤细胞系的细胞毒作用不同,其中在效靶比40∶ 1时,对BEL7402作用最强,其后依次为A549、SMMC7721、HepG2,作用最弱为Hela;在20 ∶ 1、10 ∶ 1时,对BEL7402作用最强,其后依次为SMMC7721、A549、HepG2,作用最弱为Hela.NK细胞对3株肝癌细胞的细胞毒作用也不同,其中对BEL7402作用最强,SMMC7721次之,HepG2最弱.NK细胞在效靶比40∶1时对 BEL-7402、SMCC-7721、A549、Hela 细胞的杀伤率明显高于 10∶1 时(LSD-t=2.10,2.37,2.26,5.61;P＜0.05);NK细胞在效靶比40∶1时对A549细胞的杀伤率明显高于20∶1时(LSD-t=1.14,P＜0.05).结论 效靶比40∶1时NK细胞对各肿瘤细胞系的细胞毒作用最强.NK细胞对不同肿瘤细胞系的细胞毒作用不同,对同一种肿瘤不同细胞株的细胞毒作用也不同.

Long non-coding RNAs (lncRNAs) regulate a series of physiological processes and play an important role in development, metabolism and disease. Our previous studies showed that lncRNAs involved in skeletal muscle differentiation. Here, we demonstrated that lncRNA Has2os is highly expressed in skeletal muscle and significantly elevated during skeletal cell differentiation. The knockdown of Has2os inhibited myocyte fusion and impeded the expression of the myogenic factors MyHC and Mef2C. Mechanically, Has2os regulates skeletal muscle differentiation by inhibiting the JNK/MAPK signaling pathway. Furthermore, we also revealed that Has2os is involved in the early stage of regeneration after muscle injury, and the JNK/MAPK signaling pathway is activated at both protein and mRNA levels during early repair. Our results demonstrate the new function of lncRNA Has2os, which plays crucial roles during skeletal muscle differentiation and muscle regeneration, providing a basis for the therapy of lncRNA-related muscle diseases.

目的:研究外周血自然杀伤(NK)细胞培养的扩增效率及杀伤活性的变化趋势.方法:采集10份健康供血者的外周血,采用无血清培养法对NK细胞进行扩增培养,并分别在培养的第0天、第4天、第8天、第12天、第16天、第20天进行细胞观察及计数、细胞表型分析和杀伤活性检测,总结外周血NK细胞培养的扩增效率及杀伤活性的变化趋势.结果:外周血单个核细胞(PBMCs)经诱导培养2～3 d即出现细胞集落.随着培养时间的延长,NK细胞的数量逐渐增加,不同培养时间点(第4天、第8天、第12天、第16天和第20天)NK细胞的数量相比,P<0.05;NK细胞的杀伤活性从培养第8天开始升高,至第12天达到峰值后开始下降,不同培养时间点NK细胞的杀伤活性相比,P<0.05;NK细胞的比例先增加后下降(在培养至16 d达到峰值后开始下降),其比例的下降趋势较杀伤活性的下降趋势滞后,不同培养时间点NK细胞的比例相比,P<0.05;各时间点NK细胞的存活率均大于95％.结论:采用无血清培养法对外周血NK细胞进行培养时,随着培养时间的延长,外周血NK细胞的数量呈逐渐增加的趋势.外周血NK细胞的杀伤活性在培养12 d后出现下降的趋势.在培养的第12天～第16天,NK细胞的杀伤活性较强,细胞数量较多,细胞纯度较高.采用无血清培养法体外扩增NK细胞的效率较高.

METTL3 increasing the mature miRNA levels via N6-Methyladenosine (m6A) modification of primary miRNA (pri-miRNA) transcripts has emerged as an important post-transcriptional regulation of miRNA biogenesis. Our previous studies and others have showed that muscle specific miRNAs are essential for skeletal muscle differentiation. Whether these miRNAs are also regulated by METTL3 is still unclear. Here, we found that m6A motifs were present around most of these miRNAs, which were indeed m6A modified as confirmed by m6A-modified RNA immunoprecipitation (m6A RIP). However, we surprisingly found that these muscle specific miRNAs were repressed instead of increased by METTL3 in C2C12 in vitro differentiation and mouse skeletal muscle regeneration after injury in vivo model. To elucidate the underlined mechanism, we performed reporter assays in 293T cells and validated METTL3 increasing these miRNAs at post-transcriptional level as expected. Furthermore, in myogenic C2C12 cells, we found that METTL3 not only repressed the expression of myogenic transcription factors (TFs) which can enhance the muscle specific miRNAs, but also increased the expression of epigenetic regulators which can repress these miRNAs. Thus, METTL3 could repress the muscle specific miRNAs at transcriptional level indirectly. Taken together, our results demonstrated that skeletal muscle specific miRNAs were repressed by METTL3 and such repression is likely synthesized transcriptional and post-transcriptional regulations.