技术领域Technical Field
本发明属于糖尿病诊断和治疗技术领域，具体涉及一种环状RNA及其m6A甲基化修饰的应用。The present invention belongs to the technical field of diabetes diagnosis and treatment, and specifically relates to an application of circular RNA and m6A methylation modification thereof.
背景技术Background technique
1型糖尿病（T1D）是遗传易感基因和环境因素相互作用，诱发免疫攻击破坏胰岛β细胞，导致胰岛素绝对缺乏的自身免疫性疾病。T1D患者的血糖达标率低，并发症多且重，给病人带来健康与经济上的双重打击。T1D发病机制复杂，涉及遗传、环境、免疫等多个关键环节。目前，遗传易感基因和环境因素相互作用诱发免疫攻击破坏胰岛β细胞的关键调控机制尚不清楚，这一现状严重阻碍了对T1D的早期诊断、治疗药物的开发和精准干预的开展，给糖尿病管理带来巨大的挑战。Type 1 diabetes (T1D) is an autoimmune disease caused by the interaction between genetic susceptibility genes and environmental factors, which induces immune attacks and destroys pancreatic beta cells, leading to absolute insulin deficiency. T1D patients have a low blood sugar compliance rate and multiple and severe complications, which brings a double blow to patients in terms of health and economy. The pathogenesis of T1D is complex, involving multiple key links such as genetics, environment, and immunity. At present, the key regulatory mechanism of the interaction between genetic susceptibility genes and environmental factors to induce immune attacks and destroy pancreatic beta cells is still unclear. This situation seriously hinders the early diagnosis of T1D, the development of therapeutic drugs, and the implementation of precision intervention, and brings huge challenges to diabetes management.
circRNA是一种新型非编码RNA，无5’端和3’端，而是通过共价键形成的一类具有闭合结构的RNA分子。circRNA的结构稳定，并具有多种生物学功能，研究表明，circRNA诱导的基因表达调节在免疫系统中起着重要作用，这与自身免疫性疾病的发生和发展密切相关。m6A修饰是非编码RNA产生和发挥功能的重要调节因子。circRNA is a new type of non-coding RNA. It has no 5' end and 3' end, but is a type of RNA molecule with a closed structure formed by covalent bonds. The structure of circRNA is stable and has multiple biological functions. Studies have shown that circRNA-induced gene expression regulation plays an important role in the immune system, which is closely related to the occurrence and development of autoimmune diseases. m6A modification is an important regulatory factor in the production and function of non-coding RNA.
综上，circRNA的m6A甲基化修饰在T1D中的作用研究有非常大的探索空间，本发明旨在探讨circRNA m6A甲基化修饰在T1D中的作用，有望为T1D的早期诊断提供新的分子标志物，或阐明新的调控通路以及寻找潜在治疗靶点，对寻求新的从根本上攻克T1D的预防、早期诊断和治疗方法具有重要价值。In summary, there is a lot of room for exploration in the role of circRNA m6A methylation modification in T1D. The present invention aims to explore the role of circRNA m6A methylation modification in T1D, which is expected to provide new molecular markers for the early diagnosis of T1D, or to elucidate new regulatory pathways and find potential therapeutic targets, which is of great value in seeking new prevention, early diagnosis and treatment methods to fundamentally overcome T1D.
发明内容Summary of the invention
本发明的目的在于提供一种环状RNA及其m6A甲基化修饰的应用，以解决背景技术中提出的寻求新的从根本上攻克1型糖尿病的预防、早期诊断和治疗方法的问题。The purpose of the present invention is to provide an application of circular RNA and its m6A methylation modification to solve the problem of seeking new prevention, early diagnosis and treatment methods for fundamentally overcoming type 1 diabetes proposed in the background technology.
为实现上述目的，本发明提供了环状RNA作为1型糖尿病的生物标志物在制备诊断试剂盒中的应用，所述的环状RNA为hsa_circRNA_005995。To achieve the above objectives, the present invention provides the use of circular RNA as a biomarker for type 1 diabetes in the preparation of a diagnostic kit, wherein the circular RNA is hsa_circRNA_005995.
hsa_circRNA_005995序列为：The sequence of hsa_circRNA_005995 is:
GCAAAATCCTGACAGATGACTTTGCAGACAAGGAGGGTCTGGAAATGGGTGATGAGTACTTTGCAAATCTGGACCATATCGAGAGCGTGGAGAACTTCAAAGAAGGATATGAGAGTGATGCCCCCTGTTCCTCTGACAGCAGTGGTGTAGACTTGAAGGACCAGGAAGATGGCAACAGCGGTACAGAGGACCCTGAAGAGTCCAATGATGATAGCTCAGATGATAACTTCTGTAAGGATGAGGACTTCAGCACCAGTTCAGTGTGGCGGAGCTATGCTACCCGGAGGCAGACCCGGGGCCAGAAAGAGAACGGACTCTCTGAGACAACTTCCAAGGACTCCCACCCCCCAGATCTTGGACCCCCACATATTCCTGTTCCTCCCTCAATCCCTGTAGGTGGCTGCAATCCACCTTCCTCCGAAGAGACACCCAAGAACAAGGTGGCCTCATGGTTGAGCTGCAATAGTGTCAGTGAAGGTGGTTTTGCTGACTCTGATAGCCATTCATCCTTCAAGACTAATGAAGGTGGGGAGGGCCGGGCTGGGGGAAGCCGAATGGAGGCTGAGAAGGCCTCCACCTCAGGACTAGGCATCAAGGATGAGGGAGACATCAAACAGGCCAAGAAAGAGGACACTGACGACCGAAACAAGATGTCAGT。GCAAAATCCTGACAGATGACTTTGCAGACAAGGAGGGTCTGGAAATGGGTGATGAGTACTTTGCAAATCTGGACCATATCGAGAGCGTGGAGAACTTCAAAGAAGGATATGAGAGTGATGCCCCCTGTTCCTCTGACAGCAGTGGTGTAGACTTGAAGGACCAGGAAGATGGCAACAGCGGTACAGAGGACCCTGAAGAGTCCAATGATGATAGCTCAGATGATAACTTCTGTAAGGATGAGGACTTCAGCACCAGTT CAGTGTGGCGGAGCTATGCTACCCGGAGGCAGACCCGGGGCCAGAAAGAGAACGGACTCTCTGAGACAACT TCCAAGGACTCCCACCCCCCAGATCTTGGACCCCCACATATTCCGTTCCTCCCTCAATCCCTGTAGGTGGCTGCAATCCACCTTCCTCCGAAGAGACACCCAAGAACAAGGTGGCCTCATGGTTGAGCTGCAATAGTGTCAGTGAAGGTGGTTTTGCTGACTCTGATAGCCATTCATCCTTCAAGACTAATGAAGGTGGGGAGGGCCGGGCTGGGGGAAGCCGAATGGAGGCTGAGAAGGCCTCCACCTCAGGACTAG GCATCAAGGATGAGGGAGACATCAAACAGGCCAAGAAAGAGGACACTGACGACCGAAACAAGATGTCAGT.
本发明还提供了检测环状RNA的试剂在制备1型糖尿病诊断制剂中的应用，所述的环状RNA为hsa_circRNA_005995。The present invention also provides the use of a reagent for detecting circular RNA in the preparation of a type 1 diabetes diagnostic preparation, wherein the circular RNA is hsa_circRNA_005995.
本发明还提供了一种1型糖尿病诊断制剂，含有检测环状RNA hsa_circRNA_005995的试剂。The present invention also provides a type 1 diabetes diagnostic preparation, comprising a reagent for detecting circular RNA hsa_circRNA_005995.
本发明还提供了过表达hsa_circRNA_005995的试剂在制备治疗1型糖尿病药物中的应用。The present invention also provides the use of a reagent for overexpressing hsa_circRNA_005995 in the preparation of a drug for treating type 1 diabetes.
本发明还提供了治疗1型糖尿病药物，是过表达hsa_circRNA_005995的试剂。The present invention also provides a drug for treating type 1 diabetes, which is a reagent for overexpressing hsa_circRNA_005995.
本发明还提供了环状RNA的m6A甲基化修饰作为1型糖尿病的生物标志物在制备诊断试剂盒中的应用，所述环状RNA的m6A甲基化修饰中的环状RNA为hsa_circRNA_005995。The present invention also provides the use of m6A methylation modification of circular RNA as a biomarker for type 1 diabetes in the preparation of a diagnostic kit, wherein the circular RNA in the m6A methylation modification of circular RNA is hsa_circRNA_005995.
本发明还提供了检测环状RNA的m6A甲基化修饰的试剂在制备1型糖尿病诊断制剂中的应用，所述环状RNA的m6A甲基化修饰中的环状RNA为hsa_circRNA_005995。The present invention also provides the use of a reagent for detecting m6A methylation modification of circular RNA in the preparation of a type 1 diabetes diagnostic preparation, wherein the circular RNA in the m6A methylation modification of the circular RNA is hsa_circRNA_005995.
本发明还提供了一种1型糖尿病诊断制剂，含有检测环状RNA hsa_circRNA_005995的m6A甲基化修饰的试剂。The present invention also provides a type 1 diabetes diagnostic preparation, comprising a reagent for detecting m6A methylation modification of circular RNA hsa_circRNA_005995.
本发明还提供了过表达hsa_circRNA_005995的m6A甲基化修饰的试剂在制备治疗1型糖尿病药物中的应用。The present invention also provides the use of a reagent for m6A methylation modification of overexpressed hsa_circRNA_005995 in the preparation of a drug for treating type 1 diabetes.
本发明还提供了治疗1型糖尿病药物，是过表达hsa_circRNA_005995的m6A甲基化修饰的试剂。The present invention also provides a drug for treating type 1 diabetes, which is a reagent for m6A methylation modification of overexpressed hsa_circRNA_005995.
相比于现有技术，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明对从根本上攻克T1D的预防、早期诊断和治疗方法具有重要价值。The present invention has important value in fundamentally overcoming the prevention, early diagnosis and treatment methods of T1D.
本发明收集了36例T1D患者和36例葡萄糖耐量正常对照者的外周血提取外周血单个核细胞（PBMC），抽提RNA，采用人Arraystar m6A-circRNA表观转录组芯片检测，Arraystar m6A-circRNA芯片结果显示，与正常对照组相比，T1D组中有m6A差异的circRNA共有11779个，经过筛选P<0.05且FC≥1.5的共有428个，其中，17个显著上调，411个显著下调。由于差异的circRNAs数量过多，筛选差异倍数最为显著的28个m6A-circRNAs制成了热图，以此预测有m6A差异的circRNA在T1D发生、发展中的作用。The present invention collected peripheral blood mononuclear cells (PBMC) from 36 T1D patients and 36 normal glucose tolerance controls, extracted RNA, and used human Arraystar m6A-circRNA epitranscriptome chip for detection. The results of Arraystar m6A-circRNA chip showed that compared with the normal control group, there were 11,779 circRNAs with m6A differences in the T1D group, and 428 of them were screened with P<0.05 and FC≥1.5, of which 17 were significantly upregulated and 411 were significantly downregulated. Due to the excessive number of differential circRNAs, the 28 m6A-circRNAs with the most significant difference multiples were screened to make a heat map, so as to predict the role of circRNAs with m6A differences in the occurrence and development of T1D.
初步验证：与正常对照组相比，T1D中hsa_circRNA_005995（P<0.01）m6A水平显著降低；进一步使用32例T1D和42例正常对照进行实时定量荧光PCR（qRT-PCR）验证，结果显示，与正常对照相比，T1D中hsa_circRNA_005995表达量明显下降（P<0.001）。Preliminary verification: Compared with the normal control group, the mA level of hsa_circRNA_005995 (P<0.01) in T1D was significantly reduced; further real-time quantitative fluorescence PCR (qRT-PCR) verification was performed using 32 T1D cases and 42 normal controls. The results showed that compared with the normal control, the expression level of hsa_circRNA_005995 in T1D was significantly decreased (P<0.001).
本发明明确了hsa_circRNA_005995在PBMCs各细胞成分中的表达情况。首先，通过磁珠分选法分选5个正常对照PBMCs中的CD4+T、CD8+T、CD14+单核、及CD19+B细胞，抽提RNA并通过qRT-PCR检测hsa_circRNA_005995在上述免疫细胞中的表达水平，结果显示hsa_circRNA_005995在T细胞中表达量最高。故后续细胞实验选择在Jurkat T细胞上展开。The present invention clarifies the expression of hsa_circRNA_005995 in various cell components of PBMCs. First, CD4+ T, CD8+ T, CD14+ monocytes, and CD19+ B cells in 5 normal control PBMCs were sorted by magnetic bead sorting, RNA was extracted, and the expression level of hsa_circRNA_005995 in the above immune cells was detected by qRT-PCR. The results showed that hsa_circRNA_005995 was expressed most in T cells. Therefore, subsequent cell experiments were selected to be carried out on Jurkat T cells.
RNA免疫共沉淀（RIP）实验结果显示，与阴性对照IgG相比，m6A去甲基化酶ALKBH5与hsa_circRNA_005995结合显著增多（input>3%），证明hsa_circRNA_005995与ALKBH5有结合。The results of RNA immunoprecipitation (RIP) experiments showed that compared with the negative control IgG, the binding of m6A demethylase ALKBH5 to hsa_circRNA_005995 increased significantly (input>3%), proving that hsa_circRNA_005995 was bound to ALKBH5.
分别用hsa_circRNA_005995过表达和ALKBH5敲低慢病毒感染Jurkat细胞，qRT-PCR实验表明，敲低ALKBH5之后，hsa_circRNA_005995的表达水平明显升高。放线菌素D处理后，与Vector组相比，过表达ALKBH5组hsa_circRNA_005995的稳定性明显降低（P<0.05），敲低ALKBH5之后，与对照组相比， ALKBH5敲低组hsa_circRNA_005995的稳定性显著升高（P<0.05）。Jurkat cells were infected with hsa_circRNA_005995 overexpression and ALKBH5 knockdown lentiviruses, respectively. qRT-PCR experiments showed that the expression level of hsa_circRNA_005995 was significantly increased after ALKBH5 knockdown. After actinomycin D treatment, the stability of hsa_circRNA_005995 in the ALKBH5 overexpression group was significantly reduced compared with the Vector group (P<0.05). After ALKBH5 knockdown, the stability of hsa_circRNA_005995 in the ALKBH5 knockdown group was significantly increased compared with the control group (P<0.05).
hsa_circRNA_005995过表达细胞模型增殖曲线结果显示：与对照组相比，hsa_circRNA_005995组的细胞活力显著增强。The proliferation curve results of the hsa_circRNA_005995 overexpression cell model showed that compared with the control group, the cell viability of the hsa_circRNA_005995 group was significantly enhanced.
用携带或不携带hsa_circRNA_005995的病毒载体分别感染Jurkat细胞，随后分别提取两组RNA，对Th细胞特异性转录因子T-bet（Th1）、GATA3（Th2）、RORC（Th17）、FOXP3（Treg）分别进行RT-qPCR检测，结果显示：与Vecter组相比，hsa_circRNA_005995组的Th2和Treg转录因子表达量上升，Th1和Th17转录因子表达量无明显改变。后续Elisa检测也发现，与对照组相比，过表达hsa_circRNA_005995组的白介素10（IL-10）的表达水平显著上调。Jurkat cells were infected with viral vectors carrying or not carrying hsa_circRNA_005995, and then the two groups of RNA were extracted, and RT-qPCR was performed on Th cell-specific transcription factors T-bet (Th1), GATA3 (Th2), RORC (Th17), and FOXP3 (Treg). The results showed that compared with the Vecter group, the expression of Th2 and Treg transcription factors in the hsa_circRNA_005995 group increased, and the expression of Th1 and Th17 transcription factors did not change significantly. Subsequent Elisa detection also found that compared with the control group, the expression level of interleukin 10 (IL-10) in the hsa_circRNA_005995 overexpression group was significantly upregulated.
总之，过表达hsa_circRNA_005995可以促进Jurkat细胞增殖，使得Th2和Treg在mRNA水平上升，Th1和Th17在mRNA水平无明显变化。hsa_circRNA_005995在T1D患者显著上调，过表达hsa_circRNA_005995使得Th细胞亚群比例发生改变，提示hsa_circRNA_005995作为T1D治疗靶点的潜能。In conclusion, overexpression of hsa_circRNA_005995 can promote the proliferation of Jurkat cells, increase the mRNA levels of Th2 and Treg, and have no significant changes in the mRNA levels of Th1 and Th17. hsa_circRNA_005995 is significantly upregulated in T1D patients, and overexpression of hsa_circRNA_005995 changes the proportion of Th cell subsets, suggesting the potential of hsa_circRNA_005995 as a therapeutic target for T1D.
本发明首次发现T1D患者PBMC中具有m6A修饰的hsa_circRNA_005995水平显著升高，可以影响T细胞的增殖，改变T细胞亚群比例，并且hsa_circRNA_005995可以与去甲基化酶ALKBH5相互作用，ALKBH5-hsa_circRNA_005995调节轴可能作为T1D潜在的治疗靶点。The present invention discovered for the first time that the level of hsa_circRNA_005995 with m6A modification in PBMCs of T1D patients was significantly increased, which could affect the proliferation of T cells and change the proportion of T cell subsets. In addition, hsa_circRNA_005995 could interact with the demethylase ALKBH5. The ALKBH5-hsa_circRNA_005995 regulatory axis may serve as a potential therapeutic target for T1D.
除了上面所描述的目的、特征和优点之外，本发明还有其它的目的、特征和优点。下面将参照图，对本发明作进一步详细的说明。In addition to the above-described objects, features and advantages, the present invention has other objects, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
构成本申请的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:
图1为T1D患者PBMCs中有m6A表达差异的circRNA火山图；火山图中两条垂直虚线分别表示表达水平上调以及下调1.5倍变化倍数，水平虚线表示P值为0.05，图中左侧虚线和水平虚线交叉点的左上方圆点以及图中右侧虚线和水平虚线交叉点的右上方圆点分别代表具有统计学差异，其中，左侧虚线和水平虚线交叉点的左上方圆点表示m6A表达水平≥1.5倍下调的circRNAs即甲基化水平降低的有411个，右侧虚线和水平虚线交叉点的右上方圆点表示m6A表达水平≥1.5倍上调的circRNAs即甲基化水平升高的有17个，浅灰色圆点代表不具有m6A统计学差异的circRNAs即无统计学差异的甲基化改变。Figure 1 is a volcano plot of circRNAs with different m6A expression in PBMCs of T1D patients; the two vertical dotted lines in the volcano plot represent the 1.5-fold change in expression levels, respectively, and the horizontal dotted line represents a P value of 0.05. The upper left dot at the intersection of the left dotted line and the horizontal dotted line in the figure and the upper right dot at the intersection of the right dotted line and the horizontal dotted line in the figure represent statistical differences, respectively. Among them, the upper left dot at the intersection of the left dotted line and the horizontal dotted line represents 411 circRNAs with m6A expression levels ≥1.5-fold downregulation, that is, methylation levels are reduced, and the upper right dot at the intersection of the right dotted line and the horizontal dotted line represents 17 circRNAs with m6A expression levels ≥1.5-fold upregulation, that is, methylation levels are increased. The light gray dots represent circRNAs without statistical differences in m6A, that is, methylation changes without statistical differences.
图2为差异倍数最为显著的28个m6A-circRNAs的热图；热图每行代表一个m6A-circRNA甲基化水平，每列代表一个样本的m6A-circRNA甲基化水平分布。颜色刻度显示不同样本中m6A-circRNAs相对甲基化水平。正数表示上调，负数表示下调。（T1D组：TD1、TD2、TD3、TD4、TD6、TD7；正常对照组：HC1、HC01、HC02、HC03、HC4、HC05）。Figure 2 is a heat map of the 28 m6A-circRNAs with the most significant fold differences; each row of the heat map represents the methylation level of an m6A-circRNA, and each column represents the distribution of m6A-circRNA methylation levels in a sample. The color scale shows the relative methylation levels of m6A-circRNAs in different samples. Positive numbers indicate upregulation, and negative numbers indicate downregulation. (T1D group: TD1, TD2, TD3, TD4, TD6, TD7; normal control group: HC1, HC01, HC02, HC03, HC4, HC05).
图3为hsa_circRNA_005995在MeRIP-qPCR验证实验中的m6A水平变化对比图；正常对照组，N=6；1型糖尿病组，N=6；**为P<0.01。Figure 3 is a comparison of the changes in m6A levels of hsa_circRNA_005995 in the MeRIP-qPCR validation experiment; normal control group, N=6; type 1 diabetes group, N=6; **P<0.01.
图4为hsa_circRNA_005995在qRT-PCR验证实验中的水平变化对比图；正常对照组，N=42；1型糖尿病组，N=32；*为P<0.05。Figure 4 is a comparison of the level changes of hsa_circRNA_005995 in the qRT-PCR validation experiment; normal control group, N=42; type 1 diabetes group, N=32; *P<0.05.
图5为hsa_circRNA_005995在PBMCs各细胞成分中的表达情况图；通过磁珠分选法分选3名正常对照PBMCs中的细胞，抽提RNA并通过qRT-PCR检测hsa_circRNA_005995在上述免疫细胞中的表达水平，*为P<0.05。Figure 5 shows the expression of hsa_circRNA_005995 in various cell components of PBMCs; cells from 3 normal control PBMCs were sorted by magnetic bead sorting, RNA was extracted, and the expression level of hsa_circRNA_005995 in the above immune cells was detected by qRT-PCR, *P<0.05.
图6为hsa_circRNA_005995与阴性对照IgG及ALKBH5结合的量对比图，**为P<0.01。Figure 6 is a comparison of the binding amounts of hsa_circRNA_005995 with negative control IgG and ALKBH5, **P<0.01.
图7为转染敲低慢病毒后qRT-PCR检测hsa_circRNA_005995的表达效率对比图，*为P<0.05。Figure 7 is a comparison of the expression efficiency of hsa_circRNA_005995 detected by qRT-PCR after transfection with knockdown lentivirus, * is P < 0.05.
图8为过表达后qRT-PCR检测hsa_circRNA_005995的表达效率图；Figure 8 is a graph showing the expression efficiency of hsa_circRNA_005995 detected by qRT-PCR after overexpression;
图9为敲低后qRT-PCR检测hsa_circRNA_005995的表达效率图，*为P<0.05，**为P<0.01，ns：无统计学差异。Figure 9 is a graph showing the expression efficiency of hsa_circRNA_005995 detected by qRT-PCR after knockdown, * is P < 0.05, ** is P < 0.01, ns: no statistical difference.
图10为生长曲线对比图；用酶标仪分别检测0天、1天、2天、3天、4天、5天时间点的T细胞450nm处OD值，依次绘制生长曲线，与对照组相比，*为P<0.05，**为P<0.01，***为P<0.001。Figure 10 is a growth curve comparison chart; the OD values of T cells at 450nm at 0 days, 1 day, 2 days, 3 days, 4 days, and 5 days were detected by an enzyme marker, and the growth curves were plotted in sequence. Compared with the control group, * is P<0.05, ** is P<0.01, and *** is P<0.001.
图11为不同特异性转录因子的相对表达水平对比图；横坐标表示特异性转录因子，纵坐标表示相对表达水平，与对照组相比，*为P<0.05，**为P<0.01，ns：无统计学差异。FIG11 is a comparison chart of relative expression levels of different specific transcription factors; the abscissa represents specific transcription factors, and the ordinate represents relative expression levels. Compared with the control group, * represents P<0.05, ** represents P<0.01, ns: no statistical difference.
图12为过表达hsa_circRNA_005995后IL-10的水平图，***为P<0.001，****为P<0.0001。Figure 12 is a graph showing the levels of IL-10 after overexpression of hsa_circRNA_005995, *** is P < 0.001, **** is P < 0.0001.
具体实施方式Detailed ways
以下结合附图对本发明的实施例进行详细说明，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
材料和方法：Materials and methods:
1、研究对象：1. Research subjects:
本发明中涉及的所有患者及健康对照均签署知情同意书，本研究经中南大学湘雅二医院国家代谢性疾病临床医学研究中心伦理委员会审核批准（LYF2021053）。All patients and healthy controls involved in this invention signed informed consent forms, and this study was reviewed and approved by the Ethics Committee of the National Clinical Research Center for Metabolic Diseases, Second Xiangya Hospital, Central South University (LYF2021053).
1.1、T1D患者：1.1. T1D patients:
收集2021年9月至2022年12月在中南大学湘雅二医院T1D综合门诊就诊T1D患者36名。A total of 36 T1D patients who visited the T1D Comprehensive Clinic of the Second Xiangya Hospital of Central South University from September 2021 to December 2022 were collected.
T1D 纳入标准：①符合WHO 1999年糖尿病诊断标准；②起病半年内无明显诱因出现酮症或酮症酸中毒；③起病1年内依赖胰岛素治疗；④胰岛自身抗体阳性。⑤排除肿瘤、精神病等全身性重大疾病、以及严重心、肾、肝功能不全患者。T1D inclusion criteria: ① meet the WHO 1999 diabetes diagnostic criteria; ② ketosis or ketoacidosis without obvious causes within six months of onset; ③ insulin dependence within one year of onset; ④ positive pancreatic autoantibodies. ⑤ patients with major systemic diseases such as tumors and mental illnesses, as well as severe heart, kidney, and liver dysfunction were excluded.
1.2、正常对照：1.2. Normal control:
收集2021年11月至2022年12月在中南大学湘雅二医院健康管理中心体检的正常对照36例。Thirty-six normal controls who underwent physical examinations at the Health Management Center of the Second Xiangya Hospital of Central South University from November 2021 to December 2022 were collected.
正常对照纳入标准：①经75g葡萄糖耐量试验(OGTT)筛查，空腹血糖＜6.1mmol/L，糖负荷后2小时血糖＜7.8mmol/L或糖化血红蛋白＜6.1%；②无糖尿病家族史；③排除合并其他严重自身免疫性疾病、全身性重大疾病患者。Inclusion criteria for normal controls: ① After screening by 75g glucose tolerance test (OGTT), fasting blood glucose <6.1mmol/L, blood glucose <7.8mmol/L or glycosylated hemoglobin <6.1% 2 hours after glucose load; ② No family history of diabetes; ③ Exclude patients with other severe autoimmune diseases or major systemic diseases.
2、样本收集：2. Sample collection:
所有研究对象经静脉抽取外周空腹血4.5ml于EDTA抗凝管内，并在当天分离单个核细胞（PBMC），分装至1.5ml离心管内，加1ml Trizol（invitrogen，15596026）保存于-80℃冰箱，用于后续总RNA的提取。All subjects had 4.5 ml of peripheral fasting blood drawn intravenously into EDTA anticoagulant tubes, and mononuclear cells (PBMCs) were separated on the same day and dispensed into 1.5 ml centrifuge tubes. 1 ml of Trizol (invitrogen, 15596026) was added and stored in a -80°C refrigerator for subsequent total RNA extraction.
3、实验方法：3. Experimental methods:
3.1、外周血PBMC分离：3.1. Isolation of peripheral blood PBMC:
根据密度梯度离心法使用FicollPlus（GE，17-1440-03）分离外周血中的PBMC。4.5ml的外周血用PBS（Gibco，C10010500BT），FicollPlus按1：1：1进行稀释、铺血，密度梯度离心(20℃，800G，25min，+0-0），管内液体分为三层，使用巴氏吸管将PBMC（白膜层）转移至15ml离心管中，用PBS进行PBMC的洗脱，再次离心（20℃，300G，8min，+8，-9）后进行红细胞的裂解，洗脱，最后将分离出的PBMC转移入1.5ml离心管，加1ml Trizol裂解PBMC并保存于-80℃冰箱。PBMCs were isolated from peripheral blood using FicollPlus (GE, 17-1440-03) according to density gradient centrifugation. 4.5 ml of peripheral blood was diluted with PBS (Gibco, C10010500BT) and FicollPlus at a ratio of 1:1:1, and blood was layered. Density gradient centrifugation (20°C, 800G, 25min, +0-0) was performed. The liquid in the tube was divided into three layers. The PBMCs (buffy coat layer) were transferred to a 15 ml centrifuge tube using a Pasteur pipette. The PBMCs were eluted with PBS, and the red blood cells were lysed and eluted after centrifugation again (20°C, 300G, 8min, +8, -9). Finally, the separated PBMCs were transferred to a 1.5 ml centrifuge tube, 1 ml of Trizol was added to lyse the PBMCs, and the PBMCs were stored in a -80°C refrigerator.
3.2、总RNA的提取：3.2. Extraction of total RNA:
根据制造商的说明，使用Trizol法（Invitrogen，17-1440-03）分别从三组的冷冻PBMC中提取总RNA。使用NanoDrop ND-1000仪器（NanoDropThermo）进行RNA浓度的测定和纯度的评估。A260/A280的平均值为1.8-2.0。Total RNA was extracted from frozen PBMCs of the three groups using the Trizol method (Invitrogen, 17-1440-03) according to the manufacturer's instructions. RNA concentration and purity were determined using a NanoDrop ND-1000 instrument (NanoDropThermo). The average A260/A280 value was 1.8-2.0.
3.3、人Arraystar m6A-circRNA表观转录组芯片：3.3、Human Arraystar m6A-circRNA epitranscriptome chip:
（1）m6A沉淀：将2μg抗m6A兔多克隆抗体加入300μL 1×IP缓冲液中并混匀，加入3-5μg总RNA和m6A spike-in对照混合物，4℃孵育2h。每个样品准备20μL Dynabeads™ M-280绵羊抗兔IgG悬液，用0.5%牛血清白蛋白（BSA）在4℃下阻断2h后加入300μL 1×IP缓冲液洗涤30s，重复3次，然后重悬于RNA-m6A抗体混合物中。4℃孵育2h，用500μL 1×IP缓冲液洗涤3次，500μL洗液洗涤2次。收集上清标记为“Sup”，在磁珠中加入500μL洗脱缓冲液，50℃孵育1h，洗脱后收集上清标记为“IP”。利用酸酚-氯仿和乙醇沉淀提取RNA，其中“Sup” RNA为未被m6A修饰的RNA，“IP” RNA为m6A修饰的RNA。(1) m6A precipitation: Add 2 μg of anti-m6A rabbit polyclonal antibody to 300 μL 1×IP buffer and mix well, add 3-5 μg of total RNA and m6A spike-in control mixture, and incubate at 4°C for 2 h. Prepare 20 μL of Dynabeads™ M-280 sheep anti-rabbit IgG suspension for each sample, block with 0.5% bovine serum albumin (BSA) at 4°C for 2 h, add 300 μL 1×IP buffer to wash for 30 s, repeat 3 times, and then resuspend in RNA-m6A antibody mixture. Incubate at 4°C for 2 h, wash 3 times with 500 μL 1×IP buffer, and wash 2 times with 500 μL washing buffer. Collect the supernatant and mark it as "Sup", add 500 μL elution buffer to the magnetic beads, incubate at 50°C for 1 h, collect the supernatant after elution and mark it as "IP". RNA was extracted using acid phenol-chloroform and ethanol precipitation, where "Sup" RNA was RNA not modified by m6A and "IP" RNA was RNA modified by m6A.
（2）荧光标记：分别以Cy3和Cy5标记扩增的“Sup”和“IP”RNA，用RNeasy Mini Kit试剂盒纯化合成的cRNA，cRNA的浓度和比活性（pmol染料/μg cRNA）由NanoDrop ND-1000测定。(2) Fluorescent labeling: The amplified “Sup” and “IP” RNAs were labeled with Cy3 and Cy5, respectively. The synthesized cRNA was purified using the RNeasy Mini Kit, and the concentration and specific activity (pmol dye/μg cRNA) of the cRNA were measured using NanoDrop ND-1000.
（3）芯片杂交：2.5μg Cy3与Cy5标记的cRNA混合后，加入5μL 10×阻断剂和1μL 25×Fragmentation Buffer，60°C加热30min使cRNA混合物碎片化，然后与25μL 2×Hybridization Buffer结合。将50μL杂交溶液滴入载玻片，组装到m6A-circRNA表观转录组微阵列载玻片上，将载玻片置于Agilent杂交烘箱65°C孵育17h后，清洗、固定并使用Agilent扫描仪G2505C扫描杂交阵列。(3) Chip hybridization: After 2.5 μg of Cy3 and Cy5-labeled cRNA were mixed, 5 μL of 10× blocker and 1 μL of 25× Fragmentation Buffer were added, and the cRNA mixture was fragmented by heating at 60°C for 30 min, and then combined with 25 μL of 2× Hybridization Buffer. 50 μL of hybridization solution was dropped onto the slide and assembled onto the m6A-circRNA epitranscriptome microarray slide. The slide was placed in an Agilent hybridization oven at 65°C for 17 h, then washed, fixed, and the hybridization array was scanned using an Agilent scanner G2505C.
（4）数据分析：使用Agilent Feature Extraction软件(版本11.0.1.1)对采集到的阵列图像进行分析。IP(免疫沉淀，cy5标记)和Sup(上清，cy3标记)的原始强度用log2标度的峰值RNA强度平均值归一化。在峰值归一化后，28个样品中至少有4个样品中具有Present (P)或Marginal(M) QC标志的探针信号被保留以进一步进行“m6A数量”分析。根据IP (cy5标记)归一化强度计算m6A甲基化量。通过折叠变化和统计显著性(p值)阈值筛选，确定两个对照组之间m6A甲基化环状RNA的差异。采用R软件进行分层聚类。circRNA/microRNA相互作用用Arraystar基于TargetScan和miRanda的自制miRNA靶标预测软件进行预测。(4) Data analysis: The collected array images were analyzed using Agilent Feature Extraction software (version 11.0.1.1). The raw intensities of IP (immunoprecipitation, cy5 labeling) and Sup (supernatant, cy3 labeling) were normalized by the average peak RNA intensity on a log2 scale. After peak normalization, probe signals with Present (P) or Marginal (M) QC flags in at least 4 of the 28 samples were retained for further “m6A quantity” analysis. The amount of m6A methylation was calculated based on the IP (cy5 labeling) normalized intensity. The differences in m6A methylated circular RNAs between the two control groups were determined by screening with fold change and statistical significance (p-value) thresholds. Hierarchical clustering was performed using R software. CircRNA/microRNA interactions were predicted using Arraystar’s homemade miRNA target prediction software based on TargetScan and miRanda.
3.4、MeRIP验证m6A表达水平：3.4. MeRIP verification of m6A expression level:
为检测其m6A水平的差异，对芯片中的具有m6A差异的circRNAs进行筛选及qRT-PCR验证，步骤如下：根据circRNAs纳入标准：（1）差异倍数较高的circRNAs；（2）长度在200bp-1000bp的circRNAs；（3）平均原始强度大于100的circRNAs。综合分析发现队列中T1D患者差异表达的circRNAs，最终纳入3个circRNAs。选择β-actin作为内参基因，引物由上海生工生物工程股份有限公司合成，引物序列见下表：In order to detect the difference in their m6A levels, circRNAs with m6A differences in the chip were screened and verified by qRT-PCR. The steps are as follows: According to the inclusion criteria of circRNAs: (1) circRNAs with higher difference multiples; (2) circRNAs with a length of 200bp-1000bp; (3) circRNAs with an average original intensity greater than 100. Comprehensive analysis found that circRNAs were differentially expressed in T1D patients in the cohort, and finally 3 circRNAs were included. β-actin was selected as the internal reference gene, and the primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primer sequences are shown in the table below:
表1 内参及3个验证circRNAs的qRT-PCR引物列表Table 1 List of qRT-PCR primers for internal reference and three verified circRNAs
（1）m6A沉淀：同3.3（1）。(1) m6A precipitation: same as 3.3 (1).
（2）按照表2体系配制退火混合物，加入0.2mL八联PCR管，离心混匀后65℃水浴5min，冰上放置2min。(2) Prepare the annealing mixture according to the system in Table 2, add 0.2 mL to the eight-link PCR tube, centrifuge and mix, then place in a 65°C water bath for 5 min and place on ice for 2 min.
表2 退火混合物体系配置Table 2 Annealing mixture system configuration
（3）总RNA逆转录：(3) Total RNA reverse transcription:
①在200μL EP管中加入1μg总RNA，1μL random primer和Nuclease-Free Water配成10μL体系，混匀，70℃，加热5min，立即冰上静置≥5min。① Add 1 μg total RNA, 1 μL random primer and Nuclease-Free Water to a 200 μL EP tube to make a 10 μL system, mix well, heat at 70°C for 5 min, and immediately place on ice for ≥5 min.
②按表3在冰上配置另一个10μL反应混合物（RT-Mix）。② Prepare another 10 μL reaction mixture (RT-Mix) on ice according to Table 3.
表3 总RNA逆转录10μL RT-Mix体系表Table 3 Total RNA reverse transcription 10μL RT-Mix system
③向①中加入10μL RT-Mix混合物，混匀后上机，按照25℃，退火5min；42℃，延伸1h；70℃，失活15min进行逆转录反应。③ Add 10 μL RT-Mix mixture to ①, mix well and put it on the machine for reverse transcription reaction according to the following conditions: 25℃, annealing for 5 min; 42℃, extension for 1 h; 70℃, inactivation for 15 min.
（4）qRT-PCR：(4) qRT-PCR:
qRT-PCR反应使用384孔板，每孔反应体系10μL，每个样品均设置3个复孔。冰上配置下列qPCR扩增反应体系表4。The qRT-PCR reaction was performed in a 384-well plate, with 10 μL of reaction system in each well and 3 replicate wells for each sample. The following qPCR amplification reaction system (Table 4) was prepared on ice.
表4 qPCR扩增体系表Table 4 qPCR amplification system
qRT-PCR扩增程序为预变性：95℃，10min；热循环：95℃，15s；60℃，1min(40个循环)；溶解曲线：95℃，15s；60℃，1min，95℃，15s。The qRT-PCR amplification program was as follows: pre-denaturation: 95°C, 10 min; thermal cycling: 95°C, 15 s; 60°C, 1 min (40 cycles); melting curve: 95°C, 15 s; 60°C, 1 min, 95°C, 15 s.
（5）用下列公式计算每个转录本的甲基化修饰比例：(5) Calculate the methylation modification ratio of each transcript using the following formula:
%Input=2-Ct MeRIP/（2-Ct MeRIP+2-Ct Supernatant）×Fd×100%%Input=2-Ct MeRIP / (2-Ct MeRIP +2-Ct Supernatant )×Fd×100%
注：Fd为稀释因子，如果1/30的Sup RNA和1/5的MeRIP RNA用于qPCR，则Fd=1/1。Note: Fd is the dilution factor. If 1/30 of Sup RNA and 1/5 of MeRIP RNA are used for qPCR, then Fd = 1/1.
3.5、qRT-PCR验证：3.5. qRT-PCR verification:
qRT-PCR反应使用384孔板，每孔反应体系10μl，每个样品均设置3个复孔，使用仪器为Quantstudio 6 荧光定量PCR仪。The qRT-PCR reaction used a 384-well plate, with a reaction system of 10 μl in each well. Three replicate wells were set up for each sample, and the instrument used was a Quantstudio 6 fluorescence quantitative PCR instrument.
（1）引物制备：(1) Primer preparation:
引物设计与合成均委托生工生物工程（上海）股份有限公司完成，具体序列见下表5。Primer design and synthesis were commissioned to Sangon Biotech (Shanghai) Co., Ltd. The specific sequences are shown in Table 5 below.
表5 qRT-PCR引物列表Table 5 qRT-PCR primer list
接下来步骤见3.4（4）。The next steps are shown in 3.4 (4).
3.6、磁珠分选PBMC中主要细胞成分：3.6. Magnetic bead separation of main cell components in PBMC:
（1）抽取100mL外周血，分离PBMC后用13mL Washing Buffer（autoMACS® RunningBuffer – MACS® Separation Buffer，MiltenyiBiotec，130-091-221）重悬细胞，分成5份，比例为1:2:2:4:4。(1) Draw 100 mL of peripheral blood, separate PBMCs, resuspend the cells in 13 mL of Washing Buffer (autoMACS® Running Buffer – MACS® Separation Buffer, Miltenyi Biotec, 130-091-221), and divide the mixture into 5 portions with a ratio of 1:2:2:4:4.
（2）离心后用80μL/107个细胞Washing Buffer重悬，对应各比例按20μL/107个细胞分别加入CD4磁珠（MiltenyiBiotec，130-045-101）、CD8磁珠（MiltenyiBiotec，130-045-201）、CD14磁珠（MiltenyiBiotec，130-050-021）、CD19磁珠（MiltenyiBiotec，130-050-301）、CD56磁珠（MiltenyiBiotec，130-040-401），2-8℃孵育15min。(2) After centrifugation, resuspend the cells with 80 μL/107 cells Washing Buffer and add 20 μL/107 cells of each ratio of CD4 magnetic beads (Miltenyi Biotec, 130-045-101), CD8 magnetic beads (Miltenyi Biotec, 130-045-201), CD14 magnetic beads (Miltenyi Biotec, 130-050-021), CD19 magnetic beads (Miltenyi Biotec, 130-050-301), and CD56 magnetic beads (Miltenyi Biotec, 130-040-401). Incubate at 2-8°C for 15 min.
（3）Washing Buffer清洗细胞，润磁柱（MiltenyiBiotec，130-042-401），过柱后洗柱3次，用柱塞打出阳选细胞。(3) Wash the cells with Washing Buffer, moisten the magnetic column (Miltenyi Biotec, 130-042-401), wash the column three times, and use a plunger to remove the positively selected cells.
（4）PBS清洗细胞，转入1.5mL EP管，加入1mL Trizol裂解细胞，提取总RNA并逆转录，RT-qPCR检测各细胞成分中目标基因的表达量。(4) Wash the cells with PBS, transfer them into a 1.5 mL EP tube, add 1 mL Trizol to lyse the cells, extract total RNA and reverse transcribe it, and use RT-qPCR to detect the expression levels of the target genes in each cell component.
3.7、细胞培养和病毒感染：3.7 Cell culture and virus infection:
（1）Jurkat细胞株（人急性T细胞白血病细胞Clone E6-1，广州赛库，CC1902）培养于完全培基：RPMI-1640（Gibco，C11875500BT）+10%胎牛血清（FBS，Gibco，10099-141C）FBS+1%青霉素-链霉素（Gibco，15140-122）中，置于37℃，5%CO2培养箱（Thermo）中。(1) Jurkat cell line (human acute T-cell leukemia cell clone E6-1, Guangzhou Saiku, CC1902) was cultured in complete medium: RPMI-1640 (Gibco, C11875500BT) + 10% fetal bovine serum (FBS, Gibco, 10099-141C) FBS + 1% penicillin-streptomycin (Gibco, 15140-122) and placed in a 37°C, 5%CO2 incubator (Thermo).
（2）hsa_circRNA_005995和ALKBH5慢病毒载体委托上海吉凯基因医学科技股份有限公司构建。(2) The hsa_circRNA_005995 and ALKBH5 lentiviral vectors were constructed by Shanghai GeneCare Gene Medical Technology Co., Ltd.
（3）根据感染MOI=30计算出所需对照（对照是空病毒载体）和hsa_circRNA_005995病毒体积，加入完全培基中配制病毒混合液50μL。(3) Calculate the required control (the control is an empty virus vector) and hsa_circRNA_005995 virus volumes based on infection MOI = 30, and add them to complete culture medium to prepare 50 μL of virus mixture.
（4）将430μL/孔细胞悬液接种于24孔板中，每孔细胞数量为5×105，再向每孔加入病毒转染增强液A（吉凯基因）20μL，以及对照或hsa_circRNA_005995病毒50μL。(4) 430 μL/well of the cell suspension was inoculated into a 24-well plate, with 5×105 cells per well. Then 20 μL of virus transfection enhancement solution A (Jikai Gene) and 50 μL of control or hsa_circRNA_005995 virus were added to each well.
（5）将24孔板离心：20℃，1000g，60min，随后放入恒温培养箱。(5) Centrifuge the 24-well plate at 20°C, 1000 g, 60 min, and then place it in a constant temperature incubator.
（6）24h后，向每孔加入500μL完全培基。(6) After 24 h, add 500 μL of complete culture medium to each well.
（7）96h~120h后，荧光显微镜下观察，感染效率＞80%时，PBS清洗细胞，转入1.5mLEP管，加入1mL Trizol裂解细胞，提取总RNA并逆转录，RT-qPCR检测各细胞成分中目标基因的表达量。(7) After 96 h to 120 h, observe under a fluorescence microscope. When the infection efficiency is >80%, wash the cells with PBS, transfer them into a 1.5 mL EP tube, add 1 mL of Trizol to lyse the cells, extract total RNA and reverse transcribe it, and use RT-qPCR to detect the expression of the target gene in each cell component.
3.8、RIP：3.8, RIP:
按照制造商的说明，使用Magna RIP RNA结合蛋白免疫无沉淀试剂盒（美国Millipore公司Sigma，17-701）进行RIP。RIP was performed using the Magna RIP RNA Binding Protein Immunoprecipitation Kit (Sigma, Millipore, USA, 17-701) according to the manufacturer's instructions.
（1）裂解产物的配制：为了制备总细胞裂解物，观察Jurkat细胞浓度为2×107个细胞/ml，用10mL预冷PBS，洗涤细胞2次。在等体积完全RIP裂解缓冲液（在100μL RIP裂解缓冲液中加入0.5μL蛋白酶抑制剂混合物和0.25μL核糖核酸酶抑制剂）中重悬细胞沉淀。移液管吹打混均。将裂解产物放置在冰上孵育5分钟。(1) Preparation of lysate: To prepare total cell lysate, observe the Jurkat cell concentration of 2 × 107 cells/ml and wash the cells twice with 10 mL of pre-cooled PBS. Resuspend the cell pellet in an equal volume of complete RIP lysis buffer (add 0.5 μL of protease inhibitor cocktail and 0.25 μL of ribonuclease inhibitor to 100 μL of RIP lysis buffer). Pipette to mix. Incubate the lysate on ice for 5 minutes.
（2）用于免疫沉淀的磁珠配制：通过颠倒微量离心管或移液管吹打，重悬磁珠至完全分散均匀重悬。标记用于免疫沉淀实验的适当数量微量离心管。量取50µL磁珠悬浮液，转移到每个离心管中。在每个离心管中加入500µL RIP洗涤缓冲液，短暂涡旋。离心管磁力架把离心管置于磁力架上，磁珠被磁力架吸附在管壁后，弃去上清液。从磁力架中取出离心管。(2) Preparation of magnetic beads for immunoprecipitation: Resuspend the magnetic beads by inverting the microcentrifuge tube or pipetting until they are completely dispersed and evenly suspended. Label the appropriate number of microcentrifuge tubes for immunoprecipitation experiments. Measure 50µL of the magnetic bead suspension and transfer it to each centrifuge tube. Add 500µL of RIP wash buffer to each centrifuge tube and vortex briefly. Centrifuge tube magnetic stand Place the centrifuge tube on the magnetic stand. After the magnetic beads are adsorbed to the tube wall by the magnetic stand, discard the supernatant. Remove the centrifuge tube from the magnetic stand.
在每个离心管中加入500µL RIP洗涤缓冲液，短暂涡旋。把离心管放在磁力架中，弃去上清液。从磁力架中取出离心管，使磁珠重悬于100µL RIP洗涤缓冲液中。在离心管中加入约5µg目的抗体（抗RBM15、ALKBH5抗体、兔IgG）。室温下，旋转培养30分钟。离心管短暂离心，放在磁力架中，除去上清液。从磁力架中取出离心管。在每个离心管中加入500µLRIP洗涤缓冲液，短暂涡旋。把离心管放在磁力架中，弃去上清液。再进行一次洗涤。从磁力架中取出离心管。在每个离心管中加入500µL RIP洗涤缓冲液，短暂涡旋。把离心管置于冰上备用。Add 500 µL RIP Wash Buffer to each tube and vortex briefly. Place the tubes in a magnetic rack and discard the supernatant. Remove the tubes from the magnetic rack and resuspend the beads in 100 µL RIP Wash Buffer. Add approximately 5 µg of the antibody of interest (anti-RBM15, ALKBH5 antibody, rabbit IgG) to the tubes. Incubate with rotation for 30 minutes at room temperature. Centrifuge the tubes briefly, place them in a magnetic rack, and discard the supernatant. Remove the tubes from the magnetic rack. Add 500 µL RIP Wash Buffer to each tube and vortex briefly. Place the tubes in a magnetic rack and discard the supernatant. Wash once more. Remove the tubes from the magnetic rack. Add 500 µL RIP Wash Buffer to each tube and vortex briefly. Place the tubes on ice until needed.
（3）RNA结合蛋白-RNA复合物免疫共沉淀：配制RIP免疫沉淀缓冲液。每次免疫沉淀实验需要900μL RIP免疫沉淀缓冲液。每一次反应，在860µL RIP洗涤缓冲液中加入35μL0.5M EDTA和5µL核糖核酸酶抑制剂。(3) RNA binding protein-RNA complex immunoprecipitation: Prepare RIP immunoprecipitation buffer. Each immunoprecipitation experiment requires 900 μL RIP immunoprecipitation buffer. For each reaction, add 35 μL 0.5 M EDTA and 5 μL ribonuclease inhibitor to 860 μL RIP wash buffer.
把装有磁珠的离心管放在磁力架中，弃去上清液。在每个离心管中加入900µLRIP免疫沉淀缓冲液。快速解冻之前所得的RIP裂解产物，4℃下，14000rpm，离心10分钟。取100µL上清液加至含有磁珠-抗体复合物的RIP免疫沉淀缓冲液中。免疫沉淀反应的最终体积将为1.0mL。取RIP裂解产物的上清液10µL，置于一个新的离心管中，标记为“input”。把这份input样品贮藏在-80℃，直至RNA纯化操作。这份“input”将会用于绘制标准曲线或用于RT-qPCR方法(实时或终点)的比较。将所有离心管放置在旋转器上，4℃下，孵育3小时至过夜。短暂离心免疫沉淀反应离心管，放在磁力架中，弃去上清液。从磁力架中取出离心管。在每个离心管中加入0.5mL RIP洗涤缓冲液，短暂涡旋，把离心管放在磁力架中，弃去上清液。重复五次上一步操作，再用500µL预冷的RIP洗涤缓冲液洗涤磁珠六次。Place the tubes containing the beads in the magnetic rack and discard the supernatant. Add 900 µL of RIP immunoprecipitation buffer to each tube. Thaw the RIP lysate obtained earlier and centrifuge at 14,000 rpm for 10 minutes at 4°C. Take 100 µL of the supernatant and add it to the RIP immunoprecipitation buffer containing the magnetic beads-antibody complex. The final volume of the immunoprecipitation reaction will be 1.0 mL. Take 10 µL of the supernatant from the RIP lysate and place it in a new tube and label it "input". Store this input sample at -80°C until RNA purification. This "input" will be used to generate a standard curve or for comparison with RT-qPCR methods (real-time or end-point). Place all tubes on a rotator and incubate at 4°C for 3 hours to overnight. Briefly centrifuge the immunoprecipitation tubes, place them in the magnetic rack, and discard the supernatant. Remove the tubes from the magnetic rack. Add 0.5 mL of RIP Wash Buffer to each tube, vortex briefly, place the tube on the magnetic stand, and discard the supernatant. Repeat the previous step five times, and then wash the beads six times with 500 µL of pre-cooled RIP Wash Buffer.
（4）RNA的纯化：(4) RNA purification:
①配制蛋白酶K缓冲液。每次免疫沉淀需要150µL蛋白酶K缓冲液。此缓冲液含有117µL RIP洗涤缓冲液、15µL 10%SDS和18µL 10mg/mL蛋白酶K可通过加入浓SDS，减少蛋白酶K变性的风险。① Prepare proteinase K buffer. Each immunoprecipitation requires 150µL proteinase K buffer. This buffer contains 117µL RIP wash buffer, 15µL 10% SDS, and 18µL 10mg/mL proteinase K. The risk of proteinase K denaturation can be reduced by adding concentrated SDS.
②重悬免疫沉淀物于150µL蛋白酶K缓冲液中。②Resuspend the immunoprecipitate in 150 µL proteinase K buffer.
③input样品，在离心管中加入107µL RIP洗涤缓冲液、15µL 10%SDS和18µL蛋白酶K，定容至总体积150µL。③Input sample: add 107µL RIP washing buffer, 15µL 10% SDS and 18µL proteinase K to a centrifuge tube and adjust the volume to 150µL.
④使所有离心管置于55℃下孵育30分钟，同时振荡以充分消化蛋白。④ Incubate all centrifuge tubes at 55°C for 30 minutes while shaking to fully digest the protein.
⑤孵育后，短暂离心离心管，把离心管放在磁力架上。把上清液转移到一个新的离心管中。⑤After incubation, centrifuge the tube briefly and place it on a magnetic rack. Transfer the supernatant to a new centrifuge tube.
⑥在装有上清液的离心管中，加入250µL RIP洗涤缓冲液。⑥ Add 250 µL RIP washing buffer to the centrifuge tube containing the supernatant.
⑦在每个离心管中加入400µL苯酚：氯仿：异戊醇。涡旋15秒，在室温下，14000rpm，离心10分钟，以分离各相。⑦ Add 400 µL of phenol:chloroform:isoamyl alcohol to each centrifuge tube. Vortex for 15 seconds and centrifuge at 14,000 rpm for 10 minutes at room temperature to separate the phases.
⑧小心取出350μL水相，放在一个新的离心管中。加入400µL氯仿，涡旋15秒，在室温下，14000rpm，离心10分钟，以分离各相。⑧ Carefully remove 350 μL of the aqueous phase and place it in a new centrifuge tube. Add 400 μL of chloroform, vortex for 15 seconds, and centrifuge at 14,000 rpm for 10 minutes at room temperature to separate the phases.
⑨小心取出300μL水相，放在一个新离心管中。⑨Carefully remove 300 μL of the aqueous phase and place it in a new centrifuge tube.
⑩在每个离心管中，加入50µL盐溶液I、15µL盐溶液II、5µL沉淀增强剂，然后加入850µL无水乙醇。混匀，在-80℃下保存1小时至过夜，以沉淀RNA。⑩In each centrifuge tube, add 50µL of saline solution I, 15µL of saline solution II, 5µL of precipitation enhancer, and then add 850µL of anhydrous ethanol. Mix well and store at -80℃ for 1 hour to overnight to precipitate RNA.
⑪4℃下，14000rpm，离心30分钟，小心弃去上清液。⑪ Centrifuge at 14000 rpm at 4°C for 30 minutes and carefully discard the supernatant.
⑫用80%乙醇洗涤沉淀一次。4℃下，14000rpm，离心15分钟。小心弃去上清液，使沉淀自然风干。⑫ Wash the precipitate once with 80% ethanol. Centrifuge at 14,000 rpm for 15 minutes at 4°C. Carefully discard the supernatant and allow the precipitate to air dry naturally.
⑬重悬RNA沉淀于10-20µL无酶水中，将离心管置于冰上备用。⑬ Resuspend the RNA pellet in 10-20 µL enzyme-free water and place the centrifuge tube on ice for later use.
（5）免疫沉淀RNA的分析：逆转录及qRT-PCR方法同3.4（3）（4）。(5) Analysis of immunoprecipitated RNA: Reverse transcription and qRT-PCR methods are the same as 3.4 (3) (4).
3.8、放线菌素D检测稳定性：3.8. Actinomycin D detection stability:
（1）收集细胞转移至15mL离心管，4℃，1500rpm，离心5min。(1) Collect cells and transfer to a 15 mL centrifuge tube. Centrifuge at 1500 rpm at 4°C for 5 min.
（2）加入新鲜完全培养基，轻柔吹散混匀，计数并调整至2×106/mL，将细胞接种于六孔板，每孔2.5mL。(2) Add fresh complete culture medium, gently blow to mix, count and adjust to 2×106 /mL, and inoculate the cells into a six-well plate, 2.5mL per well.
（3）用10μg/mL放线菌素D处理细胞，分别收集处理0h、2h和4h后的细胞，加入1mLTrizol吹散混匀，于4℃保存。(3) Treat the cells with 10 μg/mL actinomycin D, collect the cells after 0 h, 2 h, and 4 h of treatment, add 1 mL of Trizol, blow and mix, and store at 4°C.
（4）收集后提取RNA，进行qRT-PCR。(4) After collection, RNA was extracted and qRT-PCR was performed.
3.9、CCK8检测细胞增殖：3.9、CCK8 detection of cell proliferation:
（1）96孔板中每孔加入5×105/mL细胞悬液100μL，实验组和对照组各5个复孔，加样孔周围一圈孔加入100μL/孔PBS，铺5块板。(1) Add 100 μL of 5×105 /mL cell suspension to each well of a 96-well plate. Set up 5 replicate wells for the experimental group and the control group. Add 100 μL/well PBS to the wells around the sample wells and spread out on 5 plates.
（2）取出一块板，加样孔每孔加入10μL CCK8增殖检测试剂（DOJINDO），混匀放入培养箱中，4h后酶标仪（Bio-Tek）检测吸光度，检测波长450nm，参比波长650nm。(2) Take out a plate and add 10 μL of CCK8 proliferation detection reagent (DOJINDO) to each well. Mix well and place in the incubator. After 4 hours, use a microplate reader (Bio-Tek) to detect the absorbance. The detection wavelength is 450 nm and the reference wavelength is 650 nm.
（3）其余4块板分别于24、48、72、96后加CCK8检测。(3) The remaining four plates were tested with CCK8 after 24, 48, 72, and 96 seconds, respectively.
3.10、多因子检测：3.10. Multi-factor detection:
使用Biolegend公司的LEGEND plex人类CD8/NK多因子流式检测试剂盒进行操作：Use Biolegend's LEGEND plex human CD8/NK multi-factor flow cytometry kit to operate:
（1）实验前准备：首先，通过将Beads猛烈涡旋1分钟来确保其均匀混合，然后根据需要计算出每次反应所需的Beads量，每次25μl。接着，将Wash buffer置于室温下以促使其中的盐分完全溶解，并使用蒸馏水进行稀释。(1) Preparation before the experiment: First, vortex the beads vigorously for 1 minute to ensure that they are evenly mixed, and then calculate the amount of beads required for each reaction as needed, 25μl each time. Next, place the wash buffer at room temperature to completely dissolve the salts in it, and dilute it with distilled water.
（2）样品准备：将250μl的Assay Buffer用于溶解标准物，反复颠倒几次以确保混合均匀，然后静置10分钟。之后，将其转移到一个标记为C7的EP管中。接着取6个EP管并分别标记为C6至C1，每个管中加入37.5μl的Assay Buffer，使用四倍的比例进行稀释，从C7管中取出12.5μl进行逐级稀释，以得到C0（0 pg/ml）作为Assay Buffer。(2) Sample preparation: Use 250 μl of Assay Buffer to dissolve the standard, invert several times to ensure uniform mixing, and then let it stand for 10 minutes. After that, transfer it to an EP tube labeled C7. Then take 6 EP tubes and label them C6 to C1, add 37.5 μl of Assay Buffer to each tube, dilute it at a ratio of four times, and take 12.5 μl from C7 tube for stepwise dilution to obtain C0 (0 pg/ml) as Assay Buffer.
（3）实验操作流程：向V型底板的相应孔中加入25μl的Assay Buffer，再加入25μl的标准品或样本到对应的孔中，然后向每个管中加入25μl充分涡旋后的混合Beads。在避光条件下，将V型底板以1000rpm振荡2小时以进行孵育；之后以1000g离心5分钟，丢弃上层液体并进行一次洗涤，然后加入25μl的检测抗体。继续在避光条件下，以1000rpm振荡V型底板1小时；直接向每个孔中加入25μl的SA-PE，再次在避光条件下振荡V型底板30分钟。随后以1000g离心5分钟，小心丢弃上层液体同时避免吸走Beads，尽量去除剩余液体；接着向每个管中加入200μl稀释后的Wash buffer并涡旋混合Beads，再次离心5分钟后弃去上清；最后，向每个孔加入200-300μl稀释后的Wash buffer，将混合物转移到EP管中，再次涡旋混合Beads后，准备进行仪器分析。(3) Experimental operation process: Add 25μl of Assay Buffer to the corresponding wells of the V-bottom plate, then add 25μl of standard or sample to the corresponding wells, and then add 25μl of mixed beads after thorough vortexing to each tube. Incubate the V-bottom plate at 1000rpm for 2 hours in the dark; then centrifuge at 1000g for 5 minutes, discard the upper liquid and wash once, and then add 25μl of detection antibody. Continue to oscillate the V-bottom plate at 1000rpm for 1 hour in the dark; directly add 25μl of SA-PE to each well, and oscillate the V-bottom plate again in the dark for 30 minutes. Then centrifuge at 1000g for 5 minutes, carefully discard the upper liquid while avoiding aspirating the beads, and try to remove the remaining liquid; then add 200μl of diluted Wash buffer to each tube and vortex mix the beads, centrifuge again for 5 minutes and discard the supernatant; finally, add 200-300μl of diluted Wash buffer to each well, transfer the mixture to the EP tube, vortex mix the beads again, and prepare for instrument analysis.
（4）数据分析：使用Biolegend公司官网（https://www.biolegend.com/legendplex），导入上机后的流式数据，分析后再导出Excel表格，使用GraphPad Prism8.4.3进行统计学分析。(4) Data analysis: The flow cytometry data were imported using the Biolegend company website (https://www.biolegend.com/legendplex), and the data were exported to Excel tables after analysis. Statistical analysis was performed using GraphPad Prism 8.4.3.
4、统计学方法：4. Statistical methods:
正态分布的数据以平均值（mean）±标准差（standard deviation）表示，非正态分布的数据以四分位间距表示，分类变量的数据以频数表示。采用2^-ΔΔCT法计算qRT-PCR中所选circRNA的相对表达水平。采用Shapiro-Wilk检验数据的正态性，符合正态分布的数据使用独立样本t检验或单因素方差分析（ANOVA）进行组间差异分析，否者使用秩和检验（Mann-Whitney U检验或Kruskal-Wallis H检验）。卡方检验（Chi-square）用于分类数据组间的差异分析。统计分析采用SPSS 26.0（IBMSPSS, Inc., Chicago, IL, USA）和GraphPad Prism8.4.3（Graphpad Software Inc., San Diego, CA,USA）进行。P值<0.05被认为具有统计意义。Normally distributed data were expressed as mean ± standard deviation, non-normally distributed data were expressed as interquartile range, and categorical variables were expressed as frequency. The relative expression levels of selected circRNAs in qRT-PCR were calculated using the 2^-ΔΔCT method. The Shapiro-Wilk test was used to test the normality of the data. The data that met the normal distribution were analyzed for differences between groups using independent sample t-test or one-way analysis of variance (ANOVA), otherwise the rank sum test (Mann-Whitney U test or Kruskal-Wallis H test) was used. The Chi-square test was used to analyze the differences between categorical data groups. Statistical analysis was performed using SPSS 26.0 (IBMSPSS, Inc., Chicago, IL, USA) and GraphPad Prism8.4.3 (Graphpad Software Inc., San Diego, CA, USA). P values < 0.05 were considered statistically significant.
结果：result:
1、T1D患者PBMCs中m6A差异circRNAs表达谱：1. Expression profile of m6A differential circRNAs in PBMCs of T1D patients:
Arraystar m6A-circRNA芯片结果显示，与正常对照组相比，T1D组中有m6A差异的circRNA共有11779个，经过筛选P<0.05且FC≥1.5的共有428个，其中，17个显著上调，411个显著下调（图1）。由于差异的circRNAs数量过多，通过筛选差异倍数最为显著的28个m6A-circRNAs制成了热图（图2）。The results of Arraystar m6A-circRNA chip showed that there were 11,779 circRNAs with m6A differences in the T1D group compared with the normal control group. After screening, there were 428 circRNAs with P<0.05 and FC≥1.5, of which 17 were significantly upregulated and 411 were significantly downregulated (Figure 1). Due to the large number of differential circRNAs, a heat map was made by screening the 28 m6A-circRNAs with the most significant difference (Figure 2).
2、Hsa_circRNA_005995 m6A水平及表达水平验证：2. Hsa_circRNA_005995 m6A level and expression level verification:
MeRIP-qPCR验证实验，结果显示与正常对照组相比，T1D中hsa_circRNA_005995（P<0.01）m6A水平显著降低（图3）。下一步，使用32例T1D和42例正常对照进行qRT-PCR验证，结果显示，与正常对照相比，T1D中hsa_circRNA_005995表达量明显下降（P<0.001）（图4）。MeRIP-qPCR validation experiment, the results showed that compared with the normal control group, the m6A level of hsa_circRNA_005995 (P<0.01) in T1D was significantly reduced (Figure 3). Next, qRT-PCR validation was performed using 32 T1D cases and 42 normal controls, and the results showed that compared with the normal control, the expression of hsa_circRNA_005995 in T1D was significantly decreased (P<0.001) (Figure 4).
3.受试者基本临床特征：3. Basic clinical characteristics of the subjects:
后续验证队列一共纳入32名T1D患者和42名正常对照，受试者的人口学及临床资料如表6。A total of 32 T1D patients and 42 normal controls were included in the subsequent validation cohort. The demographic and clinical data of the subjects are shown in Table 6 .
表6 受试者人口学及临床资料Table 6 Demographic and clinical data of subjects
注：BMI为体重指数；TC为总胆固醇；TG为甘油三酯；LDL-C为低密度脂蛋白-胆固醇；HDL-C为高密度脂蛋白-胆固醇；FBG为空腹血糖；FCP为空腹C肽；HbA1c为糖化血红蛋白。Note: BMI is body mass index; TC is total cholesterol; TG is triglyceride; LDL-C is low-density lipoprotein-cholesterol; HDL-C is high-density lipoprotein-cholesterol; FBG is fasting blood glucose; FCP is fasting C-peptide; HbA1c is glycosylated hemoglobin.
4、Hsa_circRNA_005995基本特征：4. Basic characteristics of Hsa_circRNA_005995:
hsa_circRNA_005995序列为：The sequence of hsa_circRNA_005995 is:
GCAAAATCCTGACAGATGACTTTGCAGACAAGGAGGGTCTGGAAATGGGTGATGAGTACTTTGCAAATCTGGACCATATCGAGAGCGTGGAGAACTTCAAAGAAGGATATGAGAGTGATGCCCCCTGTTCCTCTGACAGCAGTGGTGTAGACTTGAAGGACCAGGAAGATGGCAACAGCGGTACAGAGGACCCTGAAGAGTCCAATGATGATAGCTCAGATGATAACTTCTGTAAGGATGAGGACTTCAGCACCAGTTCAGTGTGGCGGAGCTATGCTACCCGGAGGCAGACCCGGGGCCAGAAAGAGAACGGACTCTCTGAGACAACTTCCAAGGACTCCCACCCCCCAGATCTTGGACCCCCACATATTCCTGTTCCTCCCTCAATCCCTGTAGGTGGCTGCAATCCACCTTCCTCCGAAGAGACACCCAAGAACAAGGTGGCCTCATGGTTGAGCTGCAATAGTGTCAGTGAAGGTGGTTTTGCTGACTCTGATAGCCATTCATCCTTCAAGACTAATGAAGGTGGGGAGGGCCGGGCTGGGGGAAGCCGAATGGAGGCTGAGAAGGCCTCCACCTCAGGACTAGGCATCAAGGATGAGGGAGACATCAAACAGGCCAAGAAAGAGGACACTGACGACCGAAACAAGATGTCAGT。GCAAAATCCTGACAGATGACTTTGCAGACAAGGAGGGTCTGGAAATGGGTGATGAGTACTTTGCAAATCTGGACCATATCGAGAGCGTGGAGAACTTCAAAGAAGGATATGAGAGTGATGCCCCCTGTTCCTCTGACAGCAGTGGTGTAGACTTGAAGGACCAGGAAGATGGCAACAGCGGTACAGAGGACCCTGAAGAGTCCAATGATGATAGCTCAGATGATAACTTCTGTAAGGATGAGGACTTCAGCACCAGTT CAGTGTGGCGGAGCTATGCTACCCGGAGGCAGACCCGGGGCCAGAAAGAGAACGGACTCTCTGAGACAACT TCCAAGGACTCCCACCCCCCAGATCTTGGACCCCCACATATTCCGTTCCTCCCTCAATCCCTGTAGGTGGCTGCAATCCACCTTCCTCCGAAGAGACACCCAAGAACAAGGTGGCCTCATGGTTGAGCTGCAATAGTGTCAGTGAAGGTGGTTTTGCTGACTCTGATAGCCATTCATCCTTCAAGACTAATGAAGGTGGGGAGGGCCGGGCTGGGGGAAGCCGAATGGAGGCTGAGAAGGCCTCCACCTCAGGACTAG GCATCAAGGATGAGGGAGACATCAAACAGGCCAAGAAAGAGGACACTGACGACCGAAACAAGATGTCAGT.
为探讨hsa_circRNA_005995对细胞的功能作用，需要明确其在PBMCs各细胞成分中的表达情况。首先，通过磁珠分选法分选5个正常对照PBMCs中的CD4+T、CD8+T、CD14+单核、及CD19+B细胞，抽提RNA并通过qRT-PCR检测hsa_circRNA_005995在上述免疫细胞中的表达水平，结果显示hsa_circRNA_005995在T细胞中表达量最高（图5）。故后续细胞实验选择在Jurkat T细胞上展开。In order to explore the functional effects of hsa_circRNA_005995 on cells, it is necessary to clarify its expression in various cell components of PBMCs. First, CD4+ T, CD8+ T, CD14+ monocytes, and CD19+ B cells in 5 normal control PBMCs were sorted by magnetic bead sorting, RNA was extracted, and the expression level of hsa_circRNA_005995 in the above immune cells was detected by qRT-PCR. The results showed that hsa_circRNA_005995 was expressed most highly in T cells (Figure 5). Therefore, subsequent cell experiments were carried out on Jurkat T cells.
5、Hsa_circRNA_005995的m6A甲基化修饰对T1D的治疗潜能：5. The therapeutic potential of m6A methylation modification of Hsa_circRNA_005995 for T1D:
为探索hsa_circRNA_005995是否可以作为T1D的潜在治疗靶点，进行了接下来的功能实验。首先，RIP实验，结果显示，与阴性对照IgG相比，m6A去甲基化酶ALKBH5与hsa_circRNA_005995结合显著增多（input>3%），证明hsa_circRNA_005995与ALKBH5有结合（图6）。To explore whether hsa_circRNA_005995 can be used as a potential therapeutic target for T1D, the following functional experiments were performed. First, the RIP experiment showed that compared with the negative control IgG, the binding of m6A demethylase ALKBH5 to hsa_circRNA_005995 increased significantly (input>3%), proving that hsa_circRNA_005995 binds to ALKBH5 (Figure 6).
接下来，分别用hsa_circRNA_005995过表达和ALKBH5敲低慢病毒感染Jurkat细胞，qRT-PCR实验表明，敲低ALKBH5之后，hsa_circRNA_005995的表达水平明显升高（图7）。放线菌素D处理后，与对照组相比，过表达ALKBH5组hsa_circRNA_005995的稳定性明显降低（P<0.05）；敲低ALKBH5之后，与sh-NC组相比，sh-ALKBH5组hsa_circRNA_005995的稳定性显著升高（P<0.05）（图8、图9）。Next, Jurkat cells were infected with hsa_circRNA_005995 overexpression and ALKBH5 knockdown lentiviruses, respectively. qRT-PCR experiments showed that after ALKBH5 knockdown, the expression level of hsa_circRNA_005995 was significantly increased (Figure 7). After actinomycin D treatment, the stability of hsa_circRNA_005995 in the ALKBH5 overexpression group was significantly reduced compared with the control group (P <0.05); after ALKBH5 knockdown, the stability of hsa_circRNA_005995 in the sh-ALKBH5 group was significantly increased compared with the sh-NC group (P < 0.05) (Figures 8 and 9).
下一步，观察检测过表达hsa_circRNA_005995对Jurkat细胞增殖及T细胞亚群比例的影响。Next, we observed and detected the effect of overexpression of hsa_circRNA_005995 on Jurkat cell proliferation and the proportion of T cell subsets.
hsa_circRNA_005995过表达细胞模型增殖曲线结果显示：与对照组相比，过表达hsa_circRNA_005995组的细胞活力显著增强（图10）。The proliferation curve results of the hsa_circRNA_005995 overexpression cell model showed that compared with the control group, the cell viability of the hsa_circRNA_005995 overexpression group was significantly enhanced (Figure 10).
图11是hsa_circRNA_005995过表达对Th细胞亚群的影响，用携带或不携带hsa_circRNA_005995的病毒载体分别感染Jurkat细胞，随后分别提取两组RNA，对Th细胞特异性转录因子T-bet（Th1）、GATA3（Th2）、RORC（Th17）、FOXP3（Treg）分别进行qRT-PCR检测，结果显示：与Vecter组相比，hsa_circRNA_005995组的Th2和Treg转录因子表达量上升，Th1和Th17转录因子表达量无明显改变。后续Elisa检测也发现，与对照组相比，过表达hsa_circRNA_005995组的IL-10的表达水平显著上调（图12）。Figure 11 shows the effect of hsa_circRNA_005995 overexpression on Th cell subsets. Jurkat cells were infected with viral vectors carrying or not carrying hsa_circRNA_005995, and then two groups of RNA were extracted. Th cell-specific transcription factors T-bet (Th1), GATA3 (Th2), RORC (Th17), and FOXP3 (Treg) were detected by qRT-PCR. The results showed that compared with the Vecter group, the expression of Th2 and Treg transcription factors in the hsa_circRNA_005995 group increased, and the expression of Th1 and Th17 transcription factors did not change significantly. Subsequent Elisa detection also found that compared with the control group, the expression level of IL-10 in the hsa_circRNA_005995 overexpression group was significantly upregulated (Figure 12).
总之，过表达hsa_circRNA_005995可以促进Jurkat细胞增殖，使得Th2和Treg在mRNA水平上升，Th1和Th17在mRNA水平无明显变化。hsa_circRNA_005995在T1D患者显著上调，过表达hsa_circRNA_005995使得Th细胞亚群比例发生改变，提示hsa_circRNA_005995作为T1D治疗靶点的潜能。In conclusion, overexpression of hsa_circRNA_005995 can promote the proliferation of Jurkat cells, increase the mRNA levels of Th2 and Treg, and have no significant changes in the mRNA levels of Th1 and Th17. hsa_circRNA_005995 is significantly upregulated in T1D patients, and overexpression of hsa_circRNA_005995 changes the proportion of Th cell subsets, suggesting the potential of hsa_circRNA_005995 as a therapeutic target for T1D.
结论：in conclusion:
T1D患者PBMC中具有m6A修饰的hsa_circRNA_005995水平显著下降，可以影响T细胞的增殖，改变T细胞亚群比例，并且hsa_circRNA_005995可以与去甲基化酶ALKBH5相互作用，ALKBH5-hsa_circRNA_005995调节轴可能作为T1D潜在的治疗靶点。The level of hsa_circRNA_005995 with m6A modification in PBMCs of T1D patients is significantly decreased, which can affect the proliferation of T cells and change the proportion of T cell subsets. In addition, hsa_circRNA_005995 can interact with the demethylase ALKBH5. The ALKBH5-hsa_circRNA_005995 regulatory axis may serve as a potential therapeutic target for T1D.
以上内容是结合具体的优选实施方式对本发明作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演和替换，都应当视为属于本发明的保护范围。The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.
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