Bioinformatic Analysis for Potential Biomarkers and Therapeutic Targets of T2DM-related MI
T2DM 相关心肌梗死的潜在生物标志物和治疗靶点的生物信息学分析

Keywords: myocardial infarction, diabetes mellitus, Gene Expression Omnibus, differentially expressed genes, protein-protein interaction
关键词：心肌梗死，糖尿病，基因表达综合数据库，差异表达基因，蛋白质-蛋白质相互作用

Diabetes mellitus (DM), an increasingly common metabolic disorder, is a leading cause of morbidity and mortality and creates a significant public health burden. ${}^1$${}^1$^(1){ }^{1} The latest number of diabetes patients is expected to increase to 642 million by 2040, which will further increase the economic burden on society. ${}^2$${}^2$^(2){ }^{2} The economic burden associated with DM is primarily attributed to both macrovascular and microvascular dysfunction and complications. ${}^3$${}^3$^(3){ }^{3} Abundant studies indicated that T2DM is associated with a substantially increased risk of all-cause death, especially myocardial infarction, peripheral artery diseases, retinopathy, end-stage renal disease, and neuropathy. ${}^{4-6}$${}^{4-6}$^(4-6){ }^{4-6}
糖尿病（DM）是一种日益普遍的代谢性疾病，是导致发病率和死亡率的主要原因，并对公共健康造成重大负担。预计到 2040 年，糖尿病患者的最新数量将增加到 6.42 亿，这将进一步增加社会的经济负担。与 DM 相关的经济负担主要归因于大血管和小血管的功能障碍及其并发症。大量研究表明，2 型糖尿病（T2DM）与全因死亡风险显著增加相关，尤其是心肌梗死、外周动脉疾病、视网膜病、终末期肾病和神经病。

It is well-known that myocardial infarction (MI) is another common problem resulting in cardiovascular events, including cardiac death and heart failure. ${}^7$${}^7$^(7){ }^{7} Diabetes is an independent risk factor of MI. In fact, MI is the leading cause of death among patients with DM, and individuals with DM are at increased risk of recurrent cardiovascular events and mortality after MI. ${}^8$${}^8$^(8){ }^{8} Clinical studies indicated that mortality rates post-MI are twofold higher in DM than non-DM patients, and the rate of cardiovascular death is increased by 4.4 -fold in DM alone without other traditional cardiovascular factors compared with non-DM in the same age groups. ${}^9$${}^9$^(9){ }^{9} The FAST-MI study suggested that patients with DM had a $70\mathrm{\%}$$70\mathrm{\%}$70%70 \% increased risk of being hospitalized for nonfatal heart failure in the year following MI (adjusted hazard ratio 1.70), which shed much-needed light on the impact of DM on heart failure after acute MI. ${}^{10}$${}^{10}$^(10){ }^{10} Partial studies revealed some molecular pathways including decreased mitophagy and metabolic dysfunction that contributes to the death from MI related to DM. ${}^{11,12}$${}^{11,12}$^(11,12){ }^{11,12} However, the related genetics research for finding prognostic markers is still limited and demands further exploration.
众所周知，心肌梗死（MI）是导致心血管事件的另一个常见问题，包括心脏死亡和心力衰竭。 ${}^7$${}^7$^(7){ }^{7} 糖尿病是心肌梗死的独立危险因素。事实上，心肌梗死是糖尿病患者死亡的主要原因，糖尿病患者在心肌梗死后复发心血管事件和死亡的风险增加。 ${}^8$${}^8$^(8){ }^{8} 临床研究表明，糖尿病患者的心肌梗死后死亡率是非糖尿病患者的两倍，而在没有其他传统心血管因素的情况下，糖尿病患者的心血管死亡率比同年龄组的非糖尿病患者增加了 4.4 倍。 ${}^9$${}^9$^(9){ }^{9} FAST-MI 研究表明，糖尿病患者在心肌梗死后的第一年住院非致命性心力衰竭的风险增加（调整后的风险比为 1.70），这为糖尿病对急性心肌梗死后心力衰竭的影响提供了急需的见解。 ${}^{10}$${}^{10}$^(10){ }^{10} 部分研究揭示了一些分子通路，包括线粒体自噬减少和代谢功能障碍，这些因素导致与糖尿病相关的心肌梗死死亡。 然而，寻找预后标志物的相关遗传学研究仍然有限，需要进一步探索。

In the present study, two gene expression profiles for MI and T2DM were downloaded and analyzed. Differentially expressed genes (DEGs) between normal controls and MI or T2DM were found, and the co-expressed DEGs of the two diseases were obtained as well. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and proteinprotein interaction (PPI) analysis were conducted to detect the hub genes. Consequently, hub DEGs and the related molecular pathways may be better to understand the relationship between T2DM and MI.
在本研究中，下载并分析了心肌梗死（MI）和 2 型糖尿病（T2DM）的两个基因表达谱。发现了正常对照与 MI 或 T2DM 之间的差异表达基因（DEGs），并获得了两种疾病的共表达 DEGs。进行了基因本体（GO）、京都基因和基因组百科全书（KEGG）以及蛋白质-蛋白质相互作用（PPI）分析，以检测核心基因。因此，核心 DEGs 及相关分子通路可能更好地理解 T2DM 与 MI 之间的关系。

Gene expression profile data of peripheral blood samples from T2DM and MI patients were searched in the Gene Expression Omnibus (GEO) database (https://www.ncbi. nlm.nih.gov/geoprofiles/). GSE26168 with nine T2DM
来自 T2DM 和 MI 患者的外周血样本的基因表达谱数据在基因表达综合数据库（GEO）中被搜索（https://www.ncbi。nlm.nih.gov/geoprofiles/）。GSE26168 包含九个 T2DM
patients and eight controls, GSE15932 with eight T2DM patients and eight controls, GSE42148 with blood samples of 13 MI patients and 11 controls, and GSE61144 with 14 MI patients and 10 controls were downloaded for analyses in this study. The detailed information of these datasets was shown in Table 1. Since these datasets were obtained from public databases, ethical approval was not necessary.
本研究下载了患者和八名对照的 GSE15932，八名 T2DM 患者和八名对照的 GSE42148，13 名 MI 患者和 11 名对照的 GSE42148，以及 14 名 MI 患者和 10 名对照的 GSE61144 进行分析。这些数据集的详细信息见表 1。由于这些数据集来自公共数据库，因此不需要伦理批准。

To preprocess the data and screen DEGs between patients and controls, limma package in R-platform (R-project.org) was used and statistical difference was defined as $p$$p$pp-value $<0.05$$<0.05$< 0.05<0.05. Fold change (FC) was identified by calculating the ratio of gene expression level in patients and controls. Logarithmic operation with 2 as a base number was adopted to help with the comparison. Those with ${\log }_2\mathrm{FC}<0$${\log }_2\mathrm{FC}<0$log_(2)FC < 0\log _{2} \mathrm{FC}<0 were considered as downregulated genes, and ${\log }_2\mathrm{FC}>0$${\log }_2\mathrm{FC}>0$log_(2)FC > 0\log _{2} \mathrm{FC}>0 were considered as upregulated genes. In addition, heat maps and Venn diagrams for DEGs were created via R-platform. The relative codes were provided as Appendix 1.
为了预处理数据并筛选患者与对照组之间的差异表达基因（DEGs），在 R 平台上使用了 limma 包（R-project.org），统计差异定义为 $p$$p$pp -值 $<0.05$$<0.05$< 0.05<0.05 。通过计算患者与对照组基因表达水平的比率来识别倍数变化（FC）。采用以 2 为底数的对数运算以帮助比较。那些具有 ${\log }_2\mathrm{FC}<0$${\log }_2\mathrm{FC}<0$log_(2)FC < 0\log _{2} \mathrm{FC}<0 的被认为是下调基因，而 ${\log }_2\mathrm{FC}>0$${\log }_2\mathrm{FC}>0$log_(2)FC > 0\log _{2} \mathrm{FC}>0 的被认为是上调基因。此外，通过 R 平台创建了 DEGs 的热图和韦恩图。相关代码作为附录 1 提供。

To better understanding the function of DEGs, DAVID software (https://david.ncifcrf.gov/) was used to conduct gene ontology (GO), including biological process (BP), cellular component (CC), and molecular function (MF), and KEGG pathway enrichment analysis of T2DM- and MI-DEGs. A $p$$p$pp-value of $<0.05$$<0.05$< 0.05<0.05 was recognized as significantly enriched.
为了更好地理解差异表达基因（DEGs）的功能，使用了 DAVID 软件（https://david.ncifcrf.gov/）进行基因本体（GO）分析，包括生物过程（BP）、细胞组分（CC）和分子功能（MF），以及 T2DM 和 MI-DEGs 的 KEGG 通路富集分析。 $p$$p$pp -值为 $<0.05$$<0.05$< 0.05<0.05 被认为是显著富集的。

Table I Details of Datasets
表 I 数据集详情

Abbreviations: T2DM, type 2 diabetes mellitus; MI, myocardial infarction.
缩写：T2DM，2 型糖尿病；MI，心肌梗死。

Graph Prism 6 was applied to conduct ROC curves and calculate the area under the curve (AUC) of the hub genes of co-DEGs, which represented the diagnostic efficiency of genes. A $p$$p$pp-value of $<0.05$$<0.05$< 0.05<0.05 was regarded as statistically significant.
使用 Graph Prism 6 进行 ROC 曲线分析，并计算共表达差异基因（co-DEGs）中心基因的曲线下面积（AUC），这代表了基因的诊断效率。 $p$$p$pp 值为 $<0.05$$<0.05$< 0.05<0.05 被视为具有统计学意义。

Upregulated co-DEGs were submitted to L1000 platform (https://clue.io/), and potential drugs for T2DM-related MI were predicted. CMap connectivity score $>90$$>90$> 90>90 or CMap connectivity score $<-90$$<-90$< -90<-90 was considered to be the standard for recognizing potentially effective drugs.
上调的共同差异表达基因被提交到 L1000 平台 (https://clue.io/)，并预测了与 T2DM 相关的心肌梗死的潜在药物。CMap 连接评分 $>90$$>90$> 90>90 或 CMap 连接评分 $<-90$$<-90$< -90<-90 被认为是识别潜在有效药物的标准。

When comparing the blood samples of T2DM patients and normal controls, 1870 upregulated and 2156 downregulated genes were identified in GSE26168, while 3188 upregulated and 1400 downregulated genes were identified in GSE15932. As shown in Figure 1, 210 common upregulated and 127 common downregulated genes of GSE26168 and GSE15932 were recognized as T2DM-DEGs. As for MI patients compared with controls, 1910 upregulated and 1847 downregulated genes were identified in GSE42148, while 2046 upregulated and 3170 downregulated genes were identified in GSE61144. In addition, 264 common upregulated and 242
在比较 T2DM 患者和正常对照的血液样本时，在 GSE26168 中识别出 1870 个上调基因和 2156 个下调基因，而在 GSE15932 中识别出 3188 个上调基因和 1400 个下调基因。如图 1 所示，GSE26168 和 GSE15932 的 210 个共同上调基因和 127 个共同下调基因被认定为 T2DM-DEGs。至于与对照组相比的 MI 患者，在 GSE42148 中识别出 1910 个上调基因和 1847 个下调基因，而在 GSE61144 中识别出 2046 个上调基因和 3170 个下调基因。此外，264 个共同上调基因和 242 个下调基因。
common downregulated genes were recognized as MIDEGs. Subsequently, co-DEGs were analyzed by comparing T2DM-DEGs with MI-DEGs, where 18 upregulated DEGs and four downregulated DEGs were identified (Figure 1). T2DM, MI- and co-DEGs were listed in Supplement Table 1, while heatmaps of the DEGs with largest fold change were presented in Supplement Figures 1 and $\underset{―}{2}$$\underset{\_}{2}$2_\underline{2}.
常见的下调基因被认定为 MIDEGs。随后，通过将 T2DM-DEGs 与 MI-DEGs 进行比较，分析了共同差异表达基因（co-DEGs），识别出 18 个上调的 DEGs 和 4 个下调的 DEGs（图 1）。T2DM、MI-和共同差异表达基因列在补充表 1 中，而具有最大倍数变化的 DEGs 的热图则呈现在补充图 1 和 $\underset{―}{2}$$\underset{\_}{2}$2_\underline{2} 中。

GO analysis revealed that T2DM DEG-related biological processes (BP) mainly enriched in viral process ( $p$$p$pp-value $=0.0001$$=0.0001$=0.0001=0.0001 ), proteasome-mediated ubiquitin-dependent protein catabolic process ( $p$$p$pp-value $=0.0004$$=0.0004$=0.0004=0.0004 ), and positive regulation of peptidyl-serine phosphorylation ( $p$$p$pp-value $=0.0091$$=0.0091$=0.0091=0.0091 ). Changes in GO cellular components (CC) mainly included extracellular exosome ( $p$$p$pp-value $=0.0008$$=0.0008$=0.0008=0.0008 ), nucleoplasm ( $p$$p$pp-value $=0.0025$$=0.0025$=0.0025=0.0025 ), and cytosol ( $p$$p$pp-value $=0.0047$$=0.0047$=0.0047=0.0047 ). There was significant correlation in GABA receptor binding ( $p$$p$pp-value $=0.0019$$=0.0019$=0.0019=0.0019 ), protein binding ( $p$$p$pp-value $=0.0043$$=0.0043$=0.0043=0.0043 ), and ubiquitin conjugating enzyme activity ( $p$$p$pp-value $=0.0153$$=0.0153$=0.0153=0.0153 ) in relation to molecular function (MF) (Figure 2A).
GO 分析显示，T2DM DEG 相关的生物过程（BP）主要富集于病毒过程（ $p$$p$pp -value $=0.0001$$=0.0001$=0.0001=0.0001 ），蛋白酶体介导的泛素依赖性蛋白降解过程（ $p$$p$pp -value $=0.0004$$=0.0004$=0.0004=0.0004 ），以及肽基丝氨酸磷酸化的正调控（ $p$$p$pp -value $=0.0091$$=0.0091$=0.0091=0.0091 ）。GO 细胞成分（CC）的变化主要包括细胞外外泌体（ $p$$p$pp -value $=0.0008$$=0.0008$=0.0008=0.0008 ），核质（ $p$$p$pp -value $=0.0025$$=0.0025$=0.0025=0.0025 ），和细胞质（ $p$$p$pp -value $=0.0047$$=0.0047$=0.0047=0.0047 ）。在分子功能（MF）方面，GABA 受体结合（ $p$$p$pp -value $=0.0019$$=0.0019$=0.0019=0.0019 ），蛋白质结合（ $p$$p$pp -value $=0.0043$$=0.0043$=0.0043=0.0043 ），和泛素结合酶活性（ $p$$p$pp -value $=0.0153$$=0.0153$=0.0153=0.0153 ）之间存在显著相关性（图 2A）。

With respect to MI-DEGs, the BP terms of protein phosphorylation ( $p$$p$pp-value $<0.0001$$<0.0001$< 0.0001<0.0001 ), inflammatory response ( $p$$p$pp-value $=0.0001$$=0.0001$=0.0001=0.0001 ) and mitochondrial translational elongation ( $p$$p$pp-value $=0.0003$$=0.0003$=0.0003=0.0003 ) were significantly enriched. In the CC category, enriched GO terms were associated with mitochondrial large ribosomal subunit ( $p$$p$pp-value $=0.0001$$=0.0001$=0.0001=0.0001 ), cytosol ( $p$$p$pp-value $=0.0005$$=0.0005$=0.0005=0.0005 ), and mitochondrial inner membrane ( $p$$p$pp-value $=0.0007$$=0.0007$=0.0007=0.0007 ). In addition, the terms of protein binding ( $p$$p$pp-value $<0.0001$$<0.0001$< 0.0001<0.0001 ),
关于 MI-DEGs，蛋白质磷酸化（ $p$$p$pp -value $<0.0001$$<0.0001$< 0.0001<0.0001 ）、炎症反应（ $p$$p$pp -value $=0.0001$$=0.0001$=0.0001=0.0001 ）和线粒体翻译延伸（ $p$$p$pp -value $=0.0003$$=0.0003$=0.0003=0.0003 ）的生物过程（BP）术语显著富集。在细胞组分（CC）类别中，富集的 GO 术语与线粒体大核糖体亚基（ $p$$p$pp -value $=0.0001$$=0.0001$=0.0001=0.0001 ）、细胞质（ $p$$p$pp -value $=0.0005$$=0.0005$=0.0005=0.0005 ）和线粒体内膜（ $p$$p$pp -value $=0.0007$$=0.0007$=0.0007=0.0007 ）相关。此外，蛋白质结合（ $p$$p$pp -value $<0.0001$$<0.0001$< 0.0001<0.0001 ）的术语也被富集。

B
GSE61144

Figure I Venn diagrams for (A) upregulated differentially expressed genes (DEGs); (B) downregulated DEGs.
图 I 韦恩图 (A) 上调的差异表达基因 (DEGs)；(B) 下调的 DEGs。

Figure 2 Functional analyses of differentially expressed genes (DEGs) in type 2 diabetes mellitus (T2DM). (A) The top three enriched gene ontology (GO) terms in biological process (BP), cellular component (CC) and molecular function (MF); (B) the top nine enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
图 2 2 型糖尿病（T2DM）中差异表达基因（DEGs）的功能分析。(A) 生物过程（BP）、细胞组分（CC）和分子功能（MF）中富集的前三个基因本体（GO）术语；(B) 富集的前九个京都基因和基因组百科全书（KEGG）通路。
protein serine/threonine kinase activity ( $p$$p$pp-value $<0.0001$$<0.0001$< 0.0001<0.0001 ), and protein kinase activity ( $p$$p$pp-value $=0.00026$$=0.00026$=0.00026=0.00026 ) related MF were enriched (Figure 3A).
蛋白质丝氨酸/苏氨酸激酶活性 ( $p$$p$pp -值 $<0.0001$$<0.0001$< 0.0001<0.0001 ) 和蛋白激酶活性 ( $p$$p$pp -值 $=0.00026$$=0.00026$=0.00026=0.00026 ) 相关的分子功能被富集 (图 3A)。

KEGG pathway analysis was also performed and T2DMDEGs were majorly enriched in the pathway of ubiquitinmediated proteolysis ( $p$$p$pp-value $=0.0020$$=0.0020$=0.0020=0.0020 ), osteoclast differentiation ( $p$$p$pp-value $=0.0184$$=0.0184$=0.0184=0.0184 ), and viral carcinogenesis ( $p$$p$pp-value $=0.0256$$=0.0256$=0.0256=0.0256 ), while MI-DEGs had a strong correlation with TNF signaling pathway ( $p$$p$pp-value $=0.0051$$=0.0051$=0.0051=0.0051 ), tuberculosis ( $p$$p$pp-value $=0.0074$$=0.0074$=0.0074=0.0074 ), and VEGF signaling pathway ( $p$$p$pp-value $=0.0101$$=0.0101$=0.0101=0.0101 ), which were illustrated in Figures 2B and 3B.
KEGG 通路分析也进行了，T2DMDEGs 主要富集在泛素介导的蛋白质降解通路（ $p$$p$pp -value $=0.0020$$=0.0020$=0.0020=0.0020 ），破骨细胞分化（ $p$$p$pp -value $=0.0184$$=0.0184$=0.0184=0.0184 ）和病毒致癌（ $p$$p$pp -value $=0.0256$$=0.0256$=0.0256=0.0256 ），而 MI-DEGs 与 TNF 信号通路（ $p$$p$pp -value $=0.0051$$=0.0051$=0.0051=0.0051 ）、结核病（ $p$$p$pp -value $=0.0074$$=0.0074$=0.0074=0.0074 ）和 VEGF 信号通路（ $p$$p$pp -value $=0.0101$$=0.0101$=0.0101=0.0101 ）有很强的相关性，这在图 2B 和图 3B 中进行了说明。

We identified 253, 421, and 12 nodes, and 572, 1377, and 20 edges from PPI network of T2DM-, MI- and coDEGs, respectively. UBE3A, CDC23, WWP1, SMURF2, HACE1, CUL5, SIAH2, KBTBD8, CDC34, and UBE2H
我们分别从 T2DM、MI 和共同差异表达基因的 PPI 网络中识别了 253、421 和 12 个节点，以及 572、1377 和 20 条边。UBE3A、CDC23、WWP1、SMURF2、HACE1、CUL5、SIAH2、KBTBD8、CDC34 和 UBE2H
were recognized as hub genes in T2DM-DEGs (Figure 4), while AKT1, STAT3, TLR8, TLR2, MMP9, PTGS2, CXCL1, CCL5, MPO, and CD40LG were the top 10 hub genes of MI-DEGs (Figure 5). With respect to co-DEGs, MPO, MMP9, CAMP, LTF, AZU1, DEFA4, STAT3, and PECAM1 had much abundant connectivity (Figure 6). Details of the hub genes were presented in Table 2.
在 T2DM-DEGs 中被认定为中心基因（图 4），而 AKT1、STAT3、TLR8、TLR2、MMP9、PTGS2、CXCL1、CCL5、MPO 和 CD40LG 是 MI-DEGs 的前 10 个中心基因（图 5）。关于共同差异表达基因（co-DEGs），MPO、MMP9、CAMP、LTF、AZU1、DEFA4、STAT3 和 PECAM1 具有更丰富的连接性（图 6）。中心基因的详细信息见表 2。

ROC curves were constructed to validate the diagnostic value of the hub genes of the co-DEGs in T2DM and MI datasets, respectively. As shown in Figure 7, The AUC for AZU1, CAMP, DEFA4, LTF, MMP9, MPO, PECAM1, and STAT3 in T2DM patients were 0.986 ( $0.943-1.030$$0.943-1.030$0.943-1.0300.943-1.030 ), 0.861 ( $0.677-1.045$$0.677-1.045$0.677-1.0450.677-1.045 ), 0.931 ( $0.807-1.054$$0.807-1.054$0.807-1.0540.807-1.054 ), 0.903 ( $0.755-1.051$$0.755-1.051$0.755-1.0510.755-1.051 ), 0.986 ( $0.943-1.030$$0.943-1.030$0.943-1.0300.943-1.030 ), 0.958 ( $0.866-1.051$$0.866-1.051$0.866-1.0510.866-1.051 ), 0.833 ( $0.637-$$0.637-$0.637-0.637-
ROC 曲线被构建用于验证 T2DM 和 MI 数据集中共同差异表达基因的中心基因的诊断价值。如图 7 所示，T2DM 患者中 AZU1、CAMP、DEFA4、LTF、MMP9、MPO、PECAM1 和 STAT3 的 AUC 分别为 0.986 ( $0.943-1.030$$0.943-1.030$0.943-1.0300.943-1.030 ), 0.861 ( $0.677-1.045$$0.677-1.045$0.677-1.0450.677-1.045 ), 0.931 ( $0.807-1.054$$0.807-1.054$0.807-1.0540.807-1.054 ), 0.903 ( $0.755-1.051$$0.755-1.051$0.755-1.0510.755-1.051 ), 0.986 ( $0.943-1.030$$0.943-1.030$0.943-1.0300.943-1.030 ), 0.958 ( $0.866-1.051$$0.866-1.051$0.866-1.0510.866-1.051 ), 0.833 ( $0.637-$$0.637-$0.637-0.637-

Figure 3 Functional analyses of differentially expressed genes (DEGs) in myocardial infarction (MI). (A) The top three enriched gene ontology (GO) terms in biological process (BP), cellular component (CC) and molecular function (MF); (B) the top nine enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
图 3 心肌梗死（MI）中差异表达基因（DEGs）的功能分析。(A) 生物过程（BP）、细胞成分（CC）和分子功能（MF）中富集的前三个基因本体（GO）术语；(B) 富集的前九个京都基因和基因组百科全书（KEGG）通路。

Figure 4 Protein-protein interaction (PPI) analysis (A) and hub genes; (B) of type 2 diabetes mellitus (T2DM) related differentially expressed genes (DEGs).
图 4 蛋白质-蛋白质相互作用（PPI）分析（A）和中心基因；（B）与 2 型糖尿病（T2DM）相关的差异表达基因（DEGs）。

Figure 5 Protein-protein interaction (PPI) analysis (A) and hub genes; (B) of myocardial infarction (MI) related differentially expressed genes (DEGs).
图 5 蛋白质-蛋白质相互作用（PPI）分析（A）和中心基因；（B）与心肌梗死（MI）相关的差异表达基因（DEGs）。
1.030 ), and 0.819 ( $0.596-1.043$$0.596-1.043$0.596-1.0430.596-1.043 ), respectively. As for MI patients, the AUC for these co-DEGs were 0.757 ( $0.558-$$0.558-$0.558-0.558- 0.957 ), 0.932 ( $0.827-1.038$$0.827-1.038$0.827-1.0380.827-1.038 ), 0.829 ( $0.666-0.991$$0.666-0.991$0.666-0.9910.666-0.991 ), 0.800
1.030），以及 0.819（ $0.596-1.043$$0.596-1.043$0.596-1.0430.596-1.043 ），分别。至于 MI 患者，这些共同差异表达基因的 AUC 为 0.757（ $0.558-$$0.558-$0.558-0.558- 0.957），0.932（ $0.827-1.038$$0.827-1.038$0.827-1.0380.827-1.038 ），0.829（ $0.666-0.991$$0.666-0.991$0.666-0.9910.666-0.991 ），0.800
(0.621-0.979), 0.929 (0.829-1.028), 0.814 (0.641-0.988), 0.821 ( $0.655-0.988$$0.655-0.988$0.655-0.9880.655-0.988 ), and 0.836 ( $0.674-0.998$$0.674-0.998$0.674-0.9980.674-0.998 ), respectively (Figure 8).
(0.621-0.979), 0.929 (0.829-1.028), 0.814 (0.641-0.988), 0.821 ( $0.655-0.988$$0.655-0.988$0.655-0.9880.655-0.988 ), 和 0.836 ( $0.674-0.998$$0.674-0.998$0.674-0.9980.674-0.998 ), 分别 (图 8)。

Figure 6 Protein-protein interaction (PPI) analysis of co-expressed differentially expressed genes (co-DEGs).
图 6 共同表达的差异表达基因（co-DEGs）的蛋白质-蛋白质相互作用（PPI）分析。

Potential drugs for T2DM-related MI were analyzed by uploading the 18 upregulated co-DEGs to L1000 platform,
通过将 18 个上调的共同差异表达基因（co-DEGs）上传到 L1000 平台，分析了与 T2DM 相关的潜在药物
while the top 10 and bottom 10 chemical compounds ranked by CMap connectivity score were listed in Table 3.
在表 3 中列出了按 CMap 连接分数排名的前 10 和后 10 种化合物。

Type 2 diabetes mellitus (T2DM) and myocardial infarction (MI) represent two major health burdens with steadily increasing prevalence and mortality. ${}^{13}\mathrm{T}2\mathrm{DM}$${}^{13}\mathrm{T}2\mathrm{DM}$^(13)T2DM{ }^{13} \mathrm{~T} 2 \mathrm{DM} is a chronic disorder that is characterized by hyperglycemia and insulin resistance. It has been recognized as an independent risk factor for cardiovascular disease including atherosclerosis, myocardial infarction, nephropathy. ${}^{14}$${}^{14}$^(14){ }^{14} Much evidence has demonstrated that the pathogenesis of diabetes mellitus and atherosclerosis have a strong correlation, while the mechanisms of the connection between diabetes and atherosclerosis are not fully elucidated. The production of advanced glycation end-products (AGEs), increased oxidative stress, endothelium injury, inflammation, dyslipidemia were investigated to contribute to diabetes mellitus to
2 型糖尿病（T2DM）和心肌梗死（MI）是两种主要的健康负担，其患病率和死亡率持续上升。 ${}^{13}\mathrm{T}2\mathrm{DM}$${}^{13}\mathrm{T}2\mathrm{DM}$^(13)T2DM{ }^{13} \mathrm{~T} 2 \mathrm{DM} 是一种以高血糖和胰岛素抵抗为特征的慢性疾病。它已被认定为心血管疾病（包括动脉粥样硬化、心肌梗死、肾病）的独立危险因素。 ${}^{14}$${}^{14}$^(14){ }^{14} 大量证据表明，糖尿病和动脉粥样硬化的发病机制有很强的相关性，但糖尿病与动脉粥样硬化之间的联系机制尚未完全阐明。研究发现，晚期糖基化终产物（AGEs）的产生、氧化应激增加、内皮损伤、炎症、血脂异常等因素可能与糖尿病的发生有关。

Table 2 Hub Genes Identified in PPI Network
表 2 PPI 网络中识别的枢纽基因

Abbreviations: PPI, protein-protein interaction; DEGs, differentially expressed genes; T2DM, type 2 diabetes mellitus; MI, myocardial infarction.
缩写：PPI，蛋白质-蛋白质相互作用；DEGs，差异表达基因；T2DM，2 型糖尿病；MI，心肌梗死。

B

Figure 7 Diagnostic value of hub genes of the co-expressed differentially expressed genes (co-DEGs) in type 2 diabetes mellitus (T2DM). (A) ROC curves; (B) specific value of diagnosis efficiency.
图 7 2 型糖尿病（T2DM）中共同表达的差异表达基因（co-DEGs）中心基因的诊断价值。(A) ROC 曲线；(B) 诊断效率的特异性值。

B

Figure 8 Diagnostic value of hub genes of the co-expressed differentially expressed genes (co-DEGs) in myocardial infarction (MI). (A) ROC curves; (B) specific value of diagnosis efficiency
图 8 心肌梗死（MI）中共表达差异表达基因（co-DEGs）中心基因的诊断价值。(A) ROC 曲线；(B) 诊断效率的特异性值
atherosclerosis. ${}^{15}$${}^{15}$^(15){ }^{15} Although various drugs, adequate glycemic control, and reduction of risk factors had been applied, the complications including atherosclerosis are still inevitable in patients with diabetes mellitus. ${}^{16}$${}^{16}$^(16){ }^{16} In addition, previous studies also showed that mortality rates postMI are much higher in DM patients than the non-DM groups. ${}^{17,18}$${}^{17,18}$^(17,18){ }^{17,18}
动脉粥样硬化。 ${}^{15}$${}^{15}$^(15){ }^{15} 尽管已经应用了各种药物、适当的血糖控制和风险因素的减少，但在糖尿病患者中，动脉粥样硬化等并发症仍然不可避免。 ${}^{16}$${}^{16}$^(16){ }^{16} 此外，以前的研究还表明，心肌梗死后糖尿病患者的死亡率远高于非糖尿病组。 ${}^{17,18}$${}^{17,18}$^(17,18){ }^{17,18}

Therefore, it is necessary to explore the mechanism and discover the novel target to prevent T2DM-related MI.
因此，有必要探索机制并发现新的靶点以预防与 T2DM 相关的心肌梗死。

In this study, we searched the data sets of MI and T2DM from GEO. The results showed that 18 upregulated DEGs and four downregulated co-expressed DEGs were identified between MI and T2DM. We also performed GO
在这项研究中，我们从 GEO 搜索了 MI 和 T2DM 的数据集。结果显示，在 MI 和 T2DM 之间识别出 18 个上调的差异表达基因（DEGs）和四个下调的共表达差异表达基因（DEGs）。我们还进行了 GO 分析。

Table 3 The Top 10 and Bottom 10 Chemical Compounds Identified by LI000 Platform
表 3 LI000 平台识别的前 10 和后 10 种化合物

enrichment and KEGG pathway enrichment analyses, and a PPI network was constructed to identify the top 10 hub genes from among the common DEGs. There are eight coDEGs to demonstrate diagnostic value in MI and T2DM ( $p<0.05$$p<0.05$p < 0.05p<0.05 ), which may have an important role in early predict the risk of MI in T2DM.
富集和 KEGG 通路富集分析，并构建了 PPI 网络以识别共同 DEGs 中的前 10 个核心基因。有八个共同 DEGs 在 MI 和 T2DM 中显示出诊断价值（ $p<0.05$$p<0.05$p < 0.05p<0.05 ），这可能在早期预测 T2DM 中 MI 的风险方面发挥重要作用。

Myeloperoxidase (MPO), lactoferrin ( $LTF$$LTF$LTFL T F ), cathelicidin antimicrobial peptide (CAMP), azurocidin 1 (AUZ1), defensin alpha 4 (DEFA4) are expressed in the granules of the neutrophil, which participate in the progress of inflammatory reaction. $MPO$$MPO$MPOM P O, an abundant heme-containing peroxidase, is mainly expressed in neutrophils and to a lesser degree in monocytes. ${}^{19}$${}^{19}$^(19){ }^{19} In the presence of hydrogen peroxide and halides, MPO catalyzes the reactive oxide species including hypochlorous acid and hypohalous acid, which play an important role in inflammation and oxidative stress. ${}^{20}$${}^{20}$^(20){ }^{20} MPO and its oxidative products have been found to be enriched in human atherosclerotic lesions where they co-localized with macrophages. $MPO$$MPO$MPOM P O and its end product HOCl could activate the MMPs and inhibit TIMPs, further promoting the reduction in the fibrous cap, and lead to the destabilized atherosclerotic plaque. ${}^{21}$${}^{21}$^(21){ }^{21} In a large cohort of patients with chest pain, a single measurement of MPO on admission independently predicted acute MI. Moreover, many studies demonstrated that MPO is significantly associated with follow-up cardiovascular
髓过氧化物酶 (MPO)、乳铁蛋白 ( $LTF$$LTF$LTFL T F )、抗菌肽 (CAMP)、蓝细菌素 1 (AUZ1)、α-防御素 4 (DEFA4) 在中性粒细胞的颗粒中表达，参与炎症反应的进程。 $MPO$$MPO$MPOM P O ，一种丰富的含血红素的过氧化物酶，主要在中性粒细胞中表达，在单核细胞中表达较少。 ${}^{19}$${}^{19}$^(19){ }^{19} 在过氧化氢和卤化物存在的情况下，MPO 催化反应性氧物种，包括次氯酸和次卤酸，这在炎症和氧化应激中发挥重要作用。 ${}^{20}$${}^{20}$^(20){ }^{20} MPO 及其氧化产物已被发现富集于人类动脉粥样硬化病变中，并与巨噬细胞共定位。 $MPO$$MPO$MPOM P O 及其最终产物 HOCl 可以激活 MMPs 并抑制 TIMPs，进一步促进纤维帽的减少，导致动脉粥样硬化斑块的不稳定。 ${}^{21}$${}^{21}$^(21){ }^{21} 在一大群胸痛患者中，入院时单次测量 MPO 独立预测急性心肌梗死。此外，许多研究表明 MPO 与随访心血管事件显著相关。
events in MI patients. ${}^{22,23}$${}^{22,23}$^(22,23){ }^{22,23} Furthermore, in a study assessing neutrophil activity in T2DM patients with and without cardiovascular diseases (CVD), researchers found that plasma MPO concentration is elevated in those with CVD, suggesting plasma $MPO$$MPO$MPOM P O levels to be an additional biomarker of oxidative stress and cardiovascular risk in T2DM patients. ${}^{24}$${}^{24}$^(24){ }^{24} In addition, $LTF$$LTF$LTFL T F is a monomeric multifunctional iron-binding glycoprotein, with a single polypeptide chain of about 690 amino acid residues and two sialic acid molecules, which is mainly produced by neutrophils and epithelial cells. ${}^{25}$${}^{25}$^(25){ }^{25} It has been reported that LTF displays anti-inflammatory and antioxidant actions. ${}^{26}$${}^{26}$^(26){ }^{26} It strongly inhibits cholesteryl ester accumulation from LDL by impairing its binding to macrophages and vascular smooth muscle cells. ${}^{27}$${}^{27}$^(27){ }^{27} Though the regulating mechanism of LTF on cardiovascular diseases is not totally clear, clinical study showed that increased baseline concentration of $LTF$$LTF$LTFL T F strongly predicated the long-term risk for fatal ischemic heart disease patients with newly diagnosed DM. ${}^{28}$${}^{28}$^(28){ }^{28} MPO and LTF are present in different granules, while their behavior as the biomarker of MI is equivalent. The specific granules containing $LTF$$LTF$LTFL T F are more readily mobilized than those containing $MPO$$MPO$MPOM P O, which implies that LTF may have a wider dynamic concentration range than $MPO{.}^{29}$$MPO{.}^{29}$MPO.^(29)M P O .^{29} Once inside a lesion, neutrophils release granular proteins: CAMP1 and AZU1 stimulate recruitment of
心肌梗死患者的事件。 ${}^{22,23}$${}^{22,23}$^(22,23){ }^{22,23} 此外，在一项评估 2 型糖尿病（T2DM）患者中有无心血管疾病（CVD）的中性粒细胞活性的研究中，研究人员发现，患有 CVD 的患者血浆 MPO 浓度升高，这表明血浆 $MPO$$MPO$MPOM P O 水平是 T2DM 患者氧化应激和心血管风险的额外生物标志物。 ${}^{24}$${}^{24}$^(24){ }^{24} 此外， $LTF$$LTF$LTFL T F 是一种单体多功能铁结合糖蛋白，具有约 690 个氨基酸残基的单肽链和两个唾液酸分子，主要由中性粒细胞和上皮细胞产生。 ${}^{25}$${}^{25}$^(25){ }^{25} 有报道称 LTF 表现出抗炎和抗氧化作用。 ${}^{26}$${}^{26}$^(26){ }^{26} 它通过损害其与巨噬细胞和血管平滑肌细胞的结合，强烈抑制 LDL 的胆固醇酯积累。 ${}^{27}$${}^{27}$^(27){ }^{27} 尽管 LTF 对心血管疾病的调节机制尚不完全清楚，但临床研究表明， $LTF$$LTF$LTFL T F 的基线浓度增加强烈预测新诊断糖尿病患者的致命缺血性心脏病的长期风险。 ${}^{28}$${}^{28}$^(28){ }^{28} MPO 和 LTF 存在于不同的颗粒中，但它们作为心肌梗死的生物标志物的行为是相同的。含有 $LTF$$LTF$LTFL T F 的特定颗粒比含有 $MPO$$MPO$MPOM P O 的颗粒更容易动员，这意味着 LTF 的动态浓度范围可能比 $MPO{.}^{29}$$MPO{.}^{29}$MPO.^(29)M P O .^{29} 更广。一旦进入病变，嗜中性粒细胞释放颗粒蛋白：CAMP1 和 AZU1 刺激招募
inflammatory monocytes. During the pathogenesis of atherosclerosis, $AZU1$$AZU1$AZU1A Z U 1 is deposited on the endothelium and triggers monocyte arrest and upregulation of endothelial adhesion molecules. ${}^{30}$${}^{30}$^(30){ }^{30} A clinical trial showed that the significant positive correlation of $AZU1$$AZU1$AZU1A Z U 1 with the SYNTAX score, Grace score and Killip class in MI patients, suggesting the strong association between inflammation and disease severity. ${}^{31}$${}^{31}$^(31){ }^{31} The mechanism remains unclear and demands further investigation. CAMP (also named as LL-37) is produced in the atherosclerotic lesions, where it may function as an immune modulator by activating adhesion molecule and chemokine expression, thus increasing innate immunity in atherosclerosis. ${}^{32}$${}^{32}$^(32){ }^{32} The relationship of the $CAMP$$CAMP$CAMPC A M P in destabilization of atherosclerotic plaque and MI remains unknown. DEFA was the family of human neutrophil peptides, which was familiar with DEFA1-3. DEFA1-3 has been confirmed as a risk factor of atherosclerosis and coronary artery disease. ${}^{33}$${}^{33}$^(33){ }^{33} DEFA4 was encoded as human neutrophil peptides 4, while the role of DEFA4 in atherosclerosis and MI has not been investigated. The role of CAMP, AUZ1 and DEFA4 in T2DM or MI remains to be further studied.
炎症单核细胞。在动脉粥样硬化的发病机制中， $AZU1$$AZU1$AZU1A Z U 1 沉积在内皮上并触发单核细胞停滞和内皮粘附分子的上调。 ${}^{30}$${}^{30}$^(30){ }^{30} 一项临床试验显示， $AZU1$$AZU1$AZU1A Z U 1 与心肌梗死患者的 SYNTAX 评分、Grace 评分和 Killip 分级之间存在显著的正相关，表明炎症与疾病严重性之间的强关联。 ${}^{31}$${}^{31}$^(31){ }^{31} 其机制仍不清楚，需要进一步研究。CAMP（也称为 LL-37）在动脉粥样硬化病变中产生，可能通过激活粘附分子和趋化因子的表达，作为免疫调节因子，从而增强动脉粥样硬化中的先天免疫。 ${}^{32}$${}^{32}$^(32){ }^{32} $CAMP$$CAMP$CAMPC A M P 在动脉粥样硬化斑块不稳定和心肌梗死中的关系仍然未知。DEFA 是人类中性粒细胞肽的家族，熟悉 DEFA1-3。DEFA1-3 已被确认是动脉粥样硬化和冠状动脉疾病的风险因素。 ${}^{33}$${}^{33}$^(33){ }^{33} DEFA4 被编码为人类中性粒细胞肽 4，而 DEFA4 在动脉粥样硬化和心肌梗死中的作用尚未被研究。 CAMP、AUZ1 和 DEFA4 在 T2DM 或 MI 中的作用仍需进一步研究。

Matrix metalloproteinase-9 (MMP9) belongs to the MMP family, which is involved in matrix degradation. MMP9 has been found to be participated in the pathogenesis of diabetes and diabetic complications, including diabetic retinopathy. ${}^{34}$${}^{34}$^(34){ }^{34} Moreover, it has been demonstrated that increased MMP9 expression after MI exacerbates ischemia-induced chronic heart failure through regulating autophagic flux. ${}^{35}$${}^{35}$^(35){ }^{35} MMP9 expression and activity were increased in the fibrous cap of atherosclerotic plaques, which promotes extracellular matrix (ECM) degradation and enhances inflammation cell infiltration. ${}^{36}$${}^{36}$^(36){ }^{36} In addition, MMP9 was reported to influence the stability of plaques via promoting the migration and proliferation of vascular smooth muscle cells (VSMCs). VSMCs can further secrete vascular endothelial growth factor (VEGF), which plays an important role in neovascularization (a risk factor for plaque instability). ${}^{37}$${}^{37}$^(37){ }^{37} Circulating MMP9 has been recognized as a biomarker of predicting the plaque rupture ${}^{38}$${}^{38}$^(38){ }^{38} and increased MMP9 expression was related to the leukocyte numbers and left ventricular dimensions and impaired cardiac function. Signal transducer and activator of transcription 3 (STAT3) is an 89 KD protein and composed of six functional domains. It is a signaling molecule and transcriptional factor that plays important role, which attributes to its genomic action that upregulates genes that anti-apoptotic, pro-angiogenic. ${}^{39}$${}^{39}$^(39){ }^{39} STAT3 has been
基质金属蛋白酶-9 (MMP9) 属于 MMP 家族，参与基质降解。研究发现 MMP9 参与了糖尿病及其并发症的发病机制，包括糖尿病视网膜病变。此外，已经证明心肌梗死后 MMP9 表达增加通过调节自噬流加重缺血引起的慢性心力衰竭。MMP9 的表达和活性在动脉粥样硬化斑块的纤维帽中增加，这促进了细胞外基质 (ECM) 的降解并增强了炎症细胞的浸润。此外，有报道指出 MMP9 通过促进血管平滑肌细胞 (VSMCs) 的迁移和增殖影响斑块的稳定性。VSMCs 还可以进一步分泌血管内皮生长因子 (VEGF)，在新生血管形成中发挥重要作用（这是斑块不稳定的一个风险因素）。循环中的 MMP9 已被认定为预测斑块破裂的生物标志物，且 MMP9 表达增加与白细胞数量、左心室尺寸和心脏功能受损相关。 信号转导和转录激活因子 3（STAT3）是一种 89 KD 的蛋白质，由六个功能域组成。它是一种信号分子和转录因子，发挥着重要作用，这归因于其基因组作用，能够上调抗凋亡和促血管生成的基因。 ${}^{39}$${}^{39}$^(39){ }^{39} STAT3 已被
investigated to participate in the pathogenesis of atherosclerosis, including contributing to VSMCs proliferation, migration and phenotype transformation. Mitochondrial STAT3 is a regulator of electron transport chain and ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} homeostasis and has an effect on the mitochondrial production of oxidative stress, therefore playing a crucial role in atherosclerosis. ${}^{40}$${}^{40}$^(40){ }^{40} While whether STAT3 regulates the instability of atherosclerotic plaque remains unclear. STAT3-deficient mice are more susceptible to myocardial ischemia injury, show increased cardiac apoptosis and infarct size and have reduced cardiac function and survival. ${}^{41}$${}^{41}$^(41){ }^{41} Moreover, it has been demonstrated that N -acetylcysteine prevented myocardial injury in diabetic mice through Jak2/STAT3 ${}^{42}$${}^{42}$^(42){ }^{42} Platelet endothelial cell adhesion molecule-1 (PECAM1 or CD31), one member of the Ig gene superfamily, is composed of six extracellular Ig folds. ${}^{43}$${}^{43}$^(43){ }^{43} CD31 was reported to play a protective role in the atherosclerotic plaque. It can limit the unwanted collateral inflammatory damage and regulating leukocyte migration. ${}^{44}$${}^{44}$^(44){ }^{44} CD31 can also reduce the leakage and intraplaque hemorrhage from plaque neovessels. ${}^{45}$${}^{45}$^(45){ }^{45} Studies showed that the administration of antibodies against CD31 before reperfusion can reduce the infarct size. ${}^{46}$${}^{46}$^(46){ }^{46} A study population of cross-sectional analysis that consisted of 420 subjects with T2DM lasting more than 10 years showed that 142 patients with MI and 310 patients with no CVD. There were significant differences of PECAM1 genotype distribution in patients with MI compared with subjects in the control group, which suggest that polymorphism of PECAM1 (C373G) may be a genetic marker of MI in T2DM patients. ${}^{47}$${}^{47}$^(47){ }^{47}
研究表明，参与动脉粥样硬化的发病机制，包括促进血管平滑肌细胞（VSMCs）的增殖、迁移和表型转化。线粒体 STAT3 是电子传递链和 ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} 稳态的调节因子，并对线粒体的氧化应激产生有影响，因此在动脉粥样硬化中发挥着关键作用。 ${}^{40}$${}^{40}$^(40){ }^{40} 目前尚不清楚 STAT3 是否调节动脉粥样硬化斑块的不稳定性。缺乏 STAT3 的小鼠对心肌缺血损伤更为敏感，表现出心脏凋亡和梗死面积增加，心脏功能和生存率降低。 ${}^{41}$${}^{41}$^(41){ }^{41} 此外，已证明 N-乙酰半胱氨酸通过 Jak2/STAT3 ${}^{42}$${}^{42}$^(42){ }^{42} 预防糖尿病小鼠的心肌损伤。血小板内皮细胞粘附分子-1（PECAM1 或 CD31），是免疫球蛋白基因超家族的一个成员，由六个细胞外免疫球蛋白折叠组成。 ${}^{43}$${}^{43}$^(43){ }^{43} 有报道指出，CD31 在动脉粥样硬化斑块中发挥保护作用。它可以限制不必要的旁路炎症损伤并调节白细胞迁移。 ${}^{44}$${}^{44}$^(44){ }^{44} CD31 还可以减少斑块新生血管的渗漏和斑块内出血。 研究表明，在再灌注前施用针对 CD31 的抗体可以减少梗死面积。一项包含 420 名患有超过 10 年 T2DM 的受试者的横断面分析研究显示，142 名患者有心肌梗死（MI），310 名患者没有心血管疾病（CVD）。与对照组受试者相比，心肌梗死患者的 PECAM1 基因型分布存在显著差异，这表明 PECAM1（C373G）的多态性可能是 T2DM 患者心肌梗死的遗传标记。 ${}^{47}$${}^{47}$^(47){ }^{47}