| Objective:Type 2 diabetes mellitus(T2DM)is a physical and mental illness that has a certain association with psychological factors.Depression is one of the main psychological factors affecting the treatment of patients with T2DM,which often aggravates the T2DM process.Therefore,it is essential to provide a suitably effective treatment for T2DM patients with depressive symptoms.Baduanjin is the most important type of Chinese traditional health care Qigong,and it has a good regulatory effect on the various organs of the human body.Previous studies have found that the Qigong-Baduanjin intervention can improve blood glucose and lipid metabolism and reduce depression scores in patients with T2DM with depression(DD).However,the molecular mechanism underlying Baduanjin-mediated mitigation of the symptoms of DD patients is still unclear.In recent years,along with the development of RNA sequencing technology,lncRNAs,mRNAs,and circRNAs have found a good application prospect in disease diagnosis and treatment.Therefore,in this study,differential lncRNA,mRNA,and circRNA expression profiles were screened by high-throughput sequencing for DD-related and post-Qigong-Baduanjin interventions,and differentially expressed genes were verified by RT-PCR.The lncRNA-mRNA regulatory network following the Qigong-Baduanjin intervention was constructed,and the key nodes in the gene regulatory network were positioned.From the transcriptomics viewpoint,the effect of Qigong-Baduanjin on depressive symptoms and blood glucose levels in DD patients and the possible mechanism of action of the intervention strategy were studied.Methods:Based on the inclusion and exclusion criteria,self-rating depression scale(SDS),patient health questionnaire 9(PHQ9),and blood glucose and HbAlc assays for subjects,we divided the patients into T2DM group and DD group.A total of 10 patients were enrolled in this study with 5 patients with T2DM and 5 patients with DD.Peripheral blood was collected from the two groups of patients,and leukocytes were isolated to perform lncRNA,mRNA,and circRNA expression profiling.DD-related specific molecules were screened.Subsequently,we conducted a 12-week Health Qigong Baduanjin intervention for DD patients enrolled in the study and collected blood samples from patients in the DD group after the Baduanjin intervention to perform sequencing of lncRNA,mRNA,and circRNA expression profiles.We longitudinally compared the differential expressions of lncRNAs,circRNAs,and mRNAs in the blood of DD patients before and after the Baduanjin intervention.Subsequently,bioinformatics analysis was performed to investigate the functions of differentially expressed lncRNAs,circRNAs,and mRNAs,and of the involved molecular biological pathways.Finally,RT-PCR was performed to verify the expressions of lncRNAs and mRNAs related to the occurrence and development of DD.An lncRNA-mRNA regulatory network was constructed based on the inter-regulatory association between differentially expressed genes in the blood of DD patients following the Baduanjin intervention.Results:(1)The screening of differential expression of lncRNA,circRNA and mRNA between DD and T2DM patients.High-throughput sequencing and bioinformatics analysis were performed to compare the DD and T2DM patients.Finally,28 lncRNAs(25 upregulated,3 downregulated),107 circRNAs(32 upregulated and 75 downregulated),and 89 mRNAs(87 upregulated and 2 down-regulated)were identified in DD differential expression profiles.Stratified cluster analysis and principal component analysis showed that there were significant differences between DD and T2DM patients with respect to lncRNA,circRNA,and mRNA expression.The two groups of samples could be distinguished on the basis of lncRNA,circRNA,and mRNA expression,and all samples were classified correctly as either T2DM or DD.(2)Functional and pathway analysis of differentially expressed mRNA between DD patients and T2DM patients.Through the GO and pathway analysis of the differentially expressed genes,20 biological process(BP;e.g.,hypothalamic development),22 cellular component(CC;e.g.,neuronal cell body),13 molecular function(MF;e.g.,chemokine receptor activity)related entities and 20 pathways(e.g.,cytokine-cytokine receptor interaction pathway and T-cell receptor signaling pathway)associated with DD were identified.The analysis shows that the genes that are differentially expressed in the DD group encode proteins involved in the development of the neuropsychiatric system,immunity,and inflammation.(3)The differentially expressed lncRNA and mRNA between DD patients and T2DM patients were verified by RT-PCR.Using the same population cohort sample,screening for the important differentially expressed lncRNA(XIST,RP 11-706O15.3,and RP11-415F23.2)and mRNA(CD27)associated with DD were verified by RT-PCR experiments.The results of RT-PCR were consistent with the sequencing results,confirming the reliability of the sequencing results.(4)Effect of Qigong-Baduanjin on depression index and glucose levels of DD patients.The blood glucose level,depression index scores,and PHQ9 scores of the DD group were significantly decreased(P<0.05)after 12 weeks of Qigong-Baduanjin intervention.HbAlc levels also declined,but there was no statistically significant difference.(5)The effect of Qigong-Baduanjin on the expression of lncRNA,circRNA and mRNA in DD patients.Compared to that before the Qigong-Baduanjin intervention,207 lncRNAs(33 upregulated and 174 downregulated),266 circRNAs(96 upregulated and 170 downregulated),and 610 mRNAs(99 upregulated and 511 downregulated)that were differentially expressed were identified in the DD group.Among the above differentially expressed genes,6 differentially expressed mRNAs(CXCL8,DUSP2,OSM,CXCR4,NR4A2,and IL1B)and 1 lncRNA(NEAT1)that were significantly associated with DD were selected for RT-PCR validation,and it was found that the RT-PCR results were consistent with the sequencing results.(6)Effects of Qigong-Baduanjin on differentially expressed mRNA and pathway enrichment in DD patients.By analyzing the function and pathway enrichment of differentially expressed genes,it was found that upregulated mRNA was involved in 139 BP entries,15 CC entries and 34 MF entries,while downregulated mRNA was significantly enriched in 145 BP entries,including 60 CC entries and 37 MF bars.After the Qigong-Baduanjin intervention,22 upregulated gene-related pathways and 15 downregulated gene-related pathways were identified in the DD group.Among them,the IL-17 and TNF signaling pathways showed the highest enrichment coefficient for upregulated genes,while ribosome and splicing were the highest for downregulated genes.Therefore,the improvement of the depression index and blood glucose levels in DD patients with Qigong-Baduanjin intervention was associated with the significant enrichment of GO and pathway analysis items in the blood of DD patients treated with Baduanjin,as well as increased IL-17 and TNF signaling,ribosome and chemokine activation,and inflammatory response of the cells.(7)Effects of Qigong-Baduanjin on lncRNA-mRNA regulatory network in patients with DD.We screened 5 upregulated lncRNAs(RP13-516M14.10,NEAT1,CTD-2530H12.2,CTD-3014M21.1,and AC068580.5)to construct the lncRNA-mRNA regulatory network map and a total of 1045 related mRNA were found,in which 688 mRNAs were negatively correlated with the corresponding lncRNAs and 357 mRNAs were positively correlated with the corresponding lncRNAs.Five of our differentially expressed mRNAs were identified in the above lncRNA-mRNA regulatory network and defined as the key nodes of the lncRNA-mRNA regulatory network.The network verifies Qigong-Baduanj in-mediated regulation of the crosslinking and complex regulatory patterns between lncRNA and mRNA in DD patients.In addition,the key nodes in the lncRNA-mRNA regulatory network were screened to provide a target for the further study of the effects of Baduanjin on DD.Conclusions:1.LncRNA,circRNA,and mRNA differential expression profiles exist in T2DM patients with depression compared with patients with simple T2DM.These profiles include differentially expressed lncRNAs(XIST,RP11-706O15.3,RP11-415F23.2)and mRNA-CD27,which are important RNA molecules that we have screened related to T2DM with depression.2.The 12-week Health Qigong Duan Jin Gong method can significantly reduce blood glucose,SDS Depression Rating Scale and PHQ9 scale scores of DD patients(P<0.05);it can regulate the expressions of lncRNA,circRNA,mRNA in DD patients with dysregulated blood.The function and their involvement in pathways of differentially expressed genes between T2DM and DD are related to inflammatory responses and immunity.3.The lncRNA-mRNA regulatory network analysis confirmed the crosslinking and complex regulation patterns of lncRNA and mRNA expression and verified the authenticity of the regulatory network.This study indicates the epigenetic molecular basis of Qigong-Baduanjin intervention in DD and provides a theoretical basis for deeper research on the mechanism of action and targets of the Qigong-Baduanjin intervention in DD. |
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