| Background and Objective:Ischaemia reperfusion injury(IRI)is defined as tissue damage caused by the restoration of blood supply after ischaemia condition.Reperfusion has been demonstrated to significantly accelerate the development of ischaemiainduced necrosis by initiating the inflammatory response and oxidative stress.Myocardial ischaemia reperfusion injury(MIRI),first reported by Jennings et al.in 1960 using a coronary ligation animal model,is frequently observed in multiple clinical situations,such as myocardial ischaemia,cardiac surgery and circulatory arrest.Ischaemia reperfusion conditions might cause local myocardial inflammatory responses and apoptosis,which in turn induce irreversible myocardial damage.MIRI is considered to be one of the primary causes of death in patients with acute myocardial infarction and coronary heart disease(CHD).In the United States alone,millions of people per year suffer from CHD and myocardial infarction.Elucidating the pathogenesis of MIRI must contribute to the exploration of new therapeutic strategies for acute myocardial infarction and CHD.Long non-coding RNAs(lncRNAs),characterized by more than 200 nucleotides in length,are an important type of RNA molecule in mammalian cells.When lnc RNAs were first be identified they were considered to be a transcriptional noise.However,an increasing number of biological studies lnc RNAs have demonstrated that lnc RNAs are involved in various physiological processes,including cell proliferation,apoptosis,invasion,and development.Abnormal lnc RNA expression can result in multiple human diseases,such as metabolic disorders,neurodegenerative disorders,and cardiovascular diseases.Lnc RNA ROR,first identified as a regulator for the reprogramming of human induced pluripotent stem cells,has been demonstrated to play a role in a variety of cancers.Recently,lnc RNA ROR has been reported to aggravate MIRI;however,the downstream signalling pathway remains unknown.Micro RNAs(miRNAs),another important type of RNA molecule,are characterized by a short sequence(20?23 nucleotides)and a lack of protein-encoding capacity.Increasing number of studies have revealed that miRNAs play critical roles in the regulation of MIRI.For instance,Wang HT et al.demonstrated that miR-24-3p alleviated MIRI by inhibiting the expression of RIPK1 in mice.Inhibition of miR-874 could protect the heart from ischaemia reperfusion-induced injury by suppressing cardiomyocyte apoptosis through the STAT3 pathway in an animal model.In addition,miRNAs could mediate the biological functions of lnc RNAs by directly binding to target lnc RNAs.miR-124-3p was previously suggested to be involved in the progression of various human tumours by regulating tumour cell proliferation,apoptosis,and invasion.However,whether miR-124-3p participates in MIRI remains undetermined.To investigate the roles of lncRNA ROR and miR-124-3p in MIRI,we examined their expression in an in vivo rat MIRI rat model and H/R-treated HCM cells.Furthermore,we explored the effects of apoptosis and the inflammatory response on H/R-treated HCMs,and the underlying mechanism by searching the putative target gene of miR-124-3p.Methods:1.Construct MIRI and sham-operated rats in vivo models by surgery,and use q RT-PCR to detect the expression levels of miR-124-3p,TRAF6 and lnc RNA ROR in the myocardial tissues of rats in MIRI and sham-operated groups;TTC The staining experiment was used to evaluate the size of myocardial infarction in the MIRI group and the sham operation group;the TUNEL experiment was used to detect the changes of apoptosis in the myocardial tissue of the MIRI group and the sham operation group.2.Construct an in vitro IRI cell model by treating human cardiomyocytes(HCMs)with hypoxia and reoxygenation(H/R),and use q RT-PCR to detect the expression changes of miR-124-3p in HCM cells;in HCM cells After transfection of miR-124-3p mimics,q RT-PCR was used to detect the overexpression of miR-124-3p in HCM cells,and flow cytometry was used to detect the changes in apoptosis of H/R-treated HCM cells.q RT-PCR and ELISA experiments were used to detect the m RNA and protein expression changes of pro-inflammatory factors TNF-?,IL-6 and IL-1? in HCM cells treated with H/R.3.Analyze and predict the downstream target gene TRAF6 of miR-124-3p through bioinformatics websites micro RNA.org and star Base,and use q RT-PCR to detect the m RNA expression level of TRAF6 in the myocardial tissues of rats in the MIRI group and the sham operation group,Further verify the targeting relationship of miR-124-3p and TRAF6 through dual luciferase reporter gene experiment;after transfecting miR-124-3p mimics and miR-124-3p inhibitor in HCM cells,q RT-PCR and Western blot experiment to observe the changes of m RNA and protein expression of TRAF6;construct the pc DNA3.1-TRAF6 overexpression plasmid to transfect into HCM cells,use q RT-PCR to detect the effect of TRAF6 overexpression,and transfect the TRAF6 overexpression plasmid to stable overexpression After miR-124-3p HCM cells,flow cytometry was used to detect the changes in apoptosis of H/R-treated HCM cells,and q RT-PCR and ELISA experiments were used to detect the H/R-treated HCM cell pro-inflammatory factor TNF-The m RNA and protein expression changes of ?,IL-6 and IL-1?,and the expression changes of TRAF6,p-p65,p-I?B? and p-IKK?/? in HCM cells were detected by Western blot.4.Analyze and predict the complementary binding sites between miR-124-3p and lnc RNA ROR through bioinformatics tools,and verify the interaction between miR-124-3p and lnc RNA ROR using dual luciferase reporter gene experiments.After transfection of si-lnc RNA ROR(si-ROR)interference fragment into HCM cells,q RT-PCR was used to detect the expression changes of miR-124-3p in H/R-treated HCM cells;HCM cells were H/R-treated Afterwards,the q RT-PCR experimental method was used to detect the expression changes of lnc RNA ROR in HCM cells;the pc DNA3.1-lnc RNA ROR overexpression plasmid was constructed and transfected into HCM cells,and the effect of lnc RNA ROR overexpression was detected by q RT-PCR experiment,and pc DNA3 was also used.After the 1-lnc RNA ROR overexpression plasmid was transfected into HCM cells stably overexpressing miR-124-3p,the changes in apoptosis of H/R treated HCM cells were detected by flow cytometry,and the changes were detected by q RT-PCR and ELISA experiments Changes in the m RNA and protein expressions of pro-inflammatory factors TNF-?,IL-6 and IL-1? in HCM cells treated with H/R.Construct the pc DNA3.1-IL-1?overexpression plasmid and transfect it into HCM cells.Use q RT-PCR to detect the effect of IL-1? overexpression,and use flow cytometry to detect the changes of HCM cell apoptosis.Results:1.By constructing an MIRI rat model in vivo,it was found that the expression of miR-124-3p in the myocardial tissue of the MIRI rat model was significantly down-regulated,while the expression of lnc RNA ROR and TRAF6 was significantly increased;TTC staining results showed that the sham operation group had no myocardial infarction,while MIRI Obvious and large infarction was observed in the group;TUNEL experiment results showed that compared with the sham operation group,the apoptosis of MIRI rats was significantly increased.2.By constructing an in vitro IRI cell model,it was found that miR-124-3p was significantly down-regulated in HCM cells treated with H/R;miR-124-3p mimics was transfected in HCM cells,and miR-124-3p was found to be significantly overexpressed;Flow cytometry analysis results showed that H/R treatment significantly increased the apoptosis of HCM cells,and the overexpression of miR-124-3p reversed the apoptosis caused by H/R treatment;q RT-PCR and ELISA experiments It was found that H/R treatment significantly increased the expression of TNF-?,IL-6 and IL-1? m RNA and protein in HCM cells,while the overexpression of miR-124-3p reversed the H/R treatment-induced HCM cells The m RNA and protein expression of TNF-?,IL-6 and IL-1? are up-regulated.3.It is predicted from the bioinformatics website that miR-124-3p can bind to the 3'-UTR of the downstream target gene TRAF6,and this prediction result is confirmed by the dual luciferase reporter gene experiment;q RT-PCR and Western blot analysis The results showed that overexpression of miR-124-3p can significantly down-regulate the expression of TRAF6 m RNA and protein in HCM cells,while down-regulation of miR-124-3p expression can significantly increase the expression of TRAF6 m RNA and protein in HCM cells;transfection in HCM cells After pc DNA3.1-TRAF6,it was found that TRAF6 was overexpressed.Flow cytometry analysis showed that the overexpression of TRAF6 in HCM cells not only increased the apoptosis caused by H/R treatment,but also inhibited the overexpression of miR-124-3p.The protective effect of cell apoptosis.The results of q RT-PCR and ELISA experiments showed that H/R treatment significantly increased the m RNA and protein expression of TNF-?,IL-6 and IL-1? after overexpression of TRAF6 and reversed the overexpression of miR-124-3p leads to down-regulation of TNF-?,IL-6and IL-1? m RNA and protein expression.Western blot experiment results showed that overexpression of miR-124-3p in HCM cells can significantly down-regulate the protein expression of p-p65,p-I?B? and p-IKK?/?,but overexpression of TRAF6 in HCM cells can be seen to the contrary As a result,the overexpression of TRAF6 in HCM significantly reversed the inhibitory effects of miR-124-3p on p-p65,p-I?B?and p-IKK?/? protein expression.4.Through bioinformatics analysis,it is found that there is a complementary binding site between miR-124-3p and lnc RNA ROR,and this interaction is verified by dual luciferase reporter gene experiment,and the results of q RT-PCR experiment show that it is in HCM cells Down-regulating the expression of lnc RNA ROR can significantly increase the expression of miR-124-3p,and the expression of lnc RNA ROR in HCM cells treated with H/R increased.After transfection of pc DNA3.1-TRAF6 in HCM cells,it was found that the overexpression of lnc RNA ROR was obvious;The results of flow cytometry analysis showed that apoptosis increased significantly in H/R-treated HCM cells after overexpression of lnc RNA ROR,and overexpression of miR-124-3p could inhibit cell apoptosis caused by H/R treatment after overexpression of lnc RNA ROR.The results of q RT-PCR and ELISA showed that the m RNA and protein expression of TNF-?,IL-6 and IL-1? increased significantly in H/R-treated HCM cells after overexpression of lnc RNA ROR.Overexpression of miR-124-3p can inhibit the expression of TNF-?,IL-6 and IL-1?m RNA and protein after H/R treatment after overexpression of lnc RNA ROR;pc DNA3.1-IL-1? transfection in HCM cells The overexpression plasmid found that IL-1? was overexpressed,and the results of flow cytometry showed that IL-1?overexpression could significantly increase the apoptosis of HCM cells Conclusion:In summary,the results show that the expression of miR-124-3p was significantly down-regulated in the MIRI rat model and H/R-treated HCMs,while the expression of TRAF6 and lnc RNA-ROR was significantly up-regulated,suggesting that lnc RNA ROR,miR-124-3p and TRAF6 are in Myocardial ischemia-reperfusion injury plays an important role.Mechanism research results show that lnc RNA ROR activates the expression of its downstream target gene TRAF6 by targeting the expression of miR-124-3p,thereby promoting the inflammation and apoptosis induced by ischemia-reperfusion injury of human cardiomyocytes,revealing a key part of MIRI The functional axis of lnc RNA ROR/miR-124-3p/TRAF6 provides a new potential therapeutic target for MIRI. |
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