Role Of LncHMGCR In Diabetic Vascular Remodeling And The Underlying Mechanisms

Background Vascular remodeling is an important pathological feature of diabetic vascular disease.Abnormal changes of biological behaviors of vascular smooth muscle cells(VSMCs)in proliferation,apoptosis and migration are the key pathological mechanisms of diabetic vascular remodeling.Long non-coding RNA(Lnc RNA)refers to a class of RNA molecules that are more than 200 bases in length and cannot encode proteins.It has been documented that Lnc RNA participates in the regulation of biological processes(cell proliferation,differentiation,and apoptosis).Our previous experiment screened for differentially expressed Lnc RNAs in the aortic tissue of 12 week diabetic rats by high-throughput sequencing technology(RNA-seq).Decreased Lnc HMGCR was detected in the aortic vessels of diabetic rats and high-glucose treated VSMCs.The analysis of GO and KEGG databases showed that Lnc HMGCR is closely related to biological functions such as cell proliferation,apoptosis,and tissue metabolism,implicating that Lnc HMGCR may play an important role in the progression of diabetic vascular remodeling.In the present study,we explored the role of Lnc HMGCR in diabetic vascular remodeling and underlying mechanisms.In order to elucidate the molecular mechanisms of Lnc HMGCR,bioinformatics technique was used to predict the binding protein and binding site of Lnc HMGCR.The results showed that THO complex 5(THOC5)maybe interact with Lnc HMGCR.Recent study demonstrated that THOC5 combined with polyadenylation-specific factor 100(CPSF100)and promoted the expression of inhibitor of differentiation 3(ID3),which has been reported to promote the proliferation of aortic VSMCs.Based on the current situation and latest research progress at home and abroad,combined with bioinformatics analysis and previous experimental results,we propose the following working hypothesis: in the process of diabetic vascular remodeling,high glucose inhibits Lnc HMGCR expression in VSMCs,and participates in the regulation of VSMCs proliferation and migration by targeting THOC5/ID3 pathway.This study intends to explore the role and mechanism of Lnc HMGCR in diabetic vasculopathy at the animal and cell levels,and focus on the regulation of Lnc HMGCR/THOC5/ID3 signaling on the proliferation and migration of VSMCs.It can provide scientific data to further clarify the pathogenesis of diabetic vascular disease,and open up a new window for finding effective clinical targets for diabetic vascular disease.Method Bioinformatics Analysis: Cat RAPID database was used to screen downstream proteins and predicts binding sites of Lnc HMGCR,RCSB database was used to analyze downstream protein localization,RPISeq-RF,RPISeq-SVM and Inc Pro algorithms were used to calculate the binding score of Lnc HMGCR to downstream proteins,NCBI database was used to analyze protein conserved domains,Long Target Tool database was used to analyze and predict the binding motif and binding site of Lnc HMGCR.Animal experiment: Diabetic rats model was established by intraperitoneal injection of streptozotocin(STZ),and the experiment was randomly divided into control group and diabetic group.The weight,blood glucose and plasma insulin levels were detected;Vascular ultrasound was used to detect the aortic function of rats;EVG staining was to detect the vascular structure of rat aorta;q RT-PCR was used to detect the expression of Lnc HMGCR in the aorta;Western blot was used to detect the protein expression of THOC5 in aortic vascular tissue of diabetic rats.Cell experiments:(1)Primary vascular smooth muscle cells(VSMCs)were treated with D-glucose(30 m M)for 48 h,cell migration was detected by scratch assay;cell viability was detected by CCK-8 assay;cell proliferation was detected by Ed U kit;PCNA expression level was detected by Western blot;Lnc HMGCR expression was detected by q RT-PCR;THOC5 and ID3 m RNA expression was detected by q RT-PCR.(2)The subcellular localization of Lnc HMGCR was detected by FISH assay.(3)VSMCs were transfected with Lnc HMGCR overexpression plasmid for 24 h,then treated with D-glucose(30 m M)for 48 h.The m RNA expression of THOC5 was detected by q RT-PCR,and the protein expression of THOC5 was detected by Western blot.(4)VSMCs were transfected with THOC5 si RNA for 12 h;the transfection efficiency of THOC5 si RNA was detected by Western blot;cell migration was detected by scratch assay;cell proliferation was detected by Ed U kit;ID3 m RNA expression was detected by q RT-PCR;ID3 protein expression was detected by Western blot.Result Bioinformatics: The downstream proteins THOC5,ESRP1 and ESRP2 were obtained by Cat RAPID database;THOC5,ESRP1 and ESRP2 proteins were located in the nucleus by analysis of RCSB database;The analysis of RPISeq-RF,RPISeq-SVM and Inc Pro algorithms showed that Lnc HMGCR bound to THOC5,ESRP1 and ESRP2 at a high level.The analysis of NCBI database showed that Lnc HMGCR could bind to the conserved domains of THOC5,ESRP1 and ESRP2,and the expression abundance of THOC5 was higher than that of ESRP1 and ESRP2.Animal experiment:(1)Compared with control rats,diabetic rats showed symptoms of polydipsia and polypolyuria.The levels of blood glucose were significantly increased,and the weight and plasma levels in diabetic rats were decreased,indicating that diabetic modeling was successful.Ultrasound showed that the aorta-media thickness of diabetic rats was increased,and the inner diameter of the blood vessels during systole and diastole were decreased.EVG staining results showed that the aortic intima of diabetic rats was abnormally enlarged,wrinkled and thickened,and the middle membrane was thickened and disordered,implicating that the aorta was damaged in diabetic rats.(2)The expressions of Lnc HMGCR and THOC5(m RNA and protein)in the aorta of 12 weeks of diabetic rats were significantly decreased.Cell experiments:(1)High glucose induced proliferation and migration of VSMCs,and up-regulated the expression of PCNA protein.(2)Lnc HMGCR was mainly located in the nucleus of VSMCs.(3)High glucose inhibited Lnc HMGCR expression in VSMCs,reduced the m RNA and protein levels of THOC5,and significantly increased the m RNA and protein levels of ID3.(4)Overexpression of Lnc HMGCR could reverse decreased THOC5 induced by high glucose.(5)si RNA THOC5 significantly promoted VSMCs proliferation and migration,and elevated ID3 m RNA and protein levels.Conclusion(1)Lnc HMGCR was detected in vascular tissue,and mainly in the nucleus of VSMC.(2)Lnc HMGCR could up-regulate THOC5,and knock-out of THOC5 could up-regulate the expression of transcription factor ID3,which mediated high glucose-induced diabetic thoracic aortic remodeling.