CircHIPK3 Induce Necroptosis Of Vascular Smooth Muscle And Regulate The Formation Of Atherosclerotic Vulnerable Plaques

Objective:Atherosclerotic vulnerable plaque rupture is a cause of the acute blood syndrome.Vascular smooth muscle cells(VSMCs)is the main source of producing collagen fibrous cap,and the ability of the fibrous cap to resist stretching have an vital role,and therefore is beneficial and necessary for the stability of the plaques.Abnormal death of VSMCs results in fibrous cap thinning,necrotic core formation,and plaque calcification,all of which promote the formation of vulnerable plaques.As a hotspot in recent years,necroptosis has been confirmed to exist in the necrotic core of plaque and is closely related to plaque rupture.Circular RNA(circ RNA),as a special class of non-coding RNA molecules,is not easily degraded and has a more stable expression because its closed ring structure is not affected by RNA exonuclides.However,there are few studies on circ RNA in VSMCs necroptosis.Our study elucidates the specific molecular mechanisms by which circ HIPK3 regulates necroptosis and atherosclerotic vulnerable plaque formation through targeted proteins.Methods:1.First,we collected clinical vascular tissue for HE staining and Masson staining to determine the presence and stability of plaques.Then,NCBI database was used to screen out circ RNA with elevated expression level in plaque tissue,and the up-regulated circ RNA circ HIPK3 in vulnerable plaque tissue was verified by q RT-PCR and FISH.2.Different drugs were used to simulate pathological conditions in human aortic vascular smooth muscle cells(VSMCs),and necroptosis was detected by q RT-PCR and WB.3.Further,we synthesized circ HIPK3’s small interference sequence and overexpressed plasmid in vitro,and verified its regulation effect on necroptosis of VSMCs under physiological and pathological conditions by WB,q RT-PCR,flow cytometry and PI staining.4.Through RNA pull down,mass spectrometry and RNA immunoprecipitation,DRP1 was identified as circ HIPK3 binding protein and was positively regulated by circ HIPK3.5.By silencing DRP1,the level of necroptosis of VSMCs under physiological and pathological conditions was detected.Meanwhile,on the basis of silencing of DRP1,the regulation of circ HIPK3 on necroptosis is verified to be mediated by DRP1.6.ROS production and mitochondrial division were detected under different conditions to clarify the effect of circ HIPK3 targeting DRP1 on mitochondrial function,so as to clarify the specific molecular mechanism of circ HIPK3 regulating necroptosis at the cellular level.Results:In conclusion,circ HIPK3 was highly expressed in vulnerable plaques,and the increase of circ HIPK3 expression level promoted the degree of necroptosis of VSMCs.Circ HIPK3 targets protein DRP1,and its elevated level increases mitochondrial division,resulting in increased ROS production and impaired mitochondrial function,ultimately leading to necroptosis.Conclusion and significance:This study reveals and elucidates the specific molecular mechanism by which circ HIPK3 induces mitochondrial damage and regulates necroptosis of VSMCs and the formation of atherosclerotic vulnerable plaques by targeting DRP1 protein expression.To explore new molecular diagnostic targets for atherosclerotic plaque vulnerability from clinical and cellular levels,the results of this study can provide a potential theoretical basis for the development of accurate diagnostic biomarkers and molecular targets in the future.