| BackgroundAtherosclerosis is the fundamental cause of cardiovascular diseases and one of the top ten causes of death in the world.The morbidity and mortality of atherosclerosis have remained at a high level despite socio-economic development and the continuous innovation of medical technology.Among them,the mortality rate of ischemic stroke caused by carotid atherosclerosis(accounting for 80% of the causes of stroke)has been in the top three of all kinds of fatal diseases for years.Therefore,the search for the mechanism of atherosclerosis and the effective target of treatment is still a research hotspot.The occurrence of carotid atherosclerosis is a complex process involving multiple factors.Other studies have shown that the stability of carotid plaque greatly affects the occurrence of stroke.Currently,a large number of studies have shown that abnormalities of Long non-coding RNAs(lnc RNAs)are directly related to cardiovascular diseases.Therefore,further exploration of the regulatory mechanism between lnc RNAs and carotid plaque stability may open a new chapter in the diagnosis and treatment of carotid atherosclerosis.ObjectiveTo identify the co-expressed m RNAs associated with the long non-coding RNA Lnc-C3orf38-2:7 through apoptosis pathway.To explore the regulatory relationship between those two and further clarify the specific mechanism of the synergistic regulation of smooth muscle cell apoptosis and the decrease of carotid atherosclerotic plaque stability.Methods1.Clinical sample study: Medical history,blood samples and carotid plaque tissue samples of patients with carotid stenosis who underwent carotid endarterectomy in our center from June 2018 to now were collected under the condition that patients signed informed consent and met the ethical review.8 pairs of stable and unstable plaques of typical carotid artery stenosis were selected for RT-q PCR,PCR electrophoresis and in situ hybridization experiments to verify the co-expression of Lnc-C3orf38-2:7 m RNA related to apoptosis pathway at the nucleic acid level: the differential expression of APAF1,JUN,IKBKG and En DOG.Proteins were extracted from stable and unstable plaque 8 of typical carotid artery stenosis.Western Blot assay,immunohistochemistry and immunofluorescence were used to verify the differential expression of downstream proteins co-expressing m RNA related to apoptosis pathway in stable and unstable plaque tissues of Lnc-C3orf38-2:7.2.In vitro cell study: Lnc-C3orf38-2:7 lentivirus transfection intervention was conducted after cultured human vascular smooth muscle cells,which were divided into four experimental groups: up-regulated group,up-regulated control group,down-regulated group and down-regulated control group.RT-q PCR,Western Blot,flow cytometry,RNA in situ hybridization and Ch IRP immunoprecipitation were performed to explore the m RNA regulation relationship between Lnc-C3orf38-2:7 and the co-expressed apoptosis pathway,and further elucidate the specific pathway mechanism of the synergistic regulation of Lnc-C3orf38-2:7 and the decreased stability of carotid atherosclerotic plaque by the two proteins.ResultsClinical sample study: Lnc-C3orf38-2:7 co-expression of apoptosis pathpath-related m RNA APAF1 has the highest correlation with GO analysis(correlation=0.79,P < 0.05),and there is a clear correlation with Lnc-C3orf38-2:7 as verified by q RT-q PCR and PCR electrophoresis experiments in human tissue samples at later stage.The two are synergically upregulated in human carotid artery unstable plaques:RT-q PCR verified that m RNA APAF1 expression level was 2.07±0.46 in the human carotid unstable plaque group and 1.00±0.13 in the human carotid stable plaque group,and the m RNA APAF1 expression level in the unstable group was significantly up-regulated compared with the stable group(P < 0.001).By gray analysis PCR electrophoresis results,the m RNA APAF1 expression level in the unstable carotid plaque group was still significantly different from that in the carotid plaque stable group(P < 0.001).Lnc-C3orf38-2:7 co-expresses APAF1 m RNA associated with apoptosis pathway.APAF1 protein synthesized downstream of APAF1 is directly related to apoptosis,plays a role in regulating several apoptotic pathways,and is a key pro-apoptotic factor in mitochondrial apoptosis pathway.Western Blot assay,immunohistochemistry and immunofluorescence assay confirmed that the expression of APAF1 protein directly related to apoptosis was also increased in smooth muscle cells of unstable plaque tissues.In situ hybridization localization of Lnc-C3orf38-2:7 and co-expression of m RNA APAF1 were mainly expressed in plaque smooth muscle cells.It was predicted that it would affect the up-regulation of apoptosis of smooth muscle cells in the fibrous cap,and then reduce the stability of plaque.2.In vitro cell study: RT-q PCR results showed that the expression of m RNA APAF1 was significantly increased in the up-regulated group compared with the down-regulated group after Lnc-C3orf38-2:7 lentivirus intervention,with P value less than 0.05,which means the differential expression result was statistically significant.The experiment was repeated for many times,and the results were reproducible.RNA in situ hybridization was performed on vascular smooth muscle cells with virus upregulation group.LNC existed in both the nucleus and cytoplasm,mainly in the cytoplasm.The co-expression m RNA APAF1 only exists in cytoplasm,and the localization of APAF1 and APAF1 is highly coincident.Westernblot analysis showed that compared with the stable group,APAF1 protein was significantly increased in the upregulated group,and the P value was less than 0.05,which means the differential expression result was statistically significant.The experiment was repeated for many times,and the results were repeatable.Flow cytometry showed that APAF1 protein expression was up-regulated on smooth muscle membrane in the up-regulated group.Ch IP assay was performed to further confirm that LNC directly binds to m RNA APAF1,and the translated expression of APAF1 protein increases,leading to the up-regulation of apoptosis of smooth muscle cells in the fibrous cap,thus reducing the stability of plaque.ConclusionIn this study,the specific regulatory mechanism between Lnc-C3orf38-2:7 and its co-expression of APAF1 m RNA related to apoptosis pathway and the downstream synthesis of APAF1 protein directly related to apoptosis was explored for the first time through clinical tissue samples and in vitro cell studies.The synergistic upregulation relationship between Lnc-C3orf38-2:7 and APAF1 m RNA related to apoptosis pathway co-expressed by Lnc-C3orf38-2:7 was determined,and the expression position relationship between Lnc-C3orf38-2:7 and APAF1 m RNA was mapped in cells.Established prompt Lnc C3orf38-2:7 in smooth muscle cytoplasm through related to apoptosis pathway m RNA APAF1 directly,is directly related to the downstream synthetic more apoptosis protein-APAF1 protein,rise to smooth muscle cell apoptosis,and then make the plaque fibrous cap is easy to be broken,carotid plaques organizations tend to be unstable,the impact on the development of carotid atherosclerotic disease process and the final outcome.In this study,Lnc-C3orf38-2:7 and its co-expression of APAF1 m RNA related to apoptosis pathway may become a new target for the diagnosis and treatment of carotid atherosclerosis in the future,and will provide new pathway and strategies for the clinical carotid atherosclerosis diagnosis and future targeted treatment. |
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