An Analysis On Expression Characteristics And Significance Of MicroRNA-4284 In Patients With Arteriosclerosis Obliterans Of Lower Extremities

Objective: To explore the change characteristics of expression quantity of micro RNA-4284(miR-4284 for short)in the plasma and pathological vessels of patients with arteriosclerosis obliterans(ASO)of lower extremities and to investigate the effects of mi R-4284 on the proliferation,migration and apoptosis of arterial smooth muscle cells under serum-free and anoxic conditions.Methods: Clinical trial: The plasma of 55 patients with ASO of lower extremities and from 54 healthy controls,the pathological vessels from 24 patients with ASO of lower extremities and the normal vessels from another 24 patients with traumatic amputation who were admitted in Department of Vascular Thyroid Surgery in The First Affiliated Hospital of Southwest Medical University from 2014 to 2015 were collected.The plasma was collected from 3 patients with ASO of lower extremities and another 3normal controls with similar age and gender so as to screen 24 kinds of MicroRNAs(mi RNAs)with expression difference >2 folds between patients with ASO of lower extremities and healthy controls using microarray.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was applied to detect the expression levels of miR-4284 in the plasma of 55 patients with ASO of lower extremities and 54 normal controls,and those in the pathological vessels of 24 patients with ASO of lower extremities and inthe normal vessels of 24 healthy controls.According to Fontaine stages,the 55 patients with ASO of lower extremities were divided into stage ?,?,?and ?,and RT-qPCR was utilized to detect the expression levels of miR-4284 in patients' plasma.Cell experiment: Aortic smooth muscle and umbilical venous endothelial cells were routinely cultured,and then cultured in serum-free and anoxic conditions to simulate the ischemic and anoxic ones of lower extremities in patients with ASO of lower extremitiesclinically.Umbilical venous endothelial cells were cultured in high-glucose culture medium,and then in serum-free and anoxic conditions so as to simulate the patients with ASO of lower extremitiescomplicated with type 2 diabetes mellitus(T2DM)clinically.RT-qPCR was used to detect the expression levels of miR-4284 in cells of each group.Cell proliferation was detected via cck8 and cell migration by transwell after miR-4284 as up-regulated and down-regulated,respectively.Moreover,flow cytometry was used to detect cell apoptosis,and western blot the expressions of apoptosis-associated proteins XIAP,BCL2 and BAX.Results: The clinical trial showed that the expression level of mi R-4284 began to increase notably from Fontaine stage? and maintained persistently in a high level in stages ?,? and ? in the plasma of patients with ASO of lower extremities,when compared with that in healthy controls,and it also increased markedly in the pathological vessels in patients with ASO of lower extremities,when compared with that in healthy controls.Cell experiment: The expression level of miR-4284 increased both inlow-glucose and high-glucose serum-free and anoxic conditions in umbilical venous endothelial cells,but it decreased in aortic smooth muscle cells under serum-free and anoxic conditions.Up-and down-regulation of miR-4284 expression had the mild influence on proliferation and migration of aortic smooth muscle cells cultured in serum-free and anoxic environment,and it had a slight impact on apoptosis of aortic smooth muscle cells without culture under serum-free and anoxic conditions.In aortic smooth muscle cells cultured in serum-free and anoxic conditions,up-regulating miR-4284 expression did not have obvious influences on cell apoptosis,but down-regulating mi R-4284 expression could inhibit the cell apoptosis.Apoptosis-inhibiting-associated proteins XIAP and BCL2 increased prominently while apoptosis-promoting-associated protein BAX decreased notably after expression level of mi R-4284 was down-regulated,and there were significant differences.Conclusion: The expression level of miR-4284 increases evidently in the plasma and pathologicalvascular tissues of patients with ASO of lower extremities,and begins to increase from Fontaine stage ? in plasma.However,it decreases markedly in aortic smooth muscle cells cultured in serum-free and anoxic conditions and increased prominently in umbilical venous endothelial cells cultured serum-free and anoxic conditions.miR-4284 has the mild influence on smooth muscle cell proliferation and migration,and down-regulating miR-4284 can inhibit the cell apoptosis.Therefore,regulating miR-4284 will potentially provide a new insight in the targeted treatment ofASO of lower extremities.