MicroRNA-145 Act As A New Target For The Treatment Of Renal Vascular Lesions In Children With Lupus Nephritis

Background:Lupus nephritis?LN?is a kind of glomerulonephritis caused by systemic lupus erythematosus?SLE?.It is also one of the most common clinical manifestations and the mainly cause of death.The severity of LN which ranges from asymptomatic disease to nephrotic syndrome at last leading patients to end-stage kidney disease.Renal vascular lesion is one of the main causes of death from lupus nephritis and common in pathological.LN patients have poor quality of life,high mortality,and heavy economic pressure,which is a huge burden for patients and society.The measures we take are mainly to prevent the occurrence of the disease,delay the progression of the disease,and protect renal function.Therefore,early diagnosis and treatment is to prevent the disease from developing into chronic renal failure.The diagnosis depends on invasive detection such as renal biopsy,patients'compliance is low.Therefore,finding a reliable biomarker that can assess LN activity will help us to assess disease activity and improve prognosis.Earlier studies by our research group found that the expression of MicroRNA-145?miR-145?was down-regulated in SLE patients and showed a decreasing trend with the increase of renal pathological damage,suggesting that miR-145 is involved in the occurrence and development of SLE,and results from foreign studies Consistent.Objective:To investigate whether miR-145 is abnormally expressed in the children with LN.Find out the effect of miR-145 on the phenotype of human vascular smooth muscle cells?HVSMCs?and the mechanism of its impact.To further clarify the role of miR-145 in the human body,and provide additional ideas and methods for clinical diagnosis and clinical treatment.Methods:HVSMCs were cultured in a 37°C,5%CO₂ incubator until the cell density reached 80%-90%for passage.LV-miR-145 and LV-NC lentiviral vectors were constructed and transfected into HVSMCs.Observed with a fluorescence microscope.The cells showed green fluorescence indicating that the transfection was initially successful.Screened with puromycin for 2 weeks Cells have been transfected until the transfected cells grow steadily.After HVSMCs were treated with 10.0?g/L PDGF-BB,total RNA was extracted from cells,and cDNA was synthesized by reverse transcription.The expression of miR-145 was detected by fluorescent quantitative RT-PCR;the color was added with MTT solution and measured at 570 nm on a microplate reader.The absorbance value of each group of HVSMCs in the absolute growth period was treated with the Transwell method,stained with 0.1%crystal violet for 15 minutes,and randomly selected from 5 fields to count the number of cells passing through the membrane under a 200 X optical microscope and photographed;PDGF-BB treatment Cells in each subsequent group were tested for apoptosis by flow cytometry;protein samples were collected from the cells to be tested,processed using WB method,and subjected to chemiluminescence color development,exposure,development,fixation,and data analysis to determine the phenotypic marker?-Expression of SM-actin and OPN in HVSMCs.Data analysis was performed using SPSS 19.0software.p<0.05 indicated that the difference was statistically significant.Results:?1?After PDGF-BB treatment,with the increase of PDGF-BB concentration,the proliferation ability of HVSMCs gradually increased.?2?With the increase of PDGF-BB concentration,the expression of miR-145 of HVSMCs decreased.?3?After miR-145 was up-regulated,the proliferation ability of HVSMCs induced by PDGF-BB decreased.?4?After miR-145 was up-regulated,the migration ability of HVSMCs induced by PDGF-BB decreased.?5?after miR-145 was up-regulated,the apoptosis of HVSMCs induced by PDGF-BB increased.?6?After miR-145was up-regulated,?-SM-actin increased and osteopontin decreased.Conclusion:Our research shows that miR-145 can inhibit the proliferation and migration of HVSMCs induced by PDGF-BB,and change the phenotype of HVSMCs from proliferative to contractile.miR-145 may be involved in the occurrence and development of vascular injury in lupus nephritis,and may be a new target for the treatment of RVL in LN.