Objective To investigate the effect of Slfn1 on the proliferation of vascular smooth muscle cells (VSMCs) and its mechanism.Methods A total of56 male SD rats were sacrificed under anesthesia,and vascular segments were cut from the abdominal aorta to the aortic arch. Vascular medial membranes were isolated and cultured for 4-5 d. Immunofluorescent staining was used to detect the expression of α-smooth muscle actin (α-SMA ) to verify smooth muscle cells. VSMCs were infected with the recombinant adenoviruses for silencing Slfn1 with multiplicity ( MOI) of 1 × 102,1 × 103,and 1 × 104 for 48 hours,respectively. The percentages of VSMCs green fluorescent protein ( GFP) in total VSMCs in different MOI groups was detected by fluorescence microscopy and flow cytometry to determine the optimal MOI for subsequent experiments. The cells were divided into Slfn1 interference group ( infected with recombinant adenoviruses for silencing Slfn1 ),Slfn1 interference control group ( infected with recombinant adenoviruses for silencing Slfn1 control) and blank control group ( cultured with normal serum ) . Polymerase chain reaction ( PCR ) was used to detect Slfn1 messenger RNA expression ( mRNA) in three groups. CCK-8 assay was used to detect VSMC proliferation. Total RNA was isolated for high-throughput RNA sequencing. R language analysis was performed on RNA sequencing data to identify differentially expressed genes. Gene Ontology ( GO) and Kyoto Gene and Genome Database ( KEGG) enrichment analyses were run on differentially expressed genes. Results On the 10th to 15th day of primary culture of VSMCs,the cells showed a typical "peak-valley" growth morphology. Immunofluorescent staining showed α-SMA expression were in VSMC cytoplasm,verifying that the cells were VSMCs. After 48 hours of infecting VSMCs with recombinant adenovirus for silencing Slfn1,the infection efficiency of VSMCs in the 1 × 103 MOI group was high (90％). PCR results showed a decrease in Slfn1 mRNA expression in VSMCs in Slfn1 interference group (P<0.05),while CCK8 results showed an increase in VSMC proliferation in Slfn1 interference group ( P<0.05 ) . After infecting VSMCs with recombinant adenoviruses for silencing Slfn1 for 48 hours,RNA was extracted for high-throughput RNA sequencing. R language analysis was performed on RNA sequencing data. The results showed there were 94 upregulated genes and 181 downregulated genes among the differentially expressed genes. GO and KEGG enrichment analyses showed that the Slfn1 gene may regulate VSMC proliferation through protein glycosylation,mRNA monitoring pathway and sphingolipid signaling pathway,etc.Conclusion Silencing Slfn1 may promote VSMC proliferation,and its mechanism may be related to the regulation of protein glycosylation,mRNA monitoring pathway and sphingolipid signaling pathway.
维普
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