Prediction And Identification Of MiRNA For Regulating Neuropilin-2 In Colorectal Carcinoma

ObjectiveTo predict the possible microRNAs which might take part in regulating neuropilin-2(NRP2)by bioinformatics technique,and to identify the effect of the selected miRNA on the expression of NRP2 and its possible mechanism.Methods1.Bioinformatics technique was used to predict the possible miRNAs which might take part in regulating NRP2,select miR-486-5p which is the most possibility for further study.2.The expression of miR-486-5p in tissues of colorectal carcinoma and adjacent non-tumorous specimens were detected by quantitative real time PCR(qRT-PCR).The relationship between miR-486-5p and clinicopathologic features and prognosis was analyzed statistically.3.Construct miR-486-5p over-expressing plasmid,and the plasmid was transfected into the human colorectal carcinoma cell line SW620 by using Lipofectamine2000.qRT-PCR was used to detect the expression of miR-486-5p of the transfected cells,the expression of NRP2 protein of the transfected cells was measured by Western blot, miR-486-5p targeting NRP2 3’UTR directly was verified by dual luciferase reporter assay.4.The proliferation level of SW620 cells by MTT method, flow cytometry was used to detected the SW620 cells’apoptosis rate, and the in vitro migration abilities of SW620 cells by Transwell test.RESULTS1.There were 25 possible NRP2-regulating miRNAs predicted by bioinformatics software.Combined with the literature,we choose miR-486-5p for further study.2.There is a significantly lower expression of miR-486-5p in CRC tissues in comparison with adjacent non-tumorous tissues (P<0.05),and the expression of miR-486-5p were associated with clinicopathologic features,such as differentiation degree,tumor size,lymph node metastasis and TNM stage.Nevertheless,no significant difference was found between different gender and age.3.The expression of miR-486-5p in SW620 cells that was transfected miR-486-5p over-expressing plasmid was obviously up-regulated.Western blot revealed that miR-486-5p reduced the expression level of NRP2. miR-486-5p could directly targeting NRP2 3’UTR was verified by Dual luciferase reporter assay.4.The proliferation and migration abilities of SW620 cells were inhibited significantly,and apoptosis rate cells increased. Conclusions:miR-486-5p can effectively suppress the proliferation and in vitro migration abilities of SW620 cells,and increase the apoptosis rate indicating that miR-486-5p might be used as a target for molecular therapy of colorectal cancer.CONCLUSIONmiR-486-5p play an important role in the post-transcriptional regulation of NRP2 by binding to NRP2 3’UTR complementary sequences directly,which can interfere the normal translation of NRP2.