MiR-5002-5p Responds To Folic Acid Deficiency And Influences The Migration Of HCT-116 Cells By Targeting Gadd45α

Folate is a kind of B vitamin and a key component of one-carbon metabolism.Folate is strongly associated with DNA methylation,DNA synthesis fidelity,and DNA repair.Folate deficiency induced the abnormality on DNA metabolism and increased the risk of a variety of genetic-environmental related degenerative diseases includes the promotion and development of tumors.Gadd45α is a DNA repair and cell cycle arresting protein,its expression could be significantly increased as specific environmental stresses and DNA damage happened,resulting cell cycle arresting and apoptosis via multiple signal transduction pathways.Previous studies have shown that miRNAs can respond to folate deficiency and mediate the expression of multiple target genes to regulate a series of biological processes.This study focuses on(1)examing the effects of folate deficiency to the migration capacity and Gadd45α expression in human colorectal cancer cell line HCT-116;(2)verifying whether Gadd45α is targetly controlled by miR-5002-5p and whether this pathway is involved in regulating cell migration.This study aims to provide further scientific evidence for the viewpoint that specific miRNAs can response to folate deficiency and mediate multiple biological processes by regulating the expression of key target genes.According to the definition of the optimal folate concentration for maintaining genome stability in our laboratory,deficient(22.6 nmol/L)and sufficient(2260 nmol/L)oxidized folic acid(FA)were used to culture HCT-116 cells,respectively;Based on the effects of FA deficiency on Gadd45α expression and the optimal timeliness of Gadd45α gene expression changes after transfection of specific small interfering RNA(siGadd45α)and miR-5002-5p mimics,intervention time was set to 7 days and 60 hours,respectively.Poly(A)tailed RT-qPCR was used to detect miR-5002-5p transcription in FA deficiency,and a dual-luciferase reporter gene detection system was used to verify the targeting between miR-5002-5p and Gadd45α;RT-qPCR and Western Blot were used to analyze the expression of Gadd45α at the transcription and translation level,respectively;Under the same intervention conditions,the expressions of Gadd45α after transfection with siGadd45α and miR-5002-5p mimics were also assessed.Transwell and scratch tests were used to evaluate the effects of FA and miR-5002-5p mimics to cell migration.The results showed that:(1)FA deficiency up-regulated Gadd45α expression(P <0.05)and decreased cell migration ability(P <0.01)after 7 days and 60 hours;Under FA deficient condition,siGadd45α restored cell migration ability,suggesting that FA deficiency could up-regulate Gadd45α to inhibit the migration of tested tumor cells.(2)miR-5002-5p directly targeted Gadd45α,and its expression was down-regulated after 60 hours of FA deficiency(P <0.01);overexpression of miR-5002-5p could block FA deficiency-induced Gadd45α(P < 0.01),suggesting that Gadd45α could respond to FA deficiency mediated by miR-5002-5p.(3)In the absence of FA,transfection of miR-5002-5p mimics promoted cell migration(P <0.05),suggesting that miR-5002-5p overexpression could reverse the FA deficiency-induced inhibition of cell migration in tested tumor cells.In summary,we concluded that FA deficiency inhibits miR-5002-5p expression in HCT-116 cells,which inhibites cell migration via up-regulating Gadd45α expression.This study estimated the targeting relationship of miR-5002-5p to Gadd45α for the first time.We propose that,in colorectal cancer cells,FA deficiency could inhibit miR-5002-5p to license the expression of Gadd45α and inhibit the tumor cell migration in vitro.This model provides new insights into how tumor metastasis can be efficiently blocked and new targets for antifolate chemotherapy.