miR-144 functions as an oncomiR in KYSE-410 human esophageal carcinoma cell line in vitro and targets PURA

Priyanka Sharma,  Rinu Sharma

Abstract:

Esophageal cancer (EC) is a highly complex disease with high incidence and mortality rates. Recent studies have shown that miRNAs play critical roles in diverse biological processes including oncogenesis, and we previously reported significantly increased expression of tissue and circulating miR-144 in EC. This study evaluates the functional significance of miR-144 in esophageal squamous cell carcinoma. Herein, we analysed the role of miR-144 in ESCC by silencing it in KYSE-410 cells, and followed this with cell cycle analysis and the following assays; MTT, annexin, colony formation, scratch and matrigel invasion assay. The miR-144 knockdown significantly suppressed ESCC cell proliferation at 72 hours post transfection (p=0.029). Silencing of miR-144 significantly decreased the migration, invasion and colony formation potential of KYSE-410 cells compared to cells treated with negative control (NC). Potential targets of miR-144 were predicted by the in silico approach followed by in vitro validation in real time PCR and luciferase reporter assay. The PURA and Spred1 in silico predicted miR-144 targets were validated by qRT-PCR and luciferase reporter assay. Over-expression of miR-144 significantly decreased PURA mRNA expression by 58.85% at 24 hours post transfection (p=0.009). Further validation by dual-luciferase reporter assay confirmed it is a direct targets of miR-144. Our overall study suggests the oncogenic role of miR-144 in EC by promoting proliferation and migration of ESCC cells. To the best of our knowledge, this is the first report showing PURA as a direct miR-144 downstream target and suggests its potential as a novel therapeutic target for this disease.