miR-335-5p Inhibits EMT and PI3K/AKT Pathways via MARCH8

Ubiquitination is a highly conserved process that regulates protein stability by post-translational modification. Membrane-associated RING-CH (MARCH) proteins belong to a family of transmembrane E3 ligases which are responsible for the degradation of their target proteins. Recently, several MARCH family members, including MARCH8, have been reported to be deregulated in cancers. However, the nuances of the exact mechanism remain unexplored. Herein, we investigated regulation of PI3K/AKT and EMT pathways in esophageal cancer via miR-335-5p/MARCH8 axis. Putative miRNAs regulating MARCH8 expression were predicted using in-silico tools. Correlation between expression of miR-335-5p and MARCH8 in esophageal cancer and distant matched non-malignant tissues was evaluated using Real-time PCR. Further, luciferase assay and western blot analysis were carried out to study the direct regulation of MARCH8 via miR-335-5p in esophageal cancer cells. Expression of MARCH8 and miR-335-5p was modulated in esophageal cancer cells and its effect on PI3K/AKT and EMT pathways was evaluated using western blot analysis. Prediction tools revealed miR-335-5p to be the most promising miRNA that might regulate MARCH8 expression. Next, expression analysis of miR-335-5p and MARCH8 in esophageal cancer and distant matched non-malignant tissues revealed an inverse correlation between miR-335-5p and MARCH8 expression (r= - 0.293; p = 0.139). A significant decrease in MARCH8 expression was observed post-miR-335-5p transfection in esophageal cancer cells (p < 0.05). The direct regulation of MARCH8 via miR-335-5p was established using luciferase assay. Further, forced expression of miR-335-5p and silencing of MARCH8 in esophageal cancer cells resulted in the inhibition of PI3/AKT and EMT pathways. Our findings for the first time, demonstrate miR-335-5p mediated regulation of PI3K/AKT and EMT pathways via MARCH8.