Functional Analysis of Membrane Associates Ligase in Human Esophageal Cancer

Abstract

newline MARCH8 is a member of recently discovered Membrane Associated RING-CH (MARCH) family of E3 newlineubiquitin ligases. Though initial studies primarily focussed on its immunomodulatory role, the newly newlinediscovered targets of this E3 ligase point towards its possible role in other biological processes such as newlineembryogenesis and inhibition of apoptosis. However, its relevance in cancers is yet to be elucidated. Herein, newlinewe analysed the aberrant expression of MARCH8 in human esophageal squamous cell carcinomas (ESCC). newlineFor this, we carried out quantitative Real Time PCR to assess the levels of MARCH8 mRNA and its variants newlinein ESCC tissues. Significantly increased expression of MARCH8 mRNA was found in ESCCs as compared newlineto distant matched non-malignant tissues (p=0.024, AUC=0.654). Expression ratio of MARCH8 variant 1 to newlinevariant 2 was significantly altered in ESCCs (plt0.001). Immunohistochemical analysis revealed newlineoverexpression of MARCH8 protein in 86% of ESCC tissues (plt0.001, AUC=0.908). Interestingly, intense newlinenuclear staining of MARCH8 protein was detected in cancer cells in addition to its cytoplasmic expression. newlineAs MARCH8 expression was found to be significantly altered in ESCC tissues as compared to distant newlinematched non-malignant tissues, an in depth analysis of its role in ESCC was warranted. Hence, we further newlinesought to evaluate its role in ESCC cells (KYSE-410) by in vitro assays viz. cell proliferation assay, newlineclonogenic assay, cell cycle assay by DNA content measurement, annexin assay and migration/invasion newlineassays with MARCH8 gene knockdown. Knockdown of MARCH8 resulted in decreased proliferation, newlinemigration, invasion and clonogenic potential of esophageal cancer cells. In addition to this, silencing of newlineMARCH8 induced apoptosis in esophageal cancer cells, which was measured by cell cycle distribution assay, newlineshowed increase in subG0 and G2/M populations (cell death) and decrease in S-phase population. To further newlinecheck the type of apoptosis induced by MARCH8 silencing, annexin assay was perfor