Silencing of RegIV by shRNA causes the loss of stemness properties of cancer stem cells in MKN45 gastric cancer cells

: Regenerating islet-derived family member 4 (RegIV) is overexpressed in several types of tumours, including pancreatic and gastric cancer (GC). However, the role it plays in gastric cancer stem cells (GCSCs) remains unknown. The present study tested the hypothesis that the silencing of RegIV by shRNA in GC cells may cause the loss of their stemness properties, indicating the inhibition of growth, proliferation and increased sensitivity to chemoradiation-induced cell death. MKN45 poorly differentiated human GC cells were propagated as mammospheres in stem cell culture conditions. Mammospheres were identified as CSCs using generally acknowledged CSC markers such as CD44. A panel of 21-nucleotide shRNAs were designed to target RegIV gene expression. Several shRNA constructs were identified that led to significant reduction in RegIV mRNA expression. Furthermore, the stemness properties of control mammospheres and RegIV knockdown mammospheres were compared by tumourigenicity assay in vivo and plate colony formation assay in vitro. Finally, we evaluated the treatment response in both mammospheres which underwent chemoradiation. The knockdown expression of RegIV by shRNA deprived CSCs of their stemness properties and increased the effectiveness of cell killing following chemoradiation. Inhibition of endogenous RegIV expression may be a new therapeutic strategy for human GC.