Analysis of RHOA mutations and their significance in the proliferation and transcriptome of digestive tract cancer cells

The ras homolog family member A (RHOA) gene encodes a member of the Rho family of small GTPases and is known to function in reorganization of the actin cytoskeleton, which is associated with regulation of cell shape, attachment and motility. RHOA has been found to be recurrently mutated in gastrointestinal cancer; however, the functional significance of the mutated RHOA protein in digestive tract cancers remains to be uncovered. The aim of the present study was to understand the role of mutant RHOA in the proliferation and transcriptome of digestive tract cancer cells. Mutations of RHOA in one esophageal cancer cell line, OE19, eight gastric cancer cell lines, namely, AGS, GCIY, HGC-27, KATO III, MKN1, MKN45, SNU16 and SNU719, as well as two colon cancer cell lines, CCK-81 and SW948, were determined using Sanger sequencing. The results uncovered several mutations, including p.Arg5Gln and p.Tyr42Cys in CCK-81, p.Arg5Trp and p.Phe39Leu in SNU16, p.Gly17Glu in SW948, p.Tyr42Ser in OE19, p.Ala61Val in SNU719, p.Glu64del in AGS. Wild-type RHOA was identified in GCIY, HGC-27, KATO III, MKN1 and MKN45. Knockdown of RHOA using small interfering RNA attenuated the in vitro proliferation in the three-dimensional culture systems of GCIY, MKN1, OE19 and SW948, whereas no apparent changes were seen in CCK-81, HGC-27 and SNU719. Transcriptome analysis revealed that downregulation of the long non-coding RNA (lnc)-DERA-1 was observed in all tested cell lines following RHOA knockdown in the RHOA-mutated cell lines. Gene Ontology analysis showed that the genes associated with small molecule metabolic process, oxidation-reduction processes, protein kinase activity, transport, and cell junction were commonly downregulated in cells whose proliferation was attenuated by the knockdown of RHOA. These results suggested that certain RHOA mutations may result in upregulation of lnc-DERA-1 and genes associated with cellular metabolism and proliferation in digestive tract cancers. Keywords: RHOA; colon cancer; gastric cancer; lnc-DERA-1; metabolism; mutation; proliferation; transcriptome.