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Analysis of RHOA Mutations and Their Significance in the Proliferation and Transcriptome of Digestive Tract Cancer Cells

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Analysis of RHOA Mutations and Their Significance in the Proliferation and Transcriptome of Digestive Tract Cancer Cells ONCOLOGY LETTERS 22: 735, 2021 Analysis of RHOA mutations and their significance in the proliferation and transcriptome of digestive tract cancer cells NAOKI IKARI1,2, AKIKO SERIZAWA1, ETSUKO TANJI2, MASAKAZU YAMAMOTO1 and TORU FURUKAWA1‑3 1Department of Surgery, Institute of Gastroenterology; 2Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Shinjuku‑ku, Tokyo 162‑8666; 3Department of Investigative Pathology, Tohoku University Graduate School of Medicine, Aoba‑ku, Sendai 980‑8575, Japan Received April 23, 2021; Accepted July 14, 2021 DOI: 10.3892/ol.2021.12996 Abstract. The ras homolog family member A (RHOA) gene showed that the genes associated with small molecule metabolic encodes a member of the Rho family of small GTPases and is process, oxidation‑reduction processes, protein kinase activity, known to function in reorganization of the actin cytoskeleton, transport, and cell junction were commonly downregulated in which is associated with regulation of cell shape, attachment cells whose proliferation was attenuated by the knockdown of and motility. RHOA has been found to be recurrently mutated RHOA. These results suggested that certain RHOA mutations in gastrointestinal cancer; however, the functional signifi‑ may result in upregulation of lnc‑DERA‑1 and genes associated cance of the mutated RHOA protein in digestive tract cancers with cellular metabolism and proliferation in digestive tract remains to be uncovered. The aim of the present study was cancers. to understand the role of mutant RHOA in the proliferation and transcriptome of digestive tract cancer cells. Mutations of Introduction RHOA in one esophageal cancer cell line, OE19, eight gastric cancer cell lines, namely, AGS, GCIY, HGC‑27, KATO III, The ras homolog family member A (RHOA) gene encodes a MKN1, MKN45, SNU16 and SNU719, as well as two colon member of the Rho family of small GTPases and is known cancer cell lines, CCK‑81 and SW948, were determined using to function in reorganization of the actin cytoskeleton, which Sanger sequencing. The results uncovered several mutations, is associated with regulation of cell shape, attachment and including p.Arg5Gln and p.Tyr42Cys in CCK‑81, p.Arg5Trp motility. RHOA has been found to be recurrently mutated and p.Phe39Leu in SNU16, p.Gly17Glu in SW948, p.Tyr42Ser in gastrointestinal cancer, especially in diffuse‑type gastric in OE19, p.Ala61Val in SNU719, p.Glu64del in AGS. cancer cases (1‑3). In this cancer, residues p.Arg5, p.Gly17, Wild‑type RHOA was identified in GCIY, HGC‑27, KATO p.Tyr42 and p.Leu57 of RHOA are considered hotspot III, MKN1 and MKN45. Knockdown of RHOA using small missense mutations (1,2). However, the functional significance interfering RNA attenuated the in vitro proliferation in the of these mutations has not been consistently demonstrated. three‑dimensional culture systems of GCIY, MKN1, OE19 and Kakiuchi et al (1) suggested that the hotspot mutations were SW948, whereas no apparent changes were seen in CCK‑81, gain‑of‑function mutations because inhibiting the expression HGC‑27 and SNU719. Transcriptome analysis revealed that of the mutant RHOA induced the suppression of proliferation downregulation of the long non‑coding RNA (lnc)‑DERA‑1 of gastric cancer cells. In addition, Zhang et al (4) showed was observed in all tested cell lines following RHOA knock‑ that RHOAp.Tyr42Cys was a gain‑of‑function mutation that could down in the RHOA‑mutated cell lines. Gene Ontology analysis sufficiently induce diffuse‑type gastric cancer in a mouse model. On the other hand, Wang et al (2) indicated that these were loss‑of‑function mutations because the mutant RHOA protein showed reduced small GTPase activity and lost the ability to mediate anoikis. Sakata‑Yanagimoto et al (5) Correspondence to: Dr Toru Furukawa, Department of also reported that the RHOA p.Gly17Val mutation was a Investigative Pathology, Tohoku University Graduate School of loss‑of‑function mutation because it showed loss of GTP Medicine, 2‑1 Seiryomachi, Aoba‑ku, Sendai 980‑8575, Japan binding activity and inhibition of wild‑type RHOA function. E‑mail: [email protected] Interestingly, knockdown of RHOA in gastric cancer cells RHOA Abbreviations: RHOA, ras homolog family member A; YAP, with intrinsic abundant expression of , irrespective Yes‑associated protein 1; TAZ, WW‑domain‑containing transcription of its mutational status, results in inhibition of proliferation regulator 1 in vitro (6). Downregulation of RHOA via miR‑31 inhibits cell proliferation and invasiveness (7). Moreover, overexpres‑ Key words: RHOA, gastric cancer, colon cancer, mutation, sion of wild‑type RHOA induces immortalization of human proliferation, transcriptome, lnc‑DERA‑1, metabolism mammary epithelial cells. However, these immortalized cells were anchorage‑dependent and were unable to form tumors when implanted in nude mice (8). Although these pieces 2 IKARI et al: RHOA IN DIGESTIVE TRACT CANCER CELLS of evidence have highlighted the different aspects of the antisense, 5'‑UAC GAA UGA CGU GCG GUA CGU‑3'. For the molecular functions of RHOA, the functional role of RHOA cellular proliferation assay, 1.0x104 cells/well were seeded mutations in the digestive tract cancers are yet to be deter‑ into a 96‑well clear flat bottom ultra‑low attachment plate mined. In the present study, to understand the functional role (Corning, Inc.) with 100 µl growth medium containing 1 nM of RHOA mutations in digestive tract cancers, genotyping, of siRNA and 0.16% (vol/vol) RNAiMAX (Thermo Fisher transcriptome analysis and proliferation assays were carried Scientific, Inc.) according to the manufacture's instruction. out in cell lines expressing the mutant or wild‑type RHOA, as The cells were incubated at 37˚C with 5% CO2 in humidified well as in cells where RHOA has been knocked down. conditions, except for SW948 cells that were incubated at 37˚C with 100% air. Cells were assayed 24 h later (Day 1) and then Materials and methods every 48 h (Day 3 and 5) until Day 7. For immunoblotting, 2.5x105 cells/well were seeded into a 6‑well clear flat‑bottom Cell culture. The AGS cell line was obtained from American ultra‑low attachment plate with 1 ml medium containing 1 nM Type Culture Collection. The GCIY, KATO III, HGC‑27, siRNA and 0.16% (vol/vol) of RNAiMAX. The transfected MKN1 and MKN45 cell lines were obtained from RIKEN cells were incubated as aforementioned, and collected 48 h BioResource Center. The OE19 and SW948 cell lines were later. The low attachment plates were used to allow prolif‑ obtained from Public Health England (Salisbury, UK). SNU16 eration in three‑dimensional spheroid conditions, a method and SNU719 cell lines were obtained from Korean Cell that is more suitable for in vitro bioassays than conventional Line Bank. CCK‑81 cell line was obtained from Japanese two‑dimensional assays (14). Collection of Research Bioresources Cell Bank. All cell lines were cultured according to recommendations from suppliers. Three‑dimensional cell proliferation assay. Following AGS cells were cultured in F‑12 Ham, Kaighn's Modification RHOA‑knockdown, cell viability was assessed using the (Sigma‑Aldrich; Merck KGaA) supplemented with 10% CellTiter‑Glo® 3D Cell Viability Assay (Promega Corporation) FBS (Immuno‑Biological Laboratories Co., Ltd.). Minimum according to the manufacturer's instruction. The viability of Essential Medium (MEM; Sigma‑Aldrich; Merck KGaA) the cells transfected with NC siRNA was used as the control. supplemented with 15% FBS was used for GCIY cells. Cell viability was calculated after subtraction of background RPMI‑1640 (Sigma‑Aldrich; Merck KGaA) supplemented absorbance as follows: Cell viability (%)=(absorbance of the with 10% FBS was used for KATO III, MKN1, MKN45, OE19, sample/absorbance of the control) x100. SNU16 and SNU719 cell culture. MEM supplemented with 10% FBS was used for HGC‑27 and CCK‑81 cell maintenance. Immunoblotting. Cells were harvested and lysed in modified Leibovitz's L‑15 (Thermo Fisher Scientific, Inc.) supplemented RIPA buffer containing 1X complete mini protease inhibitor with 2 mM Glutamine (Thermo Fisher Scientific, Inc.) and cocktail (Sigma‑Aldrich; Merck KGaA) and 1X PhosSTOP 10% FBS was used for SW948 cell culture. SW948 cells were phosphatase inhibitor cocktail (Sigma‑Aldrich; Merck KGaA). maintained at 37˚C with 100% air in a humidified atmosphere; Protein concentration was determined by using the Bradford all other cell lines were cultured at 37˚C with 5% CO2 in a Protein Assay Kit (Bio‑Rad Laboratories, Inc.) according humidified atmosphere. These cell lines were selected because to the manufacture's instruction. Cell extracts containing of availability and of being characterized previously as origi‑ 40 µg protein were separated by electrophoresis on a 10‑20% nating from digestive tract tumors (1,9‑12). gradient polyacrylamide gel and blotted onto a polyvinylidene difluoride membrane (ATTO Corporation) using the XV Mutational analysis of the cell lines. DNA was extracted from Pantera MP System (DRC Co., Ltd.), according to the manu‑ the cultured cells using GenElute™ Mammalian Genomic facturer's instructions. Blocking was performed for 1 h using DNA Miniprep Kit (Sigma‑Aldrich; Merck KGaA) according the ECL Blocking Agent (Amersham Biosciences; Cytiva) at to the manufacturer's instructions. All coding exons and splice room temperature (RT), and the membrane was incubated with sites of RHOA were amplified by using AccuPrime™ Taq DNA primary antibodies overnight at 4˚C. Primary antibodies used Polymerase (Thermo Fisher Scientific, Inc.) and paired primers were the rabbit monoclonal anti‑RHOA antibody (clone 67B9; shown in Table SI. The amplified products were analyzed using 1:1,000 dilution; cat. no. 2117; Cell Signaling Technology, Inc.) Sanger sequencing, as described previously (13). To investigate and the mouse monoclonal anti‑β‑actin antibody (clone AC‑15; whether RHOAp.Arg5Gln and RHOAp.Tyr42Cys in the CCK‑81 cell 1:1,000 dilution; cat. no. A5441; Sigma‑Aldrich; Merck KGaA). line were cis‑ or trans‑compound heterozygous mutations, The membrane was incubated with a corresponding secondary mutation‑specific primers, wild‑type specific primers and antibody for 1 h at RT.
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