Ly resembles colorectal adenomas would be needed. Future research could possibly investigate functional effects of ROR2 loss in colorectal adenomas grown in in vitro organoids [51]. Another possibility would be to use an inducible mouse knockoutmodel that targeted ROR2 in the colon. Using a mouse strain that had a high prevalence for adenomas such as the APC heterozygous 57BL/6 J-ApcMin/J mouse line, would allow for the determination of whether or not early ROR2 loss potentiates adenoma growth and development.Conclusion Our study has found that ROR2 promoter hypermethylation and subsequent expression loss is an early event in CRC progression that first occurs in non-invasive adenomas. ROR2 expression was found to be downregulated in the majority of CRC cases, with subsequent in vitro experimentation indicating that the silencing of the receptor may facilitate increased cellular proliferation and migration. Although it was hypothesised that hyperactivation of the -catenin dependent Wnt signals was the cause, decreases in both -catenin dependent and independent genes following ROR2 knockdown suggested that the effects of ROR2 modulation are context dependent and that the observed effects on proliferation and migration may be influence by interactions with pathways other than catenin dependent Wnt [35, 43, 52, 53]. Future research investigating the interaction of ROR2 with various Wnt and EMT associated proteins would help elucidate the exact mechanism in which ROR2 affects cellular proliferation and migration. Examination of ROR2 loss in a more adenoma like biological model instead of in cancer cell lines would also aide in determining if the silencing of the receptor promoted CRC progression. MethodsCell linesAll colorectal cancer cells were obtained from ATCC (American Type Culture Collection, Manassas, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 VA, USA). HCT116 cells were cultured in McCoy’s media (Life Technologies, Rockville, MD) supplemented with 10 foetal bovine serum, 1?glutamine (200 mM) and penicillin/streptomycin (10 units/ml). RKO cells were cultured in RPMI media (Life Technologies, Rockville, MD) supplemented with 10 foetal bovine serum, 1?glutamine (200 mM) and penicillin/streptomycin (10 units/ml). SW620 cells were cultured in DMEM (Life Technologies, Rockville, MD) supplemented with 10 foetal bovine serum, 1?glutamine (200 mM) and penicillin/streptomycin (10 units/ml). Cells were grown in incubators with humidified atmosphere of 5 CO2 at 37 . Cells were tested on a monthly basis to ensure there was no mycoplasma contamination.ROR2 pFLAG plasmid constructionA ROR2 pFLAG plasmid (pROR2) was constructed by GSK089 solubility isolating the ROR2 cDNA transcript from the Addgene ROR2 plasmid using Primer 1 (CTGATATCGATGGCCCGGG GCTCGGCGCTCCCGC) and Primer 2 (TCCTCTAGATMa et al. BMC Cancer (2016) 16:Page 9 ofCAAGCTTCCAG CTGGACTTGG). The resulting PCR fragment then underwent restriction enzyme digestion with both EcoRV and XbaI. The DNA was then subcloned into the pFLAG-CMVTM-4 plasmid containing an N-terminal epitope tag following a similar restriction enzyme digest.ROR2 siRNA KnockdownCells were seeded at 1 ?106 cells into 60 mm plates (NuncTM, Thermo Fisher Scientific, Rockford, IL USA) and allowed to adhere over a 6 h period. Cells were then serum starved for 18 h before being transfected with either 60 pmoles of ROR2 siRNA or scrambled control siRNA (Life Technologies, Rockville, MD). siRNA were premixed in 250 l of serum free McCoy’s media (Life Technologies, Rockville, MD). s.