Detection And Analysis For Differential Expression Of ADD3Splicing Isoforms Between Colorectal Cancer And Normal Mucosa Tissues

ObjectiveTo detect the expression of ADD3and its splicing isoforms in colorectal cancer (CRC)tissue, colorectal polyps tissue, and the CRC lines SW480and SW620, and toinvestigate the correlativity between expression of these molecules and CRC, andtheir roles in the development of the cancer.MethodsTaqman real-time fluorescent quantitative reverse transcription polymerase chainreaction (FQ-RT-PCR) assays for specific detection of T-ADD3gene and itssplicing isoforms ADD3(-14) and ADD3(+14) were developed respectively. By usingthe FQ-RT-PCR method,50pairs of CRC tissues,20pairs of colorectal polyps tissuewere detected. Two CRC lines SW480and SW620were evaluated for expression ofthe moleculars after oxaliplatin and fluorouracil Intervention.Results1. The Taqman FQ-RT-PCR assays developed showed a high amplification efficiency(between95%-100%) and a stable repeatability (CV <1%) in within-run andbetween-runs).2. Expression levels of T-ADD and ADD3(-14) were found to be decreased in CRCtissues than normal mucous (P <0.001), accompanied by ADD3(+14)/ADD3ratioincreased in CRC tissues (P <0.001).3. A reduction expression was observed of T-ADD3and its splicing isoformsADD3(-14) and ADD3(+14) in colorectal polyps than normal tissues (P <0.01).There was no significant difference found in ADD3(+14)/(-14) ratio between normaltissues and colorectal polyps(P>0.05).4. Compared with the CRC SW480,the expressions of ADD3(-14) and ADD3(+14) inSW620were down-regulated (P<0.01,P<0.05) and the ADD3(+14)/(-14) ratiobecome higher (P <0.001).5.Cell intervention experiments demonstrated increase tendency of ADD3and itssplicing isoforms post treatment with oxaliplatin or fluorouracil in both of CRCSW480and SW620cells, which was more markedly in the SW620than in SW480. InSW480, only the expression of ADD3(+14) was up-regulated, while in SW620theexpressions were up-regulated both in ADD3(-14) and ADD3(+14). The expressionof T-ADD3was up-regulated in SW620only after treated with oxaliplatin.Conclusion1. In CRC there may exist a tendency that ADD3(-14) reduction leads to T-ADD3decrease, accompanied by ADD3(+14)/(-14) ratio increase. 2. In polyp tissue, the expressions of ADD3and its splicing isoforms decreased,whereas ADD3(+14)/(-14) ratio was no obvious changed.3. Changes of ADD3and its splicing isoforms in CRC may be relevant to its invasionabilities.