| BackgroudAmong diverse cancers, colorectal cancer is the third most common one and the third leading cause of cancer death in people worldwide. For the past 10 years, a class of noncoding RNA molecules known as micro RNAs(mi RNA) has been found to be associated with cancer development by acting as either tumor suppressors or oncogenes. In colorectal cancer, many mi RNAs expression patterns altered, and some have been revealed to be associated with tumorigenesis by targeting tumor-associated genes.ObjectiveTo investigate mi RNA differential expression in human colon cancer cells(SW480, HCT-15) and in oxaliplatin-resistant colon cancer cells(SW480/OXA,HCT-15/OXA) and The regulation principle of mi RNA of differences in expression were preliminary studied.We open up new areas for exploration the effective reversal of tumor resistance.Methods(1) Oxaliplatin-resistant colon cancer cells SW480/OXA, HCT-15/OXA were establishment by combining continuous dose and intermittent high dose administration(2) The differential expression of mi RNA in resistant and parental human coloncancer cell lines was detected by gene chip and confirmed using real-time fluorescence quantitative PCR. The antisense oligonucleotides and mimics of differential expression mi RNA were transfected into parental cancer cells to determine the impact on resistance.(3) Bioinformatic analyses were used to predict the target genes hsa-mi R-137 and then verified by green fluorescent protein reporter gene vector. Western blot detected target protein expression. Real-time PCR and Western blot were used to detect target gene m RNA and protein expression in parental colon cancer cells transfected with hsa-mi R-137- antisense oligonucleotides to testify the regulation of mi RNA-137.(4) Oxaliplatin resistance in human primary colon cancer cells was analyzed by MTT assay. The correlation between resistance, hsa-mi R-137 m RNA and YB-l protein was further validated by real-time fluorescence quantitative PCR and immunohistochemical analysis.Results(1) Moderate oxaliplatin-resistant colon cancer cells SW480/OXA,HCT-15/OXA were successfully induced, and both cells cross-resistant to5-fluorouracil and cis-diaminodichloroplatinum(2) Gene chip and real-time quantitative PCR both showed hsa-mi R-93,hsa-mi R-21, hsa-mi R-96, hsa-mi R-22, hsa-mi R-191 hsa-mi R-192 expression were different in parental colon cancer cells and in oxaliplatin-resistant cancer cells. MTT assay verified hsa-mi R-93, hsa-mi R-137, hsa-mi R-21 and hsa-m i R-22 have effected on regulation of oxaliplatin cytotoxicity to colon cancer cells.(3) Bioinformatics softwares predicted the direct target gene of hsa-mi R-137 was YB-l gene. Green fluorescent protein reporter gene vector and real-time PCR verified YB-l m RNA over-expressed when hsa-mi R-137 inhibitor transfected into SW480 and HCT-15. Western Blot assay confirmed hsa-mi R-137 had a negative regulation of YB-l protein in SW480 and HCT-15.(4) Real-time PCR showed that hsa-mi R-137 m RNA expression level related to sensitivity of colon cancer specimens to oxaliplatin. The higher the expression of m RNA was, the stronger the drug sensitivity it would be.Imuno-histochemi-cal analysis showed a lower percentage of YB-l over-expression in oxaliplatin sensitive specimens and a higher percentage in resistant specimens.Conclusion:(1) Abnormal expression of many mi RNAs contributed to oxaliplatin resistance in human colon cancer cells.(2) The target gene of hsa-mi R-137 was YB-l gene, and hsa-mi R-137 can negatively regulate YB-l expression in human colon cancer cells.(3) Has-mi R-137 can regulate the sensitivity of colon cancer cells to oxaliplatin through target YB-l.(4) Has-mi R-137 expression was greatly decreased while YB-l protein increased in resistant colon cancer specimens. The oxaliplatin resistance is closely related with has-mi R-137 and its target gene YB-l expression. |
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