Establishment Of An Irinotecan Resistant Human Colon Carcinoma Cell Line HT-29/CPT-11and Identification Of Differentially Expressed MicroRNAs

Objective To establish an irinotecan(CPT-11) resistance colorectal cancer cell line in vitro, then screen expression profile of microRN A (miRNA) between HT-29and HT-29/CPT-11cell lines by using microRNA microarry and examine the CPT-11resistance associated miRNAs of colorectal cancer in combination with real-time quantitative PCR. In order to discover new biomarkers that are better able to predict the cancer chemotherapy sensitivity.Method HT-29/CPT-11cell line was established by stepwise selection in increasing dose of CPT-11. The IC50and resistance index of CPT-11in HT-29and HT-29/CPT-11cell lines were detected by CCK-8assay. Cell growth curve was drawed and the doubling time was accounted. The cell cycle distribution was determined by flow cytometry. RT-PCR was used to examined the expression levels of MDR-1、MRP mRNA in HT-29and HT-29/CPT-11cell lines. The total RNA was isolated and examined,then miRNAs were isolation by Ambion’s miRNA isolation Kit.Using Human miRNA OneArray (?)v3to screen differentially expressed miRNAs. The slides were scanned by an Axon4000B scanner. The Cy5fluorescent intensities of each probe was analyzed by GenePix4.1software (Molecular Devices). Data anslysis was proceeded by Array-Pro.Fluorescent real-time quantitative PCR was applied to verify the reliability of miRNAs array result.Result (1) The resistance human colorectal cancer cell line HT-29/CPT-11was established and the resistance index was6.51.(2) The doubling time of HT-29/CPT-11was obviously longer than that of HT-29cell (26.45±0.49h vs39.79 ±0.51h).(3) Flow cytometry demonstrated that HT-29/CPT-11cells of phase G1were increased and that of S phase were reduced.(4)The expression level of MDR-1、MRP mRNA in HT-29/CPT-11cell line were higher than that of HT-29separately1.153±0.169vs0.580±0.059、1.239±0.049vs0.719±0.041P<0.05.(5) According to the miRNA microarray analysis between HT-29and HT-29/CPT-11,we select fold change>2as a criteria. Total40differential expressed miRNAs were found between HT-29and HT-29/CPT-11including6miRNAs up-regulation and34miRNAs down-regulation. Four-fold change of miRNAs were3, three-fold change of miRNAs were10and two fold change were27.(6) Among them, miR-100、miR-125b、miR-200b and miR-192were further confirmed by real-time PCR.The results of Real time RT-PCR were consistent with that of microarray.In HT-29/CPT-11,the expression of miR-100、miR-125b was decreased by (0.4482+0.006)fold and (0.3621+0.036)-fold respectively, P<0.05,the level of miR-200b and miR-192was increased by (41.4652±0.614)-fold and (18.4451±1.346)-fold comparison with HT-29,P<0.05.Conclusions Establishment of CPT-11resistance cell line HT-29/CPT-11may lay the foundation for exploring CPT-11resistance mechanism.MiRNAs differential expression profile of colorectal cencer CPT-11resistance was obtained. MiR-100、 miR-125b、miR-200b and miR-192were examined. The results of Real time RT-PCR were consistent with that of microarray.Our results suggest that these miRNAs can be used as novel biomarkers for the prediction of CPT-11chemotherapy sensitivity and provide an experimental basis for colorectal cancer individualized therapy.