2384 Categorical variables were compared by a chi-square test (Fisher’s exact probability test). Survival curves were constructed using the Kaplan-Meier product-limit method and compared by the log rank test. To evaluate the inde- pendent prognostic factors associated with patient survival (with or without gemcitabine treatment), multivariate Cox proportional hazard regression analysis was used, with miR-142-5p expression, miR-34a expression, pN status, and/or residual tumor status (R factor) as covariates. Sta- tistical signiﬁcance was deﬁned as a P value of \0.05. All statistical analyses were performed by JMP 7.01 software (SAS Institute, Cary, NC, USA). RESULTS Establishment of Gemcitabine-Resistant Pancreatic Cancer Cells Gemcitabine-resistant SUIT-2 and CAPAN-1 cells were generated by exposure to gradually increasing con- centrations of gemcitabine. The ﬁnal concentration of gemcitabine was 200 nM for SUIT-2 cells and 1 lM for CAPAN-1 cells. The viability of the parental SUIT-2 cells signiﬁcantly decreased to less than 20% after treatment with 10 nM gemcitabine, while the viability of gemcita- bine-resistant SUIT-2 cells remained unchanged after treatment with 10 nM gemcitabine. The viability of the parental CAPAN-1 cells signiﬁcantly decreased to less than 10% after treatment with 1 lM gemcitabine, while the viability of gemcitabine-resistant CAPAN-1 cells remained unchanged after treatment with 1 lM gemcitabine. miRNA Differentially Expressed between Parent and Gemcitabine-Resistant Cells We used the TaqMan miRNA array to obtain the 365 miRNA expression proﬁles from the parent and gemcita- bine-resistant pancreatic cancer cell lines. The results show that the expression of 10 miRNAs was more than 2-fold higher in both SUIT-2 and CAPAN-1 gemcitabine-resistant cells compared with the parental cells. We also identiﬁed 14 miRNAs in both SUIT-2 and CAPAN-1 gemcitabine- resistant cells that were downregulated to less than 30% of that in the parental cells. To conﬁrm our TaqMan miRNA array data, we sub- jected 11 of the differentially expressed miRNAs (5 upregulated miRNAs: miR-9, miR-27a, miR-424, miR- 449b, and miR-34a; 6 downregulated miRNAs: miR-152, miR-181c, miR-518b, miR-125a, miR-142-5p, and miR- 204), which were selected on the basis of expression levels and fold changes as promising candidate of clinical markers, to triplicate qRT-PCR analysis, and obtained consistent results (data not shown). K. Ohuchida et al. Analysis of miRNA Expression in Relation to Survival Time of Patients with Pancreatic Cancer after Curative Resection Conventional prognostic factors, such as pT category, pN category, R factor, positive vessel invasion, and adju- vant therapies, including 5-ﬂuorouracil or gemcitabine, reached signiﬁcance for overall survival (Table 1). We also found that the patients treated with gemcitabine had a better prognosis than those without, although the difference was not signiﬁcant (P = 0.058). Next, to investigate the correlation between gemcitabine response and miRNA expression identiﬁed by miRNA expression proﬁling, patients with resected pancreatic cancer were divided into two groups: patients who were treated with gemcitabine and patients who were not. Within each group, we investigated the correlation between the expression levels of the 11 miRNAs identiﬁed by miRNA expression proﬁling and the prognosis of patients with resected pancreatic cancer. miRNA expression was also divided into high- and low-level groups by recursive descent partition analysis, as described by Hoffmann et al.26 First, we investigated the correlation between prognosis and the six downregulated miRNAs: miR-152, miR-181c, miR-518b, miR-125a, miR-142-5p, and miR-204. For the gemcitabine group, the high miR-142-5p and miR-204 patients had a signiﬁcantly longer survival time than the low miR-142-5p and miR-204 patients (Figs. 1 and 2, P = 0.0077 for miR-142-5p; P = 0.0054 for miR-204). The median survival time was 45 months and 33 months, respectively, for the high miR-142-5p and high miR-204 patients, and 16.3 months in both the low miR-142-5p and low miR-204 patients (Table 2). We also found that the high miR-125a patients had signiﬁcantly longer survival times than the low miR-125a patients when the Wilcoxon test was used (P = 0.035), although the difference was not statistically signiﬁcant when the log rank test was used (Table 2, P = 0.085). However, for the nongemcitabine group, there were no differences in the survival times between the high and low miR-142-5p, miR-204, or miR- 125a patients (Table 2). Analysis of both the gemcitabine and nongemcitabine groups did not revealed any signiﬁcant differences in survival time between the high and low miR- 152, miR-181c, and miR-518b patients (data not shown). Next, we investigated the correlation between prognosis and the six upregulated miRNAs: miR-9, miR-27a, miR- 424, miR-449b, and miR-34a. For the nongemcitabine group, the high miR-34a patients had a signiﬁcantly longer survival times than the low miR-34a patients (Table 2, P = 0.012), while there were no signiﬁcant differences in survival time between the high and low miR-34a patients in the gemcitabine group (Table 2, P = 0.175). The median