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Hong-hua Peng,1 Yang-de Zhang,2 Lian-sheng Gong,2 Wei-dong Liu,2 Yang Zhang2 1Department of Oncology, Third Xiangya Hospital, Central South University, 2National Hepatobiliary and Enteric Surgery Research Center, Xiangya Hospital, Central South University, Changsha, People's Republic of China Background: MicroRNAs (miRNAs) display aberrant expression patterns and functional abnormalities in many types of cancer. However, their roles in primary gallbladder carcinoma (PGC) have not been well documented. miR-335 has been demonstrated to be involved in tumorigenesis of several cancers in the digestive system. The aim of this study was to investigate the clinical significance of miR-335 in PGC. Methods: miR-335 expression in 166 human PGC tissues and matched adjacent nondysplastic gallbladder epithelia was measured by real-time quantitative polymerase chain reaction (RT-PCR) assay. Results: The expression level of miR-335 was significantly lower in PGC tissues than that in nondysplastic gallbladder epithelia (P<0.001). Of 166 PGC patients, 96 (57.83%) had reduced expression of miR-335. Additionally, the expression of miR-335 was significantly lower in PGC tissues with high histologic grade (P=0.02), advanced pathologic T stage (P=0.009) and clinical stage (P=0.008), and with positive lymph node metastasis (P=0.001). In univariate analysis by log-rank test, histologic grade (P=0.03), pathologic T stage (P=0.008), clinical stage (P=0.01), lymph node metastasis (P<0.001), and miR-335 expression (P<0.001) were significant prognostic factors for overall survival of PGC patients. Multivariate analysis further revealed that pathologic T stage (P=0.02), lymph node metastasis (P=0.008), and miR-335 expression (P=0.006) maintained independent prognostic influence on overall survival. Conclusion: This study offers convincing evidence for the first time that miR-335 was downregulated in a majority of PGC patients and may be associated with the aggressive