The precise functions and mechanisms of microRNAs (miR) in gallbladder cancer (GBC) remain elusive. In this study, we found that miR-135a-5p expression is often dampened and correlated with neoplasm histologic grade in GBC. MicroRNA-135a-5p introduction clearly inhibited GBC cell proliferation in vitro and in vivo. Moreover, very low density lipoprotein receptor (VLDLR), which is often upregulated in GBC tissues, was identified as a direct functional target of miR-135a-5p. Furthermore, the p38 MAPK pathway was proven to be involved in miR-135a-VLDLR downstream signaling. Together, these results suggested that the miR-135a–VLDLR–p38 axis may contribute to GBC cell proliferation.

Gallbladder cancer (GBC) is an aggressive disease in which epithelial-mesenchymal transition (EMT) plays a critical role. Whether inhibition of mTOR effects via EMT reversal in GBC remains unclear. Using genetic and pharmacologic inhibitions of mTOR, we investigated the changes of EMT levels in GBC cells. Expressions of EMT related genes were also studied. Migration and invasion assays were carried out and in vivo tumour metastasis mouse models were established. Circulating tumour DNA was quantified. We used EMT index (ratio of Vimentin/Ecadherin expression) to profile EMT levels. We found that inhibition of mTOR using shRNAs and rapamycin inhibited EMT in GBC-SD gallbladder cancer cells. Inhibition of mTOR inhibited EMT in GBC-SD cells in TGF-β-dependent manner, which was contributed majorly by mTORC2 inhibition. Rapamycin decreased invasiveness and migration of GBC-SD cells in vitro and in vivo. We have in the current study shown that rapamycin diminishes the ability of invasion and migration of GBC via inhibition of TGF-β-dependent EMT. Our findings contribute to the understanding of the carcinogenesis of GBC.

Abstract. MicroRNAs (miRNAs) are a class of small, single-stranded, non-coding RNA molecules which can act as oncogenes or tumor suppressor genes in human cancer. However, the possible functions and mechanisms of miRNA action in gallbladder cancer (GBC) have not been elucidated. In the present study, it was found that miR-26a was often downregulated in GBC and the expression of miR-26a was associated with neoplasm histological grade. miR-26a significantly inhibited the proliferation of GBC cells based on the gain-of-function assays. Furthermore, we demonstrated that high mobility group AT-hook 2 (HMGA2) was a direct target of miR-26a. The results showed that HMGA2 mRNA levels and miR-26a levels were negatively correlated. In addition, we confirmed that reintroduction of HMGA2 antagonized the inhibition of miR-26a to GBC cell proliferation and all these effects were achieved through the cell cycle. Together, all these results suggest that miR-26a expression contributes to GBC proliferation by targeting HMGA2. miR-26a shows promise as a prognosis factor and therapeutic target of GBC patients. IntroductionMicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNAs which act as post-trancriptional regulators via binding to the 3' untranslated regions (3'-UTRs) of the target gene mRNA, and have vital functions in many of physiological and pathological processes, as well as in carcinogenesis (1-5). It has been reported that miRNAs can act as oncogene or tumor suppressor gene in various cancers, including lung, colorectal, and liver cancer (6,7).Gallbladder cancer (GBC) with poor prognosis is one of the most prevalent and aggressive malignant types of cancer in China. GBC is the tenth most common cancer in Shanghai and the average survival of patients is ~9 months (8). Many patients have no chance to accept radical operation when they are diagnosed. Despite its poor prognosis and high morbidity, the miRNA function in GBC remains largely unknown.The miRNA microarray assays performed on 4 pairs of GBC and paracancerous tissues, revealed that miR-26a is significantly downregulated in GBC tissues. It has been reported that miR-26a was downregulated in pancreatic, nasopharyngeal, breast and lung cancer, and miR-26a can acts as tumor suppressor gene via directly targeting enhancer of Zeste homolog 2 (EZH2) (9-12). Also in hepatocellular carcinoma cells, miR-26a can inhibit cell proliferation by regulating cyclin D2,14). It has been demonstrated that expression levels of miR-26a are lower in the tumors of renal cell carcinoma patients who developed tumor relapse, moreover, the lowest levels of miR-26a are observed in primary metastatic tumors (15). These findings suggested that miR-26a might play a vital role in GBC physiological and pathological processes.In the present study, we demonstrated that miR-26a is significantly downregulated in GBC tissues compared with paracancerous tissues and miR-26a is closely correlated with the histologic grade of the neoplasm. Enforced, highly expressed miR-26a was able to inhibit G...