Bai Ruan

Downregulation of miR-200a Induces EMT Phenotypes and CSC-like Signatures through Targeting the β-catenin Pathway in Hepatic Oval Cells

Hepatocellular carcinoma (HCC) can be derived from malignant transformed adult hepatic progenitor cells. However, the regulatory factors and molecular mechanisms underlying the process are not well defined. Our previous microRNA (miRNA) microarray analysis revealed a significant decrease of miR-200a level in F344 rat HCC side population (SP) fraction cells versus their normal counterparts. In the present study, we further investigated the effect of miR-200a on hepatic oval cell (HOC) phenotypes. We first confirmed downregulated miR-200a levels in rat hepatoma cells compared with WB-F344 cells. Next, by lentivirus-mediated loss-of-function studies, we showed that stable knockdown of miR-200a confers a mesenchymal phenotype to WB-F344 cells, including an elongated cell morphology, enhanced cell migration ability and expression of epithelial mesenchymal transition (EMT)-representative markers. Concomitantly, several cancer stem cell (CSC)-like traits appeared in these cells, which exhibit enhanced spheroid-forming capacity, express putative hepatic CSC markers and display superior resistance to chemotherapeutic drugs in vitro. Furthermore, bioinformatics analysis, luciferase assays and western blot analysis identified β-catenin (CTNNB1) as a direct and functional target of miR-200a. Knockdown of miR-200a partially activated Wnt/β-catenin signaling, and silencing of β-catenin functionally attenuated anti-miR-200a effects in vitro in WB-F344 cells. At length, in vivo xenograft assay demonstrated the acquisition of tumorigenicity of WB-F344 cells after miR-200a siliencing. Collectively, our findings indicate that miR-200a may function as an important regulatory factor in neoplastic transition of HOCs by targeting the β-catenin pathway.

Downregulation of lncRNA-ATB correlates with clinical progression and unfavorable prognosis in pancreatic cancer.

Long noncoding RNAs (lncRNAs) have been shown to play critical roles in the development and progression of diseases. lncRNA activated by transforming growth factor beta (TGF-β) (lncRNA-ATB) was discovered as a prognostic factor in hepatocellular carcinoma, gastric cancer, and colorectal cancer. However, little is known about the role of lncRNA-ATB in pancreatic cancer. This study aimed to assess lncRNA-ATB expression in pancreatic cancer and explore its role in pancreatic cancer pathogenesis. Quantitative real-time polymerase chain reaction was performed to detect lncRNA-ATB expression in 150 pancreatic cancer tissues and five pancreatic cancer cell lines compared to paired adjacent normal tissues and normal human pancreatic ductal epithelial cell line HPDE6c-7. The correlations between lncRNA-ATB expression and clinicopathological characteristics and prognosis were also analyzed. We found that lncRNA-ATB expression was decreased in pancreatic cancer tissues and pancreatic cancer cell lines. Low lncRNA-ATB expression levels were significantly correlated with lymph node metastases (yes vs. no, P = 0.009), neural invasion (positive vs. negative, P = 0.049), and clinical stage (early stage vs. advanced stage, P = 0.014). Moreover, patients with low lncRNA-ATB expression levels exhibited markedly worse overall survival prognoses (P < 0.001). Multivariate analysis indicated that decreased lncRNA-ATB expression was an independent predictor of poor prognosis in pancreatic cancer patients (P = 0.005). In conclusion, lncRNA-ATB may play a critical role in pancreatic cancer progression and prognosis and may serve as a potential prognostic biomarker in pancreatic cancer patients.

MicroRNA-150 suppresses cell proliferation and metastasis in hepatocellular carcinoma by inhibiting the GAB1-ERK axis

MicroRNA-150 (miR-150) is frequently dysregulated in cancer and is involved in carcinogenesis and cancer progression. In this study, we found that miR-150 was significantly downregulated in hepatocellular carcinoma (HCC) tissues compared to adjacent noncancerous tissues. Low levels of miR-150 were significantly associated with worse clinicopathological characteristics and a poor prognosis for patients with HCC. miR-150 overexpression inhibited cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo. Further experiments indicated that Grb2-associated binding protein 1 (GAB1) was a direct target of miR-150 in HCC cells. In addition, GAB1 expression was increased in HCC tissues and inversely correlated with miR-150 levels. Knockdown of GAB1 mimicked the tumor-suppressive effects of miR-150 overexpression on HCC cells, whereas restoration of GAB1 expression partially abolished the inhibitory effects. Moreover, miR-150 overexpression decreased GAB1 expression, subsequently downregulated phospho-ERK1/2 and suppressed epithelial-mesenchymal-transition (EMT). These effects caused by miR-150 overexpression were alleviated by exogenous GAB1 expression. Taken together, this study demonstrates that miR-150 may be useful as a prognostic marker and that the identified miR-150-GAB1-ERK axis is a potential therapeutic target for HCC.

RACK1 modulates apoptosis induced by sorafenib in HCC cells by interfering with the IRE1/XBP1 axis

Sorafenib is one of the preferred drugs for the treatment of advanced primary hepatocellular carcinoma (HCC). However, its side-effects and acquired resistance limit its use. The unfolded protein response (UPR) induced by chemotherapeutics has been demonstrated to be required for tumor cells to maintain malignancy and therapy resistance. Activation of the IRE1/XBP1 pathway during the UPR is important for tumor survival under pathophysiological conditions. In the present study, we found that the UPR was activated and RACK1 was overexpressed in three human HCC cell lines and in HCC samples. Activation of the IRE1/XBP1 signaling pathway plays a protective role when HCC cells encounter endoplasmic reticulum (ER) stress due to in vitro sorafenib treatment. We then found that the interaction between IRE1 and RACK1 was essential for the activation of IRE1 signaling in sorafenib-treated cells. Exogenous overexpression of RACK1 enhanced the phosphorylation level of IRE1 and increased XBP1 mRNA splicing activity, which protected the HCC cells from sorafenib-induced apoptosis. However, the re-expression of RACK1 led HCC cells to regain susceptibility to sorafenib-induced apoptosis. Taken together, the present study suggests that the RACK1/IRE1 complex may contribute to activation of the UPR in HCC cells. Targeting RACK1 in combination with sorafenib administration is a potential strategy for clinical trials of advanced HCC treatment.

RRAD inhibits aerobic glycolysis, invasion, and migration and is associated with poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer worldwide. However, the mechanism underlying the HCC development remains unclear. Ras-related associated with diabetes (RRAD) is a small Ras-related GTPase which has been implicated in metabolic disease and several types of cancer, yet its functions in HCC remain unknown. A tissue microarray constructed by 90 paired HCC tissues and adjacent non-cancerous liver tissues was used to examine the protein levels of RRAD, and the messenger RNA (mRNA) expression of RRAD was also detected in a subset of this cohort. The prognostic significance of RRAD was estimated by the Kaplan-Meier analysis and Cox regression. The glucose utilization assay and lactate production assay were performed to measure the role of RRAD in HCC glycolysis. The effect of RRAD in HCC invasion and metastasis was analyzed by transwell assays. Our results suggested that the expression of RRAD was downregulated in HCC tissues compared to the adjacent non-tumorous liver tissues both in mRNA and protein levels and lower RRAD expression served as an independent prognostic indicator for the survival of HCC patients. Moreover, RRAD inhibited hepatoma cell aerobic glycolysis by negatively regulating the expression of glucose transporter 1 (GLUT1) and hexokinase II (HK-II). In addition, RRAD inhibition dramatically increased hepatoma cell invasion and metastasis. In conclusion, our study revealed that RRAD expression was decreased in HCC tumor tissues and predicted poor clinical outcome for HCC patients and played an important role in regulating aerobic glycolysis and cell invasion and metastasis and may represent potential targets for improving the treatment of HCC.

Overexpression of miR-200a suppresses epithelial-mesenchymal transition of liver cancer stem cells

Due to high incidence of invasion and intrahepatic metastasis, hepatocellular carcinoma (HCC) is one of the most aggressive tumors in the world, which is also associated with the acquisition of epithelial-mesenchymal transition (EMT). Increasing evidence suggests that cancer cells with EMT traits share many biological characteristics with cancer stem cells. And miR-200a has been known as a powerful regulator of EMT. Here, we sought to investigate the role of miR-200a in regulation of EMT phenotype of liver cancer stem cells (LCSCs). We used side population (SP) sorting to obtain cancer stem-like cells from HCC cell lines and identified that the SP fraction could be enriched with LCSCs. Then, we detected the expression of miR-200a and EMT makers in SP and non-SP cells. Our results suggested that miR-200a was down-regulated in SP cells, along with relatively low epithelial marker and high mesenchymal marker. In order to find the role of miR-200a in the manipulation of EMT, we transfected miR-200a mimic into LCSCs and found that overexpression of miR-200a resulted in down-regulation of N-cadherin, ZEB2, and vimentin, but up-regulation of E-cadherin. Moreover, overexpression of miR-200a resulted in decreased migration and invasion ability in LCSCs. In conclusion, our study revealed that miR-200a played an important role in linking the characteristics of cancer stem cells with EMT phenotype in HCC, and targeting miR-200a might be an effective strategy to weaken the invasive behavior of LCSCs.