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Although thousands of long noncoding RNAs (lncRNAs) have been annotated, only a limited number of them have been functionally characterized. Here, we identified an oncogenic lncRNA, named lnc‐UCID (lncRNA up‐regulating CDK6 by interacting with DHX9). Lnc‐UCID was up‐regulated in hepatocellular carcinoma (HCC), and a higher lnc‐UCID level was correlated with shorter recurrence‐free survival of HCC patients. Both gain‐of‐function and loss‐of function studies revealed that lnc‐UCID enhanced cyclin‐dependent kinase 6 (CDK6) expression and thereby promoted G1/S transition and cell proliferation. Studies from mouse xenograft models revealed that tumors derived from lnc‐UCID‐silenced HCC cells had a much smaller size than those from control cells, and intratumoral injection of lnc‐UCID small interfering RNA suppressed xenograft growth. Mechanistically, the 850‐1030‐nt domain of lnc‐UCID interacted physically with DEAH (Asp‐Glu‐Ala‐His) box helicase 9 (DHX9), an RNA helicase. On the other hand, DHX9 post‐transcriptionally suppressed CDK6 expression by binding to the 3′‐untranslated region (3′UTR) of CDK6 mRNA. Further investigation disclosed that lnc‐UCID enhanced CDK6 expression by competitively binding to DHX9 and sequestering DHX9 from CDK6‐3′UTR. In an attempt to explore the mechanisms responsible for lnc‐UCID up‐regulation in HCC, we found that the lnc‐UCID gene was frequently amplified in HCC. Furthermore, miR‐148a, whose down‐regulation was associated with an increase of lnc‐UCID in HCC, could bind lnc‐UCID and inhibit its expression. Conclusion: Up‐regulation of lnc‐UCID, which may result from amplification of its gene locus and down‐regulation of miR‐148a, can promote HCC growth by preventing the interaction of DHX9 with CDK6 and subsequently enhancing CDK6 expression. These findings provide insights into the biological functions of lncRNAs, the regulatory network of cell cycle control, and the mechanisms of HCC development, which may be exploited for anticancer therapy.

Hepatocellular carcinoma (HCC) is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. There is frequent down-regulation of miR-195 expression in HCC tissues. In this study, the role of miR-195 in HCC angiogenesis and metastasis was investigated with in vitro capillary tube formation and transwell assays, in vivo orthotopic xenograft mouse models, and human HCC specimens. Reduction of miR-195 in HCC tissues was significantly associated with increased angiogenesis, metastasis, and worse recurrence-free survival. Both gain-of-function and loss-of-function studies of in vitro models revealed that miR-195 not only suppressed the ability of HCC cells to promote the migration and capillary tube formation of endothelial cells but also directly repressed the abilities of HCC cells to migrate and invade extracellular matrix gel. Based on mouse models, we found that the induced expression of miR-195 dramatically reduced microvessel densities in xenograft tumors and repressed both intrahepatic and pulmonary metastasis. Subsequent investigations disclosed that miR-195 directly inhibited the expression of the proangiogenic factor vascular endothelial growth factor (VEGF) and the prometastatic factors VAV2 and CDC42. Knockdown of these target molecules of miR-195 phenocopied the effects of miR-195 restoration, whereas overexpression of these targets antagonized the function of miR-195. Furthermore, we revealed that miR-195 down-regulation resulted in enhanced VEGF levels in the tumor microenvironment, which subsequently activated VEGF receptor 2 signaling in endothelial cells and thereby promoted angiogenesis. Additionally, miR-195 down-regulation led to increases in VAV2 and CDC42 expression, which stimulated VAV2/Rac1/CDC42 signaling and lamellipodia formation and thereby facilitated the metastasis of HCC cells. Conclusion: miR-195 deregulation contributes to angiogenesis and metastasis in HCC. The restoration of miR-195 expression may be a promising strategy for HCC therapy. (Hepatology 2013;58:642-653)

Based on microarray data, we have previously shown a significant down-regulation of miR-29 in hepatocellular carcinoma (HCC) tissues. To date, the role of miR-29 deregulation in hepatocarcinogenesis and the signaling pathways by which miR-29 exerts its function and modulates the malignant phenotypes of HCC cells remain largely unknown. In this study, we confirmed that reduced expression of miR-29 was a frequent event in HCC tissues using both Northern blot and real-time quantitative reverse-transcription polymerase chain reaction. More interestingly, we found that miR-29 down-regulation was significantly associated with worse disease-free survival of HCC patients. Both gain- and loss-of-function studies revealed that miR-29 could sensitize HCC cells to apoptosis that was triggered by either serum starvation and hypoxia or chemotherapeutic drugs, which mimicked the tumor growth environment in vivo and the clinical treatment. Moreover, introduction of miR-29 dramatically repressed the ability of HCC cells to form tumor in nude mice. Subsequent investigation characterized two antiapoptotic molecules, Bcl-2 and Mcl-1, as direct targets of miR-29. Furthermore, silencing of Bcl-2 and Mcl-1 phenocopied the proapoptotic effect of miR-29, whereas overexpression of these proteins attenuated the effect of miR-29. In addition, enhanced expression of miR-29 resulted in the loss of mitochondrial potential and the release of cytochrome c to cytoplasm, suggesting that miR-29 may promote apoptosis through a mitochondrial pathway that involves Mcl-1 and Bcl-2. Conclusion: Our data highlight an important role of miR-29 in the regulation of apoptosis and in the molecular etiology of HCC, and implicate the potential application of miR-29 in prognosis prediction and in cancer therapy. (HEPATOLOGY 2010.)