Shi-Mei Zhuang

Identification of miRNomes in Human Liver and Hepatocellular Carcinoma Reveals miR-199a/b-3p as Therapeutic Target for Hepatocellular Carcinoma

The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for ∼88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumor-promoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.

Effects of MicroRNA‐29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma

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.)

A functional polymorphism in the miR-146a gene is associated with the risk for hepatocellular carcinoma

A G > C polymorphism (rs2910164) is located in the stem region opposite to the mature miR-146a sequence, which results in a change from G:U pair to C:U mismatch in the stem structure of miR-146a precursor. Here, we elucidated the biological significance of this polymorphism, based on cancer association study and cell model system. The cancer association study included 479 hepatocellular carcinoma (HCC) and 504 control subjects. We found that the genotype distribution of this polymorphism in HCC cases was significantly different from that in control subjects (P = 0.026). The association between the genotype and the risk of HCC was further analyzed using multivariate unconditional logistic regression, with adjustment for sex, age and hepatitis B virus status. The results revealed that male individuals with GG genotype were 2-fold more susceptible to HCC (odds ratio = 2.016, 95% confidence interval = 1.056-3.848, P = 0.034) compared with those with CC genotype. We next examined the influence of this polymorphism on the production of mature miR-146a and found that G-allelic miR-146a precursor displayed increased production of mature miR-146a compared with C-allelic one. Further investigations disclosed that miR-146a could obviously promote cell proliferation and colony formation in NIH/3T3, an immortalized but non-transformed cell line. These data suggest that the G > C polymorphism in miR-146a precursor may result in important phenotypic traits that have biomedical implications. Our findings warrant further investigations on the relation between microRNA polymorphism and human diseases.

MicroRNA‐195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells

Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down‐regulation of miR‐195 has been observed in various types of cancers. However, the biological function of miR‐195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR‐195. Our results showed that miR‐195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR‐195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR‐195 blocked G1/S transition, whereas inhibition of miR‐195 promoted cell cycle progression. Subsequent investigation characterized multiple G1/S transition‐related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR‐195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR‐195, whereas overexpression of these proteins attenuated miR‐195‐induced G1 arrest. In addition, miR‐195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR‐195 may block the G1/S transition by repressing Rb‐E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR‐195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR‐195 in cancer therapy. (HEPATOLOGY 2009.)

MicroRNA‐29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. We previously found that microRNA‐29b (miR‐29b) down‐regulation was significantly associated with poor recurrence‐free survival of HCC patients. Therefore, the role of miR‐29b in tumor angiogenesis, invasion, and metastasis was further investigated in this study using in vitro capillary tube formation and transwell assays, in vivo subcutaneous and orthotopic xenograft mouse models, and Matrigel plug assay, and human HCC samples. Both gain‐ and loss‐of‐function studies showed that miR‐29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro. Using mouse models, we revealed that tumors derived from miR‐29b‐expressed HCC cells displayed significant reduction in microvessel density and in intrahepatic metastatic capacity compared with those from the control group. Subsequent investigations revealed that matrix metalloproteinase‐2 (MMP‐2) was a direct target of miR‐29b. The blocking of MMP‐2 by neutralizing antibody or RNA interference phenocopied the antiangiogenesis and antiinvasion effects of miR‐29b, whereas introduction of MMP‐2 antagonized the function of miR‐29b. We further disclosed that miR‐29b exerted its antiangiogenesis function, at least partly, by suppressing MMP‐2 expression in tumor cells and, in turn, impairing vascular endothelial growth factor receptor 2‐signaling in endothelial cells. Consistently, in human HCC tissues and mouse xenograft tumors miR‐29b level was inversely correlated with MMP‐2 expression, as well as tumor angiogenesis, venous invasion, and metastasis. Conclusion: miR‐29b deregulation contributes to angiogenesis, invasion, and metastasis of HCC. Restoration of miR‐29b represents a promising new strategy in anti‐HCC therapy. (HEPATOLOGY 2011;)

MicroRNA-26a/b and their host genes cooperate to inhibit the G1/S transition by activating the pRb protein

The functional association between intronic miRNAs and their host genes is still largely unknown. We found that three gene loci, which produced miR-26a and miR-26b, were embedded within introns of genes coding for the proteins of carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase (CTDSP) family, including CTDSPL, CTDSP2 and CTDSP1. We conducted serum starvation-stimulation assays in primary fibroblasts and two-thirds partial-hepatectomies in mice, which revealed that miR-26a/b and CTDSP1/2/L were expressed concomitantly during the cell cycle process. Specifically, they were increased in quiescent cells and decreased during cell proliferation. Furthermore, both miR-26 and CTDSP family members were frequently downregulated in hepatocellular carcinoma (HCC) tissues. Gain- and loss-of-function studies showed that miR-26a/b and CTDSP1/2/L synergistically decreased the phosphorylated form of pRb (ppRb), and blocked G1/S-phase progression. Further investigation disclosed that miR-26a/b directly suppressed the expression of CDK6 and cyclin E1, which resulted in reduced phosphorylation of pRb. Moreover, c-Myc, which is often upregulated in cancer cells, diminished the expression of both miR-26 and CTDSP family members, enhanced the ppRb level and promoted the G1/S-phase transition. Our findings highlight the functional association of miR-26a/b and their host genes and provide new insight into the regulatory network of the G1/S-phase transition.

MicroRNA-125b promotes apoptosis by regulating the expression of Mcl-1, Bcl-w and IL-6R

The microRNA miR-125b is multi-faceted, with the ability to function as a tumor suppressor or an oncogene, depending on the cellular context. To date, the pro-apoptotic role of miR-125b and its underlying mechanisms are unexplored. In this study, both gain- and loss-of-function experiments revealed that miR-125b expression not only induced spontaneous apoptosis in various cell lines derived from the liver, lung and colorectal cancers, but also sensitized cancer cells to diverse apoptotic stimuli, including nutrient starvation and chemotherapeutic treatment. Furthermore, downregulation of miR-125b was a frequent event in hepatocellular carcinoma (HCC) tissues, and the miR-125b level was positively associated with the rate of apoptosis in HCC tissues. Subsequent investigations identified Mcl-1, Bcl-w and interleukin (IL)-6R as direct targets of miR-125b. Restoration of miR-125b expression not only diminished the expression of Mcl-1 and Bcl-w directly but also indirectly reduced the Mcl-1 and Bcl-xL levels by attenuating IL-6/signal transducer and activator of transcription 3 signaling. Consistent with these findings, introduction of miR-125b reduced the mitochondrial membrane potential and promoted the cleavage of pro-caspase-3. These data indicate that miR-125b may promote apoptosis by suppressing the anti-apoptotic molecules of the Bcl-2 family and miR-125b downregulation may facilitate tumor development by conferring upon cells the capability to survive under conditions of nutrient deprivation and chemotherapeutic treatment. Our findings highlight the importance of miR-125b in the regulation of apoptosis and suggest miR-125b as an attractive target for anti-cancer therapy.

MicroRNA‐195 Suppresses Angiogenesis and Metastasis of Hepatocellular Carcinoma by Inhibiting the Expression of VEGF, VAV2, and CDC42

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)

Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data

We present the analysis of the evolution of tumors in a case of hepatocellular carcinoma. This case is particularly informative about cancer growth dynamics and the underlying driving mutations. We sampled nine different sections from three tumors and seven more sections from the adjacent nontumor tissues. Selected sections were subjected to exon as well as whole-genome sequencing. Putative somatic mutations were then individually validated across all 9 tumor and 7 nontumor sections. Among the mutations validated, 24 were amino acid changes; in addition, 22 large indels/copy number variants (>1 Mb) were detected. These somatic mutations define four evolutionary lineages among tumor cells. Separate evolution and expansion of these lineages were recent and rapid, each apparently having only one lineage-specific protein-coding mutation. Hence, by using a cell-population genetic definition, this approach identified three coding changes (CCNG1, P62, and an indel/fusion gene) as tumor driver mutations. These three mutations, affecting cell cycle control and apoptosis, are functionally distinct from mutations that accumulated earlier, many of which are involved in inflammation/immunity or cell anchoring. These distinct functions of mutations at different stages may reflect the genetic interactions underlying tumor growth.

A novel GSK-3 beta–C/EBP alpha–miR-122–insulin-like growth factor 1 receptor regulatory circuitry in human hepatocellular carcinoma†

miR‐122 is a highly abundant, hepatocyte‐specific microRNA. The biomedical significance and regulatory mechanisms of miR‐122 remain obscure. We explored the role of miR‐122 in tumorigenesis in the context of gene regulatory network. The miR‐122 promoter and its transactivator were identified by way of luciferase reporter system, electrophoretic mobility shift, and chromatin immunoprecipitation assays. The miR‐122 regulatory circuitry and its implication in hepatocarcinogenesis were identified using livers of different development stages, human hepatocellular carcinoma (HCC) tissues and cell lines, and aflatoxin B1 (AFB1)‐transformed cells. We characterized the −5.3 to −4.8 kb region upstream of miR‐122 precursor as miR‐122 promoter. Further investigation revealed that deletion of predicted CCAAT/enhancer‐binding protein alpha (C/EBPα) binding sites C/EBPα knockdown significantly reduced miR‐122 promoter activity and endogenous miR‐122 expression; and C/EBPα directly interacted with the miR‐122 promoter in vitro and in vivo. These data suggest that C/EBPα is a transactivator for miR‐122 transcription. We further demonstrated that miR‐122 suppressed insulin‐like growth factor 1 receptor (IGF‐1R) translation and sustained glycogen synthase kinase‐3 beta (GSK‐3β) activity. The activated GSK‐3β not only repressed cell proliferation, but also activated C/EBPα, which maintained miR‐122 levels and thereby enforced IGF‐1R suppression. Interestingly, down‐regulation of miR‐122 and C/EBPα, and up‐regulation of IGF‐1R were frequently observed in HCC tissues, and decreased miR‐122 levels were associated with worse survival of HCC patients. Moreover, AFB1 exposure resulted in decreased activity in GSK‐3β, C/EBPα, and miR‐122 and increased levels of IGF‐1R, whereas restoration of miR‐122 suppressed the tumorigenicity of HCC and AFB1‐transformed cells. Conclusion: We have identified a novel GSK‐3β–C/EBPα–miR‐122–IGF‐1R regulatory circuitry whose dysfunction may contribute to the development of HCC. Our findings provide new insight into miR‐122s function and the mechanisms of hepatocarcinogenesis. (Hepatology 2010;52:1702‐1712)