MicroRNA-148a suppresses the epithelial-mesenchymal transition and metastasis of hepatoma cells by targeting Met/Snail signaling.

Metastasis is responsible for the rapid recurrence and poor survival of malignancies. Epithelial-mesenchymal transition (EMT) has a critical role in metastasis. Increasing evidence indicates that EMT can be regulated by microRNAs (miRNAs). miR-148a is a liver-abundant miRNA. However, the role of miR-148a in the development of liver cancer remains largely unknown. In this study, we found that, compared with normal livers, miR-148a was significantly decreased in hepatocellular carcinoma (HCC) tissues, especially in those with the portal vein tumor thrombus. An in vitro transwell assay and an in vivo orthotopic liver xenograft model showed that the restoration of miR-148a expression significantly repressed the migration and pulmonary metastasis of hepatoma cells. Linear regression analysis revealed a positive correlation between the expression of miR-148a and the mRNA level of E-cadherin gene in human HCC tissues. Both gain- and loss-of-function studies disclosed that miR-148a promoted the expression of epithelial marker (E-cadherin) and reduced the levels of mesenchymal markers (N-cadherin, fibronectin or vimentin) in hepatoma cells. These data suggest that miR-148a may suppress EMT and cancer metastasis. Further mechanistic investigations showed that miR-148a directly inhibited Met expression by binding to its 3'-UTR. Moreover, the reintroduction of miR-148a attenuated the downstream signaling of Met, like activated phosphorylation of AKT-Ser473 and inhibitory phosphorylation of GSK-3ß-Ser9, and consequently reduced the nuclear accumulation of Snail, a transcription factor that promotes EMT. Taken together, miR-148a may negatively regulate Met/Snail signaling and therefore inhibit the EMT and metastasis of hepatoma cells. These findings highlight the significance of miR-148a downregulation in tumor progression and implicate miR-148a as an attractive candidate for cancer therapy.

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.

Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen.

The introduction of the Simian virus 40 (SV40) early region, the telomerase catalytic subunit (hTERT) and an oncogenic allele of H-Ras directly transforms primary human cells. SV40 small T antigen (ST), which forms a complex with protein phosphatase 2A (PP2A) and inhibits PP2A activity, is believed to have a critical role in the malignant transformation of human cells. Recent evidence has shown that aberrant microRNA (miRNA) expression patterns are correlated with cancer development. Here, we identified miR-27a as a differentially expressed miRNA in SV40 ST-expressing cells. miR-27a is upregulated in SV40 ST-transformed human bronchial epithelial cells (HBERST). Suppression of miR-27a expression in HBERST cells or lung cancer cell lines (NCI-H226 and SK-MES-1) that exhibited high levels of miR-27a expression lead to cell growth arrested in the G(0)-G(1) phase. In addition, suppression of miR-27a in HBERST cells attenuated the capacity of such cells to grow in an anchorage-independent manner. We also found that suppression of the PP2A B56γ expression resulted in upregulation of miR-27a similar to that achieved by the introduction of ST, indicating that dysregulation of miR-27a expression in ST-expressing cells was mediated by the ST-PP2A interaction. Moreover, we discovered that Fbxw7 gene encoding F-box/WD repeat-containing protein 7 was a potential miR-27a target validated by dual-luciferase reporter system analysis. The inverse correlation between miR-27a expression levels and Fbxw7 protein expression was further confirmed in both cell models and human tumor samples. Fbxw7 regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates, including c-Myc, c-Jun, cyclin E1 and Notch 1. Thus, promotion of cell growth arising from the suppression of Fbxw7 by miR-27a overexpression might be responsible for the viral oncoprotein ST-induced malignant transformation. These observations demonstrate that miR-27a functions as an oncogene in human tumorigenesis.

Identification of Siah-interacting protein as a potential regulator of apoptosis and curcumin resistance.

The mechanism underlying curcumin (diferuloylmethane) resistance is still largely unknown. Here we employed proteomic approach to identify the Siah-interacting protein (SIP) as a candidate for detailed study, because the spot intensity of SIP on a two-dimensional gel displayed 70-90% reduction in curcumin-sensitive cells, but remained unchanged in curcumin-resistant sublines, after curcumin treatment. Both gain- and loss-of-function studies revealed that SIP promoted curcumin-induced apoptosis. Moreover, SIP underwent phosphorylation and nuclear translocation in curcumin-sensitive but not resistant cells, upon curcumin exposure. The nuclear translocation of SIP was remarkably impaired when a putative nuclear localization sequence (NLS, amino acid (aa) 143-159) was deleted or the serine 141 was mutated into alanine, whereas truncation of the N-terminal region (aa 1-43) obviously increased the nuclear import of SIP. In accordance with their nuclear localization, N-terminal truncation significantly enhanced the proapoptotic effect of SIP, whereas NLS deletion or Ser141Ala mutation attenuated the apoptosis-promoting activity of both wild-type- and N-terminal truncated-SIP. These data suggest that SIP plays a role in apoptosis and curcumin resistance, and the function of SIP may be regulated by different motifs, such as the NLS, N-terminal region and serine 141. Our findings provide new insights into the biological significance of SIP and the mechanisms of drug resistance.

Mutation analysis of the pRb pathway in 2',3'-dideoxycytidine- and 1, 3-butadiene-induced mouse lymphomas.

The pRb pathway plays a key role in controlling the G1/S transition in cell cycle progression. Aberrations of various components of the pRb pathway, such as retinoblastoma protein and its upstream actors including cyclin D1, cyclin dependence kinase-4 and p16/p15 cyclin dependent kinase inhibitors, have been reported in a variety of human tumors. Furthermore, the alterations of retinoblastoma protein and its upstream components often occur in a reciprocal manner. Previously, we have reported frequent inactivation of the Cdkn2a/Cdkn2b loci encoding p16/p15 cyclin dependent kinase inhibitors in a subset of 2',3'-dideoxycytidine- and 1, 3-butadiene-induced mouse lymphomas (S.-M. Zhuang, A. Schippert, A. Haugen-Strano, R.W. Wiseman, P. Söderkvist, Inactivation of p16(INK4a)-alpha, p16(INK4a)-beta and p15(INK4b) genes in 2', 3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas, Oncogene 16 (1998) 803-808), indicating the involvement of pRb pathway in lymphomagenesis. To investigate whether alteration of other components in pRb pathway is an alternative mechanism underlying the development of these chemically induced lymphomas, we have examined the genetic status of Rb1, Ccnd1 and Cdk4 genes that encode retinoblastoma protein, cyclin D1 and cyclin dependence kinase-4, respectively. Gross alterations of the Rb1, Ccnd1, and Cdk4 genes were not detected by Southern analysis in any of the tumors examined. In addition, single-strand conformation analysis failed to reveal point mutations in the Cdk4 amino terminal domain that is important for its association with Cdkn2a gene products. These results indicate that the mechanisms underlying the development of 2', 3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas involve inactivation of p16/p15 cyclin-dependent kinase inhibitors but not genomic alterations of the Rb1, Ccnd1 and Cdk4 genes.

Inactivations of p16INK4a-alpha, p16INK4a-beta and p15INK4b genes in 2',3'-dideoxycytidine- and 1,3-butadiene-induced murine lymphomas.

The p16INK4a (alpha and beta form) and p15INK4b genes were analysed for homozygous deletion, hypermethylation and point mutation in B6C3F1 mouse lymphomas induced by 2',3'-dideoxycytidine or 1,3-butadiene. Although the p16INK4a-alpha gene appeared normal in DNA from 2',3'-dideoxycytidine-induced lymphomas, Southern analyses revealed homozygous deletions or rearrangements of the p16INK4a-beta and/or p15INK4b genes in four of 16 tumours. Surprisingly, two of these lymphomas showed exclusive deletions of the p16INK4a EIbeta exon. The p15INK4b promoter region was hypermethylated in two additional 2',3'-dideoxycytidine-induced lymphomas. In contrast, homozygous deletions spanning the p16INK4a and p15INK4b loci were observed in only two of 31 1,3-butadiene-induced tumours. Thus, these cyclin dependent kinase inhibitor genes may play a significant role in chemically induced mouse lymphomas and support the contention of tumour suppressor activity for the p19ARF protein encoded by the p16INK4a-beta gene. Different genetic pathways may be involved in the development of these chemically induced tumours since we have previously shown that mutations in p53 and ras genes are common in 1,3-butadiene- but not 2',3'-dideoxycytidine-induced lymphomas.

Genetic alterations of p53 and ras genes in 1,3-butadiene- and 2',3'-dideoxycytidine-induced lymphomas.

Mutations of p53 and ras genes were analyzed in 40 and 31 1,3-butadiene (BD)-induced lymphomas of B6C3F1 mice (BLFs), respectively, and in 63 2',3'-dideoxycytidine-induced lymphomas, which were collected from B6C3F1 (n = 16) or NIH Swiss mice (DLSs; n = 47). The frequencies of K- and N-ras mutations in BLFs (32 and 13%, respectively) were higher than those in DLSs (13 and 2%, respectively). Seven of 10 K-ras-mutated BLFs contained codon 13 CGC mutations, whereas no mutation in K-ras codon 13 was detected in DLSs, suggesting that the codon 13 CGC mutation is specific for BD exposure. Interestingly, 8 of 13 BLFs with ras mutations were from low-dose (< or = 200 ppm) or stop-exposure (26 weeks) groups. These results suggest that ras mutations play an important role in the development of BD-induced lymphoma and may represent an early event. Analysis of genetic alterations in exons 5-8 of the p53 gene revealed mutations in seven of the BLFs and three of the DLSs. All seven BLFs carrying p53 mutations were collected from the high-dose (625 ppm) continuous exposure group, which might indicate that p53 is involved in the progression of BD-induced lymphoma and in late stage of lymphomagenesis. Mutations in ras and p53 genes are relatively infrequent in 2',3'-dideoxycytidine-induced lymphomas, suggesting that other genes must be involved.

Allelotype analysis of 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice.

To identify potential tumor suppressor genes involved in lymphoma development, we generated allelotypes of 16 2',3'-dideoxycytidine (ddC and 31 1,3-butadiene (BD)-induced lymphomas from C57BL/6 x C3H/He F1 (hereafter called B6C3F1) mice. Two or more anonymous simple sequence length polymorphisms per autosome were examined for loss of heterozygosity (LOH). Allelic losses throughout the genome were generally infrequent, except for markers on chromosome 2, 4, 11 and 12. The highest frequency of allelic losses was observed on chromosome 12, with 38 and 39% in ddC and BD-induced lymphomas, respectively. The most prevalent LOH was localized to the distal region bounded by markers D12Mit263 and D12Nds2. No known tumor suppressor genes have been mapped to this region, and no obvious candidates could be identified, suggesting the presence of novel suppressor gene(s). LOH on chromosome 2 was observed in 31% of ddC-induced lymphomas but in only 3% (1/31) of BD-induced lymphomas, suggesting a ddC-specific genetic effect. Detailed analysis localized a potential tumor suppressor gene residing on the distal region of chromosome 2, between markers D2Mit147 and D2Mit148. Twenty-five % of ddC-induced and 23% of BD-induced lymphomas showed LOH on chromosome 4, and two discrete regions were identified. One of the regions includes the IFN gene cluster and is syntenic to human chromosome 9p2l-22. Candidate tumor suppressor genes, Mts1 (multiple tumor suppressor 1) and Mts2 have been mapped to this region. The second region is located on the distal part of chromosome 4, which is homologous to human chromosome 1p35-36, a region that is frequently deleted in various types of human tumors. Finally, 19% of ddC-induced and 29% of BD-induced lymphomas revealed LOH on chromosome 11 at the Acrb locus, which lies within 1 cM of p53, suggesting that the p53 tumor suppressor gene also plays a role in lymphomagenesis. These results suggest that multiple potential suppressor loci contribute to lymphoma development in B6C3F1 mice.

Differential sensitivity to Ad5 E1B-21kD and Bcl-2 proteins of apoptin-induced versus p53-induced apoptosis.

Apoptin, a small protein derived from chicken anemia virus (CAV), induces apoptosis in human tumor cell lines regardless of whether these express p53 or not. We examined whether the small adenovirus 5 E1B protein of 21 kDa (E1B-21kD), also called E1B-19kD) and Bcl-2 could inhibit apoptin-induced apoptosis in human tumor cell lines and compared this with p53-induced apoptosis. E1B-21kD, but not Bcl-2 was found to inhibit apoptin-induced apoptosis in the osteosarcoma cell lines U2OS and Saos-2. However, neither expression of E1B-21kD nor of Bcl-2 resulted in inhibition of apoptin-induced apoptosis in Hep3B hepatoma cells and kidney rhabdoid tumor G401 cells. Both Bcl-2 and Ad5 E1B-21kD were able to inhibit p53-induced apoptosis in the human tumor cell lines Saos-2 and Hep3B. In Saos-2 and U2OS, but not in Hep3B and G401, expression of E1B-21kD leads to retention of apoptin in the cytoplasm, in that way preventing its nuclear function. These results indicate that proteins inhibiting the p53-induced apoptotic pathway do not block apoptin-induced apoptosis or do so only in a cell type-specific manner. The apoptin-induced apoptotic pathway is distinct from that induced by p53 and, therefore, apoptin is a potential antitumor agent.

Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro.

Apoptin, a small protein encoded by chicken anemia virus (CAV) was expressed in various human hematologic malignant cell lines derived from leukemias and lymphoma. Three of these cell lines contain bcl-2 or BCR-ABL proteins, known to block apoptosis induced by chemotherapeutic compounds. By immunofluorescence and propidium-iodide staining apoptin was shown to induce apoptosis in all analysed cell lines. Early after expression, apoptin exhibited a fine-granular distribution in the still intact nucleus. Later, apoptin became aggregated and the nucleus segmented. The data with truncated apoptin indicate that for optimal induction of apoptosis apoptin has to be located in the nucleus.

Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells.

Previously, we have shown that the chicken anemia virus-derived VP3 ("apoptin") protein induces apoptosis in chicken mononuclear cells. Here, we report that apoptin also induces apoptosis in human osteosarcoma cells, regardless of whether they expressed wild-type, mutant p53, or no p53 at all. Moreover, the nuclear location of apoptin appears to be important for its optimal induction of apoptosis. The fact that apoptin can induce p53-independent apoptosis in human tumor cells makes apoptin a potential candidate for treatment of frequently occurring types of cancer cells that do not contain functional p53.

An epidemic of encephalitis and meningoencephalitis in children caused by echovirus type 30 in Shanghai.

An outbreak of viral central nervous system infection occurred from May to September, 1991 in Shanghai, China. Totally 376 cases were admitted into our hospital, 54% with encephalitis and 46% with meningoencephalitis. Echovirus type 30 was isolated from CSF and stools of the patients. Only 3 of the patients were under 1 year of age. CSF examination revealed the cell count from 0-1600 x 10(6)/L. Interestingly, 9 had CSF PMNs over 90% and 13 had low sugar levels. EKG was randomly done in 20 patients and 15 demonstrated myocarditis. The outcome was uneventful in all except 1 who had psychological problem at the time of discharge.