Study On Molecule Mechanism Of MiR-34a Inhibiting Invasion And Metastasis In Hepatocellular Carcinoma Cells And Live Attenuated Influenza A Virus As Vector To Elicit Broadly Neutralizing Antibody Of HIV-1

Hepatocellular Carcinoma (HCC) and Acquired Immune Deficiency Syndrome (AIDS) are two seriously disease of threaten health of humanity. Our investigation introduced functions of microRNA (miRNA) in HCC and revealed the effect of miR-34a on cell migration and invasion as well as the function of regulating gene expression in normal hepatocyte and HCC. At the same time, in efforts to develop AIDS vaccine components, we also introduced the molecule mechanism that live attenuated influenza A virus as vector elicited broadly neutralizing antibody of HIV-1.1. miR-34a inhibits invasion and metastasis of HCC cellsHepatocellular carcinoma is a highly malignant tumor with very poor prognosis and distant invasion and migration. Although invasion and migration is the overwhelming cause of mortality in patients with solid tumors, our understanding of its molecular determinant is limited. Studies on tumor invasion and metastasis have revealed a critical role of miRNA in these processes, since miRNA can post-transcriptionally regulate a variety of genes pivotal for invasion or metastasis.miRNA are a class of 17–25 nucleotides small non-coding RNA. In mammals, mature miRNA are integrated into an RNA-inducing silencing complex (RISC) and associate with 3′-untranslated regions (3′UTR) of specific target messenger RNA (mRNA) to suppress translation and occasionally also induce their degradation. miRNA play important regulatory roles in processes such as cell differentiation and proliferation. They can function as tumor suppressors or oncogenes, depending on whether they specifically target oncogenes or tumor suppressor genes. In this regard, oncogenic miRNA are usually overexpressed in tumors. For instance, miR-21, miR-10b, miR-373, and miR-520c are overexpressed in tumors or tumor cell lines inducing cell migration and invasion. By contrast, suppressive miRNA, such as miR-126*, miR-335, miR146a, and miR-29c are downregulated leading to tumor growth, carcinogenesis, and invasion.Recently, miR-34a has been demonstrated to be a direct transcriptional target of p53 and it is commonly deleted in various types of cancers. Decreased expression of miR-34a is partly due to the inactivating mutations of p53 in tumors. For example, loss of miR-34a expression was observed in neuroblastoma, which may be due to the relatively common deletion of a region on chromosome 1p36, which encompasses miR-34a. Moreover, the expression of miR-34a was low or undetectable in 11 of 15 pancreatic cancer cell line. Nine of 25 human colon cancers (36%) also showed downregulation of miR-34a compared with counterpart normal tissues. The biological targets of miR-34a have been recently identified. miR-34a induces G1 arrest, apoptosis, and senescence by regulation of critical cell cycle motors or apoptosis inhibitors including CDK4/6, cyclin E2 (CCNE2), cyclin D1 (CCND1), E2F3, Bcl-2, and MYCN. Interestingly, miR-34a also directly represses the expression of the oncogenic tyrosine kinase receptor c-Met. c-Met is well-characterized as the receptor for hepatocyte growth factor (HGF). HGF-stimulated activation of c-Met triggers phosphorylation of its signal transduction intermediate molecules such as ERK1/2, the key factors influencing the tumor invasion and migration. Indeed, c-Met is implicated in cell scattering, migration and invasion. These evidence suggest that miR-34a might also possess a role in invasion and metastasis. In the present study we provided the evidence that miR-34a regulated cell scattering, migration and invasion in HCC tissues and HepG2 cell line, at least in part, by targeting the metastasis-related gene c-Met. Furthermore, expression levels of miR-34a and c-Met in human HCC tissues were also determined and their possible role in human HCC development was discussed.In the present study, we identified the role of miR-34a in the regulation of tumor cell scattering, migration, and invasion. Down-regulation of miR-34a expression was highly significant in 19 of 25 (76%) human hepatocellular carcinoma (HCC) tissues compared with adjacent normal tissues and associated with the metastasis and invasion of tumors. Furthermore, resected normal/tumor tissues of 25 HCC patients demonstrated an inverse correlation between miR-34a and c-Met-protein. In HepG2 cells, ectopic expression of miR-34a potently inhibited tumor cell migration and invasion in a c-Met-dependent manner. miR-34a directly targeted c-Met and reduced both mRNA and protein levels of c-Met; thus, decreased c-Met-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2).In conclusion, our present study demonstrates previously uncharacterized biological functions of miR-34a, with the ability to inhibits cell scattering, migration and invasion in HepG2 cell line. This together with our correlational data in downregulation of miR-34a expression in human HCC tissues, implicates that introduction strategies for miR-34a, strategies interfering with the HGF/c-Met interaction, or inhibiting c-Met expression, will have a strong rationale for therapeutic applications in HCC in the future.2.Live attenuated chimeric influenza A virus as vector elicits broadly neutralizing antibody of HIV-1Human influenza viruses are a major concern as a potential global pandemic. So choice of a cell line to propagate influenza virus is an essential tool for the production of influenza vaccines. Vero cells have been widely used for human vaccine production over the past 30 years and are the only continuous cell line fully accepted by regulatory authorities for production of whole virus vaccine. Influenza A and B viruses bind to sialyloligosaccharides on host cell surface glycolipids or glycoproteins via the hemagglutinin (HA) protein. Human influenza viruses preferentially bind to sialyloligosaccharides containing terminal N-acetyl sialic acid linked to galactose by anα2,6-linkage (NeuAcα2,6Gal), while avian influenza viruses mainly bind to those containing anα2,3-linkage (NeuAcα2,3Gal). Although NeuAcα2,6Gal is present in Vero cells, the amount of NeuAcα2,6Gal may not be as high as that of epithelial cells in human airway, leading to suboptimal growth in these cells.In this study, we stably transfected Vero cells with cDNA of human 2,6-sialyltransferase (SIAT1), whose product catalyzesα2,6-sialylation of galactose on glycoproteins. Vero cells were transfected with a plasmid containing the SIAT1 and neomycin resistance gene and selected with antibiotic G418 sulfate. After transfection, overexpression of the SIAT1 in the transfected Vero cells (Vero-SIAT1 cells) was confirmed by Western blot and Immunofluorescence microscopy. Vero-SIAT1 cells expressed 7 times higher amounts of NeuAcα2,6Gal but 3 times lower amounts of NeuAcα2,3Gal as compared to parental Vero cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. Furthermore, the influenza viruses A (H1N1 and H3N2) and B grew in Vero-SIAT1 cells to the higher titers than in Vero cells. Taken together, these results imply that Vero-SIAT1 cells are useful not only for the propagation of human influenza viruses, but also for the increasing output of live attenuated chimeric influenza A virus as vector to elicit broadly neutralizing antibody of HIV-1The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. Here we try to use live attenuated influenza as vector that bear this region to elicit antibody in animal. Live attenuated influenza has some advantages: firstly it can be immunized by intranasal and produce mucosal immunity; secondly influenza virus has more HA protein on the surface and has more chance to contact with BCR; thirdly, like HIV-1, influenza virus can change confirmation when it entry into cell.In efforts to enhance the exposure of these epitopes and develop AIDS vaccine components, we generated combinatorial libraries in which HA-MPER (IDGVKLESMGVYQ) corresponding region was replaced by HIV-1 MPER (NEQELLELDKWASLWNWFNITNWLWYIK),2F5 (ELDKWA) or 4E10 (NWFNIT) epitope. In the present study, we constructed 17 chimeric influenza viruses vector that can assembly into particle and replicate in HEK293T+Vero cell. The results showed that two of 17 live attenuated chimeric influenza A viruses can highly efficient assemblage, named as Flu-HIV-MPER (including 2F5 and 4E10 epitopes) and Flu-HIV-4E10-3 (including 4E10 epitope). RT-PCR and Western Blot show that this two recombinant viruses are attenuated and can be recognized by 2F5 and 4E10 antibody.Guinea pigs immunized with different chimeras, or some primed with ELDKWA/NWFNIT based peptides and boosted with different chimeras. The results showed that guinea pigs immunized with different chimeras (Flu-HIV-MPER or Flu-HIV-4E10-3) produced strong anti-2F5/4E10 epitopes antibody titers, as shown by binding to a synthetic peptide containing the ELDKWA/NWFNIT sequence in an ELISA. Sera showed 50% neutralization of strains SF162(B),SC19-15(BC),11018(BC) and YN78-8(BC) at titers ranging from 107～132. In addition, guinea pigs primed with ELDKWA/NWFNIT based peptides, boosted with different chimeras, and Western blot showed that these sera can be recognized by 2F5 and 4E10 antibody. These guinea pigs produced strong anti-2F5/4E10 epitopes antibody titers, as shown by binding to a synthetic peptide containing the ELDKWA/NWFNIT sequence in an ELISA. Sera showed 50% neutralization of strains SF162(B),SC19-15(BC),11018(BC) and YN78-8(BC) at titers ranging from 90～350. In a word, these live attenuated chimeric influenza A viruses immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 pseudoviruses, and are among the few reported that elicit broad neutralization directed against any recombinant HIV-1 epitope, providing a critical advance in developing effective AIDS vaccine components.