Down-regulation of the miRNA miR-338-3p correlates with the invasive ability of hepatocellular carcinoma (HCC) cells. However, it is currently unclear whether down-regulation of miR-338-3p induces epithelial-mesenchymal transition (EMT), which may be the underlying mechanism governing HCC invasion. Here, we demonstrate that restoration of miR-338-3p expression via transfection of a miR-338-3p mimic reversed EMT and inhibited the motility and invasiveness of HCC cells. Conversely, silencing of endogenous miR-338-3p expression with a miR-338-3p-specific inhibitor induced EMT and enhanced HCC cell motility. Additionally, Snail1 (an upstream regulatory protein of EMT) and Gli1 (a key transcription factor in the sonic hedgehog (SHH) signaling pathway) expression was up-regulated in cells treated with the miR-338-3p inhibitor and down-regulated by the miR-338-3p mimic. Further analyses demonstrated that miR-338-3p inhibitor-induced EMT in HCC cells was blocked by treatment with a small interfering RNA (siRNA) targeting Snail1, that the SHH signaling pathway was required for both miR-338-3p inhibitor-induced EMT and up-regulation of Snail1, and that miR-338-3p targeted a sequence within the 3′-untranslated region of N-cadherin mRNA. Notably, miR-338-3p expression was significantly down-regulated in HCC samples from patients with metastases and was associated with poor metastasis-free survival rates. Lastly, correlations between the expression levels of miR-338-3p and E-cadherin, Smoothened (SMO), Gli1, Snail1, N-cadherin, and vimentin were confirmed in HCC xenograft tumors and HCC patient specimens. Our findings suggest that miR-338-3p suppresses EMT and metastasis via both inhibition of the SHH/Gli1 pathway and direct binding of N-cadherin. miR-338-3p is a potential therapeutic target for metastatic HCC.
Applying reverse vaccinology strategy, we employed a sequence encoding an enolase from Taenia asiatica to search its homolog in the expression sequence tag (EST) database of Echinococcus granulosus and found two EST sequences (Access number: CN653186 and CN649593) of a clone Eg_PSGRS_13B09 from E. granulosus protoscolex full-length cDNA library, which are responding for the 5' and 3' partial cds of E. granulosus enolase, respectively. Primers are designed according to the 5' end and 3'end of the putative encoding sequence to amplify the genomic DNA of E. granulosus strain isolated from sheep in Qinghai province of China by polymerase chain reaction (PCR). A sole product of 1,449 bp in length was obtained, which contains two little introns of 78 bp and 69 bp, respectively. The introns were excised by unsymmetrical PCR with combined flank sequences of introns as primers. The structural, functional, and immunological characteristics of putative amino acid sequence were predicted by bioinformatics analysis. The complete coding sequence was predicted to encode 433 residues and contain a transmembrane region aa(104-124), with the N terminus outside and C terminus inside. The inside part is quite the functional domain. SWISS-MODEL modulated its 3D structure as a barrel which constitutes of alternatively arranged alpha helix-beta sheet, with the key sites such as substrate binding region, active sites, Mg(2+)-binding sites closely located at the center. The protein contains a potential nuclear localization sequence aa(190-199) and several linear B cell epitopes and CTL T cell epitopes, of which the outside epitope aa(49-57) and inside epitope aa(228-236) are facultative T cell and B cell epitope, and the linear B cell epitope aa(206-213) contains the active center site Glu(210), suggesting the putative protein is a potential membrane with strong immunogenicity. The complete cds was expressed in Escherichia coli, and the recombinant protein can be recognized by the serum from patient infected with E. granulosus. Reverse vaccinology process identified E. granulosus tegumental membrane protein enolase as vaccine candidate.
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