Transfection of C/EBPα-saRNA inhibits metastasis by targeting EGFR/β-catenin signaling pathway.

<p>(A) C/EBPα binding sites in EGFR and β-catenin genomic regions. (B) Western blot shows decreased ADAM17, EGFR protein and phosphorylation levels and decreased β-catenin in hepatoma cells transfected with C/EBPα-saRNA. (C) Reduced gene expression of c-Myc, Axin2, CCDN1, and Lgr5 in C/EBPα-saRNA transfected HepG2 cells compared with scramble-saRNA transfected as the control. (D) Immunohistochemistry analysis of EGFR, phosphorylation of EGFR, and β-catenin in liver tissues. (E) Western blot shows inhibited ADAM17, EGFR protein and phosphorylation levels and suppressed β-catenin in liver tissue of C/EBPα-saRNA-dendrimer-treated mice (n = 9).</p

Transfection of C/EBPα-saRNA in hepatoma cells inhibited migration, invasion and EMT.

<p>(A-B) Wound healing assays of SMMC-7721 (A) and HepG2 (B) cells transfected with scramble-saRNA or C/EBPα-saRNA at 0 h, 12 h, and 24 h. Magnification, 40×. (C) Transwell assays show suppressed invasion of cells transfected with C/EBPα-saRNA compared with scramble-saRNA as the control after 24 h. Magnification, 100×. (D-E) Western blot analysis shows decreased N-cadherin, Slug, and Vimentin and up-regulation of E-cadherin, albumin, and C/EBPα in hepatoma cells transfected with C/EBPα-saRNA compared with the control. Data represent mean±SD.</p

Primers and product size.

<p>Primers and product size.</p

Transfection of C/EBPα-saRNA in SMMC-7721 and HepG2 cells resulted in inhibition of migration.

<p>(A–B) Gene expression of C/EBPα (A) and albumin (B) in SMMC-7721 and HepG2 cells transfected with C/EBPα-saRNA or scramble-saRNA as the control for 48 h. (C) Transwell assays show reduced migration of SMMC-7721 and HepG2 cells transfected with C/EBPα-saRNA compared with the control after 18 h. Magnification, 100×. Data represent mean±SD.</p

Intravenous injection of C/EBPα-saRNA-dendrimer inhibited tumor metastasis and improved liver function in nude mice with tumors from HepG2-RFP cells.

<p>(A) Imaging of liver orthotopic xenograft tumors after injection of C/EBPα-saRNA on day 40 post-injection of HepG2-RFP cells and scramble-saRNA as the control. Higher fluorescence intensity and intrahepatic metastasis was observed in the control group. (B) C/EBPα-saRNA-dendrimer was tested for nuclease sensitivity in nude mice for indicated times. (C) Survival analysis of C/EBPα-saRNA-injected mouse groups and the control (n = 9) (log rate: 0.0167). (D) Statistical table of tumor formation, intrahepatic and distant metastasis. (E) Hematoxylin and eosin staining of liver and lung from C/EBPα-saRNA injected mice and the control. (F) Expression of C/EBPα was determined by RT-PCR and western blotting in liver tissue of mice injected with C/EBPα-saRNA-dendrimer or the control. (G-I) C/EBPα-saRNA-dendrimer injected nude mice showed significant changes in circulating levels of serum albumin (G), AST (H) and ALT (I). Data represent mean±SD.</p

A Designed Tryptophan- and Lysine/Arginine-Rich Antimicrobial Peptide with Therapeutic Potential for Clinical Antibiotic-Resistant <i>Candida albicans</i> Vaginitis

New therapeutic agents for <i>Candida albicans</i> vaginitis are urgently awaiting to be developed because of the increasing antibiotic resistance of <i>C. albicans</i>. Antimicrobial peptides (AMPs) are one of the most promising choices for next-generation antibiotics. In this study, novel peptides were designed based on snake venom antimicrobial peptide cathelicidin-BF to promote anti-<i>C. albicans</i> activity and decrease side-effects. The designing strategies include substitutions of charged or hydrophobic amino acid residues for noncharged polar residues to promote antimicrobial activity and insertion of a hydrophobic residue in the hydrophilic side of the helix structure to reduce hemolysis. A designed tryptophan and lysine/arginine-rich cationic peptide <b>4</b> (ZY13) (VKRWKKWR­WKWKKWV-NH₂) exhibited excellent antimicrobial activity against either common strain or clinical isolates of antibiotic-resistant <i>C. albicans</i> with little hemolysis. Peptide <b>4</b> showed significant therapeutic effects on vaginitis in mice induced by the infection of clinical antibiotic-resistant <i>C. albicans</i>. The approaches herein might be useful for designing of AMPs

Sequences of qRT-PCR primers used for mRNA analysis.

<p>Sequences of qRT-PCR primers used for mRNA analysis.</p

Expression levels of miR-200a and validation for stable miR-200a knockdown in WB cells.

<p>(A) QRT-PCR analysis of the relative miR-200a levels in WB cells and three hepatoma cells (H-4-II-E, CBRH-7919, RH-35) compared with the normal liver cell line BRL. (B) Validation of miR-200a levels in WB cells lentivirally transfected with miR-200a antagomir (WB-anti-miR-200a) or negative control (WB-miR-NC) by qRT-PCR analysis. (C and D) Functional evaluation of down-regulated miR-200a on its validated target ZEB2 in WB cells using qRT-PCR (C) and western blot analysis (D). For A and B, data are normalized to U6 and represented as the mean ± SD; n = 5; **, p<0.01. For C, data are normalized to β-actin and presented as the mean ± SD; n = 4.</p

Stable knockdown of miR-200a facilitates CSC-like phenotypes in WB cells.

<p>(A) Growth curve of WB-miR-NC and WB-anti-miR-200a cells determined by cell counting. Data are expressed as the mean ± SD; n = 3; *, p<0.05. (B) Representative images of spheroids formed by WB-miR-NC and WB-anti-miR-200a cells in the spheroid formation assay (left, magnification×100). Number of spheroids formed in the primary, secondary and tertiary generations of suspension cultured WB-miR-NC or WB-anti-miR-200a cells (right). Data represent means from four randomly selected fields under the microscope, and error bars represent SD. *, p<0.05; **, p<0.01. (C and D) Apoptosis of WB-miR-NC and WB-anti-miR-200a cells measured by caspase-3/7 assay and Annexin V and PI staining. Data are expressed as the mean ± SD (C) and representative dot plots of apoptosis tests are shown (D); n = 5. (E) Expression of EpCAM, CD133, ABCG2, CK19, AFP, ALB and c-myc in WB-anti-miR-200a cells measured by qRT-PCR. Data are normalized to β-actin, shown relative to the level in WB-miR-NC cells and expressed as the mean ± SD; n = 3; *, p<0.05; **, p<0.01. (F) WB-miR-NC and WB-anti-miR-200a cells were treated with 10 ng/ml paclitaxel or 30 ng/ml doxorubicin for 48 h and then subjected to FACS with Annexin V and PI staining, respectively. Representative dot plots (left) and the mean percentage of apoptotic cells (± SD) from three independent experiments (right) are shown; *, p<0.05; **, p<0.01.</p

Stable knockdown of miR-200a confers mesenchymal characteristics to WB cells.

<p>(A) Morphological changes in WB-miR-NC and WB-anti-miR-200a cells (magnification×200). (B) Evaluation of in vitro migration abilities of WB-miR-NC and WB-anti-miR-200a cells by transwell migration assay. Representative images (upper, magnification×200) and the mean number of migrated cells (± SD) in five randomly selected fields counted under the microscope (lower) are shown; **, p<0.01. (C) Western blot analysis of epithelial (E-cadherin) and mesenchymal (N-cadherin and vimentin) markers in WB-miR-NC and WB-anti-miR-200a cells.</p