Overexpression of miR-127 inhibits MMP13 and TGFβ-mediated induction of HCC cell migration.

<p>(<i>A</i>) Cell migration assay. MHCC97H cells were transfected with MMP13 siRNA (siMMP13) (20 nM) in the absence or presence of pTarget or pTarget-miR-127 (2 µg). After cells were seeded onto the inserts, lower chamber media were treated with MMP13 peptide (50 ng/ml) as indicated. Cells that had migrated through the membrane were fixed and stained with crystal violet (left). Quantitative results on the right. (<i>B-C</i>) qPCR analysis of MMP13 (<i>B</i>) and miR-127 (<i>C</i>) expression under the same experimental conditions as (<i>A</i>). (<i>D</i>) Cell migration assay. MHCC97H and Hepa-1 cells were transfected with pTarget or pTarget-miR-127 (2 µg), in the absence or presence of TGFβ (5 ng/ml). Migrated cells were stained with crystal violet and visualized by microscopy (left). Quantitative results on the right. (<i>A-D</i>): *p<0.01, miR-127 <i>vs.</i> pTarget in control group; ^¥p<0.01, pTarget in MMP13 group <i>vs.</i> pTarget in control group;^‡p<0.01, miR-127 <i>vs.</i> pTarget in MMP13 group; ^§p<0.01, miR-127 <i>vs.</i> pTarget in siMMP13 group.</p

TGFβ enhances c-Jun-mediated repression of miR-127 via ERK and JNK pathways.

<p>(<i>A-C</i>) qPCR analysis of c-Jun (<i>A</i>), miR-127 (<i>B</i>), and MMP13 (<i>C</i>) mRNA expression in MHCC97H cells treated with TGFβR inhibitor (SB 431542, 10 µM), NFκB inhibitor (BAY-11-7082, 5 µM, ERK inhibitor (PD 98059, 50 µM), p38 inhibitor (SB 203580, 10 µM) and JNK inhibitor (SP 600125, 50 µM),) in the absence (−) or presence of TGFβ (+) (5 ng/ml). Statistical results represent the mean ± SD. *p<0.01, TGFβ (+) <i>vs.</i> TGFβ (−) group.</p

Model of negative-feedback regulation of miR-127 expression by TGFβ/c-Jun that controls MMP13 expression and stability in HCC.

<p>TGFβ activates the oncogene c-Jun through ERK and JNK pathways. The activation of c-Jun serves a dual function, which involves induction of MMP13 gene expression but repression of miR-127 gene transcription by inhibiting miR-127 promoter activity. c-Jun also antagonizes p53 activation of the miR-127 promoter and gene transcription. On the other hand, overexpression of miR-127 decreases MMP13 protein levels by binding to its 3′UTR and causing MMP13 degradation, thus diminishing TGFβ-mediated HCC migration.</p

Overexpression of miR-127 inhibits HCC cell migration and tumor growth.

<p>(<i>A</i>) Wound healing assay. MHCC97H cells were transfected with either an empty vector pTarget (-) or a miR-127 expression vector in pTarget (+) (2 µg). Wound closure was photographed 48 hr later (left) and the residual gap between the migrating cells was quantified (right), which is expressed as a percentage of the initial scraped area. (<i>B</i>) Cell migration assay. MHCC97H cells were transfected with pTarget or pTarget-miR127 (2 µg), a negative (neg) control or miR-127 inhibitor (anti-miR-127) (20 nM). Cells that had migrated through the membrane of Transwell inserts were fixed, stained with crystal violet, and visualized by microscopy. (<i>C</i>) Cell invasion assay. MHCC97H cells were transfected with pTarget or pTarget-miR127 (2 µg), and seeded onto pre-coated inserts with BME solution and incubated for 16 hr. The invaded cells were dissolved in Cell Dissociation Solution/Calcein-AM and absorbance was measured. (<i>D</i>) Tumorigenesis assay. Control 97HLuc-Sico or 97HLuc-miR-127 cells were grafted subcutaneously in the dorsa of athymic mice, and tumor growth was monitored using the Xenogen bioluminescent imaging system by day 9. The number represents tumor images detected (photons/s/cm²/steridian). *p<0.01, miR-127 <i>vs.</i> pTarget control; ^¥p<0.01, anti-miR-127 <i>vs.</i> negative control.</p

TGFβ represses miR-127 expression by enhancing c-Jun activity.

<p>(<i>A</i>) qPCR analysis of MMP13 (left), c-Jun (middle), and miR-127 (right) expression in MHCC97H cells treated with TGFβ. *p<0.01, TGFβ (+) <i>vs.</i> TGFβ (-) group. (<i>B</i>) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter (pro) luciferase (luc) reporter, and c-Jun or c-Fos expression plasmids. *p<0.01, c-Jun (+) <i>vs.</i> c-Jun (-) group. (<i>C)</i> Transient transfection assay. Hela cells were co-transfected with miR-127 or AP1 promoter reporter along with c-Jun/c-Fos plasmid (100 ng) in the absence or presence of TGFβ (5 ng/ml). *p<0.01, c-Jun/Fos <i>vs.</i> control without (-) or with (+) TGFβ; ^¥p<0.01, control with (+) TGFβ <i>vs.</i> control without (-) TGFβ. (<i>D</i>) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter reporter, c-Jun (100 ng) and/or p53 plasmids (100 ng). *p<0.01, c-Jun or p53 <i>vs.</i> control; ^¥p<0.01, c-Jun/p53 <i>vs.</i> p53. (<i>B-D</i>) Luciferase (Luc) activity was normalized by Renilla activity (act). (<i>E</i>) qPCR analysis of miR-127 and p53 expression in HepG2 cells transfected with control (con) or p53 siRNAs (si-p53) (20 nM), or in HCT116<i>p53^+/+</i> (+) and HCT116<i>p53^−/−</i> (-) cells. *p<0.01, si-p53 <i>vs.</i> control. (<i>F</i>) qPCR analysis of miR-127 expression in MHCC97H cells that were overexpressed with c-Jun and/or p53. *p<0.01, p53 <i>vs.</i> control (-); ^¥p<0.01, c-Jun/p53 <i>vs.</i> p53.</p

miR-127 is downregulated in a subset of HCC specimens.

<p>(<i>A-B</i>) qPCR analysis of miR-127 expression (<i>A</i>) and MMP13 mRNA (<i>B</i>, left), and Western blot (WB) analysis of MMP13 protein (<i>B</i>, right) in 5 pairs of surrounding controls and HCC specimens. *p<0.01, tumor <i>vs.</i> non-tumor.</p

Optimization of Zn_<i>x</i>Fe_3–<i>x</i>O₄ Hollow Spheres for Enhanced Microwave Attenuation

We report here the composition optimization of Zn_<i>x</i>Fe_3–<i>x</i>O₄ hollow nanospheres for enhancing microwave attenuation. Zn_<i>x</i>Fe_3–<i>x</i>O₄ hollow nanospheres were synthesized through a simple solvothermal process. The maximum magnetization moment of 91.9 emu/g can be obtained at <i>x</i> = 0.6. The composite filled with Zn_0.6Fe_2.4O₄ exhibited the bandwidth of 3.21–8.33 GHz for RL < −10 dB and a maximum relative bandwidth (<i>W</i>_p,max) of 88.6% at optimized thickness <i>t</i>₀ = 0.34 cm. The enhancement should be attributed to the enhanced permeability resonance at high frequency. This optimized hollow material is very promising to be used as a mass efficient and broadband microwave attenuation material

SHP does not interfere p53 and Mdm2 interaction.

<p>A–B: GST pull down to determine the <i>in vitro</i> interaction of SHP with p53. Flag-p53 was <i>in vitro</i> translated and used to interact with various GST-SHP fusion proteins, including GST-FL (full length), GST-2 (interaction domain), GST-1+2 (repression domain deletion), GST-3 (repression domain), and GST-2+3 (N-terminal domain deletion), that were expressed from bacterial Escherichia coli BL21/DE3/RIL. C: Immunoprecipitation and Western blots. Plasmids expressing Myc-p53 were cotransfected with Flag-SHPWT, Flag-SHP38H, Flag-SHP170N, and Flag-SHP171A plasmids in Hela cells. Co-IP and WB were performed with corresponding antibodies as indicated. D–E: Western blots to determine the effect of SHP on p53 and Mdm2 interaction. D: HA-Mdm2 (2 µg), Myc-p53 (4 µg) and Flag-SHP (2 µg) plasmids were cotransfected in 293T cells, and their proteins were detected using anti-HA, anti-Myc and anti-Flag antibodies, respectively. E: HA-Mdm2 (2 µg), Flag-p53 (4 µg) and Flag-SHP (2 µg) plasmids were cotransfected in Hela cells, and their proteins were detected using anti-HA and anti-Flag antibodies, respectively.</p

Downregulation of SHP protein by p53 is mediated by proteosome degradation.

<p>A: Western blots to determine the effect of p53 on SHPWT, SHPK170R, or SHPK170N protein expression. Flag-SHPWT, Flag-SHPK170R or Flag-SHPK170N expression vectors were transfected in 293T cells without or with Flag-p53 co-expression, and p53 and SHP proteins detected simultaneously using anti-Flag antibodies on the same blot. B: Immunoprecipitation and Western blots to determine the association of SHP proteins with p53 protein. Flag-SHPWT, Flag-SHPK170R or Flag-SHPG171A expression vectors were co-transfected with GST-p53 expression vector. Whole cell lysates were immunoprecipitated with the anti-GST antibodies, and SHP and p53 proteins were detected using anti-Flag or anti-GST antibodies, respectively. C: Western blots to determine the effect of p53 on SHPK170R protein levels. Flag-SHPK170R and Flag-p53 plasmids were cotransfected in 293T cells, and SHP and p53 proteins were detected using anti-Flag antibodies. p53 and SHP protein can be easily distinguished because of different molecular weight. D: Western blots to determine the effect of p53 on SHPG171A protein levels. Myc-p53 and Flag-SHPG171A plasmids were cotransfected in 293T cells, and p53 and SHPG171A proteins were detected using anti-Myc and anti-Flag antibodies, respectively. E–F: Western blots to determine the effect of p53 on SHP protein expression in the presence of MG132 or TSA (E) or on the phosphorylated and acetylated SHP protein expression (F). Flag-SHP expression vectors were transfected alone or together with Myc-p53 vectors, and both proteins were detected using antibodies as indicated in the figures. CON, control. G: <i>In vitro</i> Ubiquitination assays to determine the effect of p53 on SHP protein ubiquitination. Flag-SHP and HA-Ub expression vectors were transfected without or with GFP-p53 vectors. Whole cell lysates were immunoprecipitated with the anti-Flag antibodies, and ubiquitinated SHP was detected using anti-HA antibodies (indicated by a solid line).</p

p53 causes rapid SHP protein degradation independent of Mdm2.

<p>A: Western blots to determine the effects of p53 and Mdm2 on SHP protein expression in <i>p53^−/−Mdm2^−/−</i> MEF cells lacking p53 and Mdm2. The <i>p53^−/−Mdm2^−/−</i> MEF cells were transfected with Flag-SHP, Flag-p53 or Mdm2 expression vectors, and Mdm2, p53 or SHP proteins were detected using anti-Mdm2 and anti-Flag antibodies, as indicated. The sizes of p53 and SHP proteins are distinct, thus both proteins were detected simultaneously using anti-Flag antibodies on the same blot. B: Western blots to determine the effect of p53 on endogenous SHP protein expression. Flag-p53 expression vectors were transfected in HepG2 cells, and p53 (both endogenous and exogenous), SHP (endogenous), and Mdm2 (endogenous) proteins were detected using anti-p53, anti-SHP and anti-Mdm2 antibodies, respectively.</p