Journal: Nature Communications

Article Title: Differential TGFβ pathway targeting by miR-122 in humans and mice affects liver cancer metastasis

doi: 10.1038/ncomms11012

Figure Lengend Snippet: ( a ) Western blot analysis of TGFβ1 and TGFβR1 in HepG2, Huh7 or Hepa1-6 cells when treated with an miR-122 expression plasmid (122), miR-122 sponge (122sp) or scramble sequence as an negative control (NC), respectively. Quantitative analysis is shown on the right, and three independent repeats are performed in each experiment. HepG2 and Huh7 are human liver cancer cells, while Hepa1-6 is mouse cell line. ( b ) Real-time PCR analysis of TGFβ1 and TGFβR1 mRNAs in either HepG2, Huh7 or Hepa1-6 cells treated with miR-122, miR-122sp or NC. Three independent repeats are performed in each experiment. ( c ) Western blot analysis of TGFβ1 and TGFβR1 in PANC-1 or MCF-7 cells as well as NIT-1 cells transfected with a miR-122 expression plasmid or NC, respectively. Quantitative analysis is shown on the right. n =3. PANC-1 or MCF-7 are human non-liver cell line, while NIT-1 is a mouse pancreatic β-cell line. ( d ) Real-time PCR analysis of TGFβ1 and TGFβR1 mRNAs in PANC-1 or MCF-7 cells as well as NIT-1 cells treated with miR-122 or NC. Three independent repeats are performed in each experiment. ( e ) Western blot analysis of intracellular TGFβ1, Smad2 and p-Smad2 in HepG2 cells transfected with miR-122, NC or miR-122 together with TGFβ1, respectively. Quantitative analysis is shown on the right. Three independent repeats are performed in each experiment. ( f ) Western blot analysis of intracellular TGFβ1, Smad2 and p-Smad2 in SMC-7721 cells transfected with miR-122, NC or miR-122 together with TGFβ1. Quantitative analysis is shown on the right, and three independent repeats are performed in each experiment. ( g ) Western blot analysis of intracellular TGFβR1, Smad2 and p-Smad2 in NIT-1 cells transfected with miR-122, NC or miR-122 together with TGFβR1. Quantitative analysis is shown on the right, and three independent repeats are performed in each experiment. Error bars, ±s.d. ** P <0.01; *** P <0.001 by two-sided Student's t -test. β-Actin was used as a loading control.

Article Snippet: The membranes were probed with antibodies against TGFβ1 (R&D, cat. # MAB240), TGFβ2 (R&D, cat. # MAB612), TGFβ3 (R&D, cat. # MAB643), TGFβR1 (R&D, cat. # MAB5871), Smad2 (Cell Signaling Technology, cat. # 3103S), Phospho-Smad2 (Cell Signaling Technology, cat. # 3101S), E-cadherin (Cell Signaling Technology, cat. # 3195S), vimentin (Abcam, cat. # Ab8978) or β-actin (Santa Cruz, cat. # SC-7210).

Techniques: Western Blot, Expressing, Plasmid Preparation, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Transfection

Journal: Nature Communications

Article Title: Differential TGFβ pathway targeting by miR-122 in humans and mice affects liver cancer metastasis

doi: 10.1038/ncomms11012

Figure Lengend Snippet: ( a ) Evidence for the different versions of human TGFβ1 mRNA, which include gene annotation from UCSC Genes and ENCODE/GENCODE, the PacBio sequencing data from mixed 20 tissues , human mRNA from GenBank (NCBI) or PolyA sequencing data of different tissues . ( b – e ) Luciferase activity was measured after transfection of the indicated reporter constructs in Hela cells. TGFβR1 5′UTR was cloned into the promoter region. Hum, human, Mou, mouse. The TGFβ1 3′UTRs were cloned into the 3′UTR of luciferase in a pGL plasmid. The 3′UTR of human TGFβ1 was divided into two parts, H1 or H2, because of its long length. Six independent repeats are performed in each experiment. ( f ) Luciferase activity was measured after transfection of the indicated reporter constructs. The human TGFβ1 5′UTR was divided into three different fragments and then each truncated 5′UTR was cloned into the promoter region of pGL plasmid. Six independent repeats are performed in each experiment. ( g ) Diagram depicting the miR-122 targeting region of the TGFβ1 5′UTR and the mutated site. Luciferase reporter activity containing the different constructs was shown below. Error bars, ±s.d. * P <0.05; ** P <0.01; *** P <0.001 by two-sided Student's t -test.

Article Snippet: The membranes were probed with antibodies against TGFβ1 (R&D, cat. # MAB240), TGFβ2 (R&D, cat. # MAB612), TGFβ3 (R&D, cat. # MAB643), TGFβR1 (R&D, cat. # MAB5871), Smad2 (Cell Signaling Technology, cat. # 3103S), Phospho-Smad2 (Cell Signaling Technology, cat. # 3101S), E-cadherin (Cell Signaling Technology, cat. # 3195S), vimentin (Abcam, cat. # Ab8978) or β-actin (Santa Cruz, cat. # SC-7210).

Techniques: Sequencing, Luciferase, Activity Assay, Transfection, Construct, Clone Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Differential TGFβ pathway targeting by miR-122 in humans and mice affects liver cancer metastasis

doi: 10.1038/ncomms11012

Figure Lengend Snippet: ( a ) Luciferase activity was measured after transfection of the indicated reporter constructs. TGFβ1 5′UTR was cloned into the promoter region of a pGL plasmid. Rhesus, Rhesus monkey. Six independent repeats are performed in each experiment. ( b ) The evolutionary trajectory of the miR-122 target site in the TGFβ1 5′UTR in animals. The gain of the miR-122 target site occurs in the common ancestor of the manatee and humans as well as other primates (black arrow), while the loss of this site in the pig, dog, rat or mouse due to the insertion of a few of bases between the 11th and 12th bases (red arrow). The dot means the nucleotide is identical to the one in humans, and the red line means the insertion of one or a few of bases. For the predicted miR-122 target site in each species, the luciferase assay was performed. ‘+' denotes the silence effect, and ‘−' denotes no silence effect. Experimental data were shown in . ( c ) Expression levels of TGFβ1 or TGFβR1 in C5.18 (rat cells) or LLC-PK1 (pig cells) transfected with an miR-122 expression plasmid or NC. Quantitative analysis is shown below and three independent repeats are performed in each experiment. ( d ) Luciferase activity was measured after transfection with the indicated reporter constructs. The candidate sequences (see for the sequences) were cloned in the CDS of a fusion protein of luciferase and eGFP. NC, negative control; PC, positive control; Rhes., Rhesus monkey. Six independent repeats are performed in each experiment. ( e ) The evolutionary trajectory of the miR-122 target site in the TGFβR1 CDSs in animals. Three events are involved in the gain and loss of target sites, that is, a G–>A mutation (red), A–>G mutation (blue) and a G–>A mutation (green). The dot means the nucleotide is identical to the one in humans. For the predicted miR-122 target site in each species, the luciferase assay was performed. ‘+' denotes the silence effect, and ‘−' denotes no silence effect. Experimental data were shown in . Error bars, ±s.d. ** P <0.01; *** P <0.001 by two-sided Student's t -test.

Article Snippet: The membranes were probed with antibodies against TGFβ1 (R&D, cat. # MAB240), TGFβ2 (R&D, cat. # MAB612), TGFβ3 (R&D, cat. # MAB643), TGFβR1 (R&D, cat. # MAB5871), Smad2 (Cell Signaling Technology, cat. # 3103S), Phospho-Smad2 (Cell Signaling Technology, cat. # 3101S), E-cadherin (Cell Signaling Technology, cat. # 3195S), vimentin (Abcam, cat. # Ab8978) or β-actin (Santa Cruz, cat. # SC-7210).

Techniques: Luciferase, Activity Assay, Transfection, Construct, Clone Assay, Plasmid Preparation, Expressing, Negative Control, Positive Control, Mutagenesis

Journal: Nature Communications

Article Title: Differential TGFβ pathway targeting by miR-122 in humans and mice affects liver cancer metastasis

doi: 10.1038/ncomms11012

Figure Lengend Snippet: ( a ) Quantitative analysis of miR-122 levels in hepatocellular tumours in HBx gene knock-in transgenic mice or normal liver tissue (eight independent repeats) as well as human hepatocellular tumour tissues or normal adjacent tissue (six independent repeats). ( b ) Western blot assay showing the expression levels of TGFβ1/TGFβR1 in tissues, as indicated. Representative images are shown in the inset. Eleven independent repeats are performed in each experiment. ( c ) Quantitative analysis of vessel numbers in the indicated tissues by CD31 staining. ( d ) Kaplan–Meier curves for overall survival in HCC cohorts from the Liver Cancer Institute (LCI) and Zhongshan Hospital. Expression value=log2 of Robust multi-array analysis (RMA)-calculated signal intensity. miR-122 expression value>0.39 was designed as the high expression group, while miR-122 expression value<-0.58 was designed as the low expression group. n =165 patients; P value based on the Mantel–Cox log-rank test. ( e ) Kaplan–Meier curves for overall survival in HCC cohorts from the LCI and Zhongshan Hospital. Expression value=log2 of RMA-calculated signal intensity. TGFβ1 expression value <4 was designed as the low expression group, while TGFβ1 expression value>5 was designed as the high expression group. n =244 patients; P value based on the Mantel–Cox log-rank test. Error bars, ±s.d. ** P <0.01; *** P <0.001 by two-sided Student's t -test. ND, no difference.

Article Snippet: The membranes were probed with antibodies against TGFβ1 (R&D, cat. # MAB240), TGFβ2 (R&D, cat. # MAB612), TGFβ3 (R&D, cat. # MAB643), TGFβR1 (R&D, cat. # MAB5871), Smad2 (Cell Signaling Technology, cat. # 3103S), Phospho-Smad2 (Cell Signaling Technology, cat. # 3101S), E-cadherin (Cell Signaling Technology, cat. # 3195S), vimentin (Abcam, cat. # Ab8978) or β-actin (Santa Cruz, cat. # SC-7210).

Techniques: Gene Knock-In, Transgenic Assay, Western Blot, Expressing, Staining

Journal: Nature Communications

Article Title: Differential TGFβ pathway targeting by miR-122 in humans and mice affects liver cancer metastasis

doi: 10.1038/ncomms11012

Figure Lengend Snippet: ( a ) Schematic diagram showing the screening strategy used to investigate the miRNA target sites along the 3′UTR of three pairs of ligands/receptors. ( b ) Cutoff value was set according to 50 control experiments. ( c ) SAMcell assay demonstrated the consistent results in the luciferase assay. Eight randomly selected miRNAs, including miR26a, 222, 365, 99b*, 496, 369, 99a or 216, were examined to target HGF/HGFR in humans and mice. ( d ) Column diagram showing the number of four types of miRNA target sites in HGF/HGFR , TGFβ1/TGFβR1 or FGF/FGFR , including unique, common, cooperative and orthogonal type. Cooperative type refers to the situation in which a pair of a ligand and receptor was simultaneously targeted by one certain miRNA in one species.

Article Snippet: The membranes were probed with antibodies against TGFβ1 (R&D, cat. # MAB240), TGFβ2 (R&D, cat. # MAB612), TGFβ3 (R&D, cat. # MAB643), TGFβR1 (R&D, cat. # MAB5871), Smad2 (Cell Signaling Technology, cat. # 3103S), Phospho-Smad2 (Cell Signaling Technology, cat. # 3101S), E-cadherin (Cell Signaling Technology, cat. # 3195S), vimentin (Abcam, cat. # Ab8978) or β-actin (Santa Cruz, cat. # SC-7210).

Techniques: Luciferase