Journal: Experimental and Therapeutic Medicine

Article Title: Knockdown of ALDH1A3 reduces breast cancer stem cell marker CD44 via the miR-7-TGFBR2-Smad3-CD44 regulatory axis

doi: 10.3892/etm.2021.10527

Figure Lengend Snippet: miR-7 directly targets TGFBR2 and Smad3 binds to the CD44 promoter region. (A) Lenti-miR-7 and lentivector MDA-MB-231 cells were respectively incubated with TGF-β1 and TGF-β1 + SB431542, and then CD44 mRNA relative expression levels were measured by RT-qPCR. (B) The relative expression levels of Smad2, Smad3, and Smad4 were measured by RT-qPCR in lenti-miR-7 and lentivector MDA-MB-231 cells. (C) TGFBR2 mRNA relative expression levels were measured by RT-qPCR. (D) TGFBR2 3'UTR with the miR-7 binding site was predicted, and complementary sequences of miR-7 to TGFBR2 3'UTR mutated are presented in red. (E) Cells were harvested and luciferase activities were measured after 48-h transfection. (F) The expression levels of TGFBR2, Smad3, and CD44 were analyzed by western blotting. (G) TGFBR2 mRNA relative expression levels were measured by RT-qPCR in BCSC-siALDH1A3, BCSC-lenti-miR-7, and control cells. (H) ChIP-PCR assay in MDA-MB-231 cells. * P<0.05, ** P<0.01 and *** P<0.001. miR-7, microRNA-7; TGFBR2, transforming growth factor-β receptor 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BCSC, breast cancer stem cell; si, small interfering.

Article Snippet: The antibodies used for western blotting included CD44 (1:2,000 dilution; cat. no. 60224-1-Ig), Smad3 (1:2,000 dilution; cat. no. 66516-1-Ig), GAPDH (1:10,000 dilution; cat no. 60004-1-Ig) and TGFBR2 (1:2,000 dilution; cat. no. 66636-1-Ig) from ProteinTech Group, Inc. IRDye ® 680RD donkey anti-mouse IgG secondary antibody (1:10,000 dilution; cat. no. P/N 926-68072) was from LI-COR Biosciences.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Transfection, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Knockdown of ALDH1A3 reduces breast cancer stem cell marker CD44 via the miR-7-TGFBR2-Smad3-CD44 regulatory axis

doi: 10.3892/etm.2021.10527

Figure Lengend Snippet: Detection of miR-7 and BCSC-related molecular expression in breast cancer surgical specimens. (A) Relative expression levels of miR-7, Smad3, CD44, and TGFBR2 in breast cancer postsurgery samples analyzed by RT-qPCR. (B-E) Relative expression levels of miR-7 and TGFBR2, miR-7 and Smad3, Smad3 and CD44, and miR-7 and CD44, respectively. The green points represent adjacent noncancerous tissues; the red points represent tumor tissues (n=12). * P<0.05. miR-7, microRNA-7; BCSC, breast cancer stem cell; TGFBR2, transforming growth factor-β receptor 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: The antibodies used for western blotting included CD44 (1:2,000 dilution; cat. no. 60224-1-Ig), Smad3 (1:2,000 dilution; cat. no. 66516-1-Ig), GAPDH (1:10,000 dilution; cat no. 60004-1-Ig) and TGFBR2 (1:2,000 dilution; cat. no. 66636-1-Ig) from ProteinTech Group, Inc. IRDye ® 680RD donkey anti-mouse IgG secondary antibody (1:10,000 dilution; cat. no. P/N 926-68072) was from LI-COR Biosciences.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Knockdown of ALDH1A3 reduces breast cancer stem cell marker CD44 via the miR-7-TGFBR2-Smad3-CD44 regulatory axis

doi: 10.3892/etm.2021.10527

Figure Lengend Snippet: Detection of the molecular expression in SK-BR-3, MCF-7, and LD breast cancer cell lines. (A-C) TGFBR2, Smad3, and CD44 expression levels in SK-BR-3, MCF-7, and LD cells were measured by RT-qPCR analysis after miR-7 mimic transfection. (D-F) TGFBR2, Smad3, and CD44 relative expression levels in SK-BR-3, MCF-7, and LD cells were detected after the inhibitor transfection by RT-qPCR. * P<0.05, ** P<0.01 and *** P<0.001. TGFBR2, transforming growth factor-β receptor 2; RT-qPCR, reverse transcription-quantitative polymerase chainreaction; miR-7, microRNA-7.

Article Snippet: The antibodies used for western blotting included CD44 (1:2,000 dilution; cat. no. 60224-1-Ig), Smad3 (1:2,000 dilution; cat. no. 66516-1-Ig), GAPDH (1:10,000 dilution; cat no. 60004-1-Ig) and TGFBR2 (1:2,000 dilution; cat. no. 66636-1-Ig) from ProteinTech Group, Inc. IRDye ® 680RD donkey anti-mouse IgG secondary antibody (1:10,000 dilution; cat. no. P/N 926-68072) was from LI-COR Biosciences.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Reverse Transcription

Journal: Bioscience Reports

Article Title: miR-200-3p suppresses cell proliferation and reduces apoptosis in diabetic retinopathy via blocking the TGF-β2/Smad pathway

doi: 10.1042/BSR20201545

Figure Lengend Snippet: ARPE-19 cells were treated with 30 mM HG for 24 h. ( A ) The expression of miR-200a-3p in ARPE-19 cells was determined by RT-qPCR. ( B ) RT-qPCR was performed to examine the expression of miR-200a-3p in cells transfected with miR-200a-3p mimic and NC-mimic, ** P <0.01, compared with the NC group; ### P <0.001, compared with the mimic-NC group. ( C ) Cell proliferation was detected by MTT assay in ARPE-19 cells transfected with miR-200a-3p mimic and then incubated with 30 mM HG, ** P <0.01, compared with the NC group; ▲▲ P <0.01, compared with the HG group. ( D,E ) Cell apoptosis was determined by flow cytometry in ARPE-19 cells transfected with miR-200a-3p mimic and then incubated with 30 mM HG, *** P <0.001, compared with the NC group; ▲▲▲ P <0.001, compared with the HG group. ( F,G ) The protein levels of Bax, caspase-3, Bcl-2, and VEGF in ARPE-19 cell treated with HG or HG plus miR-200a-3p mimic were detected using Western blotting, ** P <0.01, compared with the NC group; ▲▲ P <0.01, compared with the HG group. ( H ) The mRNA levels of IL-6, TNF-α and IL-1β were measured using RT-qPCR, ** P <0.01, compared with the NC group; *** P <0.001, ▲▲ P <0.01, ▲▲▲ P <0.001, compared with the HG group.

Article Snippet: Briefly, ARPE-19 cells transfected with miR-200a-3p mimic, pcDNA3.1-pcDNA-3.1-TGF-β2, TGF-β2+miR-200a-3p mimic or pretreatment with 10 nM SRI-011381 (SRI; TGF-β2/Smad pathway activator, MedChem Express, Monmouth Junction, U.S.A.) were seeded in 96-well plates and then exposed to HG treatment.

Techniques: Expressing, Quantitative RT-PCR, Transfection, MTT Assay, Incubation, Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: miR-200-3p suppresses cell proliferation and reduces apoptosis in diabetic retinopathy via blocking the TGF-β2/Smad pathway

doi: 10.1042/BSR20201545

Figure Lengend Snippet: ( A ) Bioinformatics analysis indicated the putative binding sites and corresponding mutant region for miR-200a-3p within TGF-β2. ( B ) Dual-luciferase reporter gene assay for WT and MUT TGF-β2 3′UTR luciferase vectors in HEK-293T cells transfected with control or miR-200a-3p mimic. ( C ) The expression of TGF-β2 protein in ARPE-19 cells transfected with miR-200a-3p mimic was determined by Western blotting. ** P <0.01, compared with the NC group.

Article Snippet: Briefly, ARPE-19 cells transfected with miR-200a-3p mimic, pcDNA3.1-pcDNA-3.1-TGF-β2, TGF-β2+miR-200a-3p mimic or pretreatment with 10 nM SRI-011381 (SRI; TGF-β2/Smad pathway activator, MedChem Express, Monmouth Junction, U.S.A.) were seeded in 96-well plates and then exposed to HG treatment.

Techniques: Binding Assay, Mutagenesis, Luciferase, Reporter Gene Assay, Transfection, Control, Expressing, Western Blot

Journal: Bioscience Reports

Article Title: miR-200-3p suppresses cell proliferation and reduces apoptosis in diabetic retinopathy via blocking the TGF-β2/Smad pathway

doi: 10.1042/BSR20201545

Figure Lengend Snippet: ARPE-19 cells were treated with HG (30 mM), HG (30 mM)+pcDNA3.1-TGF-β2, HG (30 mM)+pcDNA3.1 TGF-β2+miR-200a-3p mimic, HG (30 mM)+SRI (5 mM)+miR-200a-3p mimic. ( A,B ) The protein levels of TGF-β2, Smad2, and Smad3 were detected using Western blotting. ( C ) MTT assay was used to measure cell viability. ( D,E ) Flow cytometry was performed to examine cell apoptosis. ( F ) Western blotting was applied to detect the protein levels of Bax, caspase-3, Bcl-2, and VEGF. ( G ) The mRNA levels of IL-6, IL-1β, and TNF-α were measured using RT-qPCR. ** P <0.01, *** P <0.001, compared with the NC group; ▲ P <0.05, compared with the HG group; # P <0.05, ## P <0.01, compared with the HG+TGF-β2 group. Abbreviation: SRI, TGF-β2/Smad pathway activator SRI-011381.

Article Snippet: Briefly, ARPE-19 cells transfected with miR-200a-3p mimic, pcDNA3.1-pcDNA-3.1-TGF-β2, TGF-β2+miR-200a-3p mimic or pretreatment with 10 nM SRI-011381 (SRI; TGF-β2/Smad pathway activator, MedChem Express, Monmouth Junction, U.S.A.) were seeded in 96-well plates and then exposed to HG treatment.

Techniques: Western Blot, MTT Assay, Flow Cytometry, Quantitative RT-PCR

Journal: Bioscience Reports

Article Title: miR-200-3p suppresses cell proliferation and reduces apoptosis in diabetic retinopathy via blocking the TGF-β2/Smad pathway

doi: 10.1042/BSR20201545

Figure Lengend Snippet: DR model group, all rats received an intraperitoneal injection of STZ (60 mg/kg) which was dissolved in freshly prepared sodium citrate buffer (0.1 mM, pH 4.5); DR+miR-200a-3p group, given agomir (miR-200a-3p; 3 mg/kg) through tail intravenous injection and then received an intraperitoneal injection of STZ; DR+miR-200a-3p+SRI group, all rats were given SRI (1 mg/kg) through tail intravenous injection and then received an intraperitoneal injection of STZ. ( A ) The body weight of all mice was measured, * P <0.05, ** P <0.01, compared with the NC group. ( B ) The level of blood glucose in all mice was examined, ** P <0.01, *** P <0.001, compared with the NC group. ( C ) The levels of inflammatory cytokines were detected using ELISA. ( D ) Immunohistochemical staining was performed to detect the expression of VEGF. ( E – G ) The protein levels of Bax, caspase-3, bcl-2, MMP-2, MMP-9, TGF-β2, and VEGF were measured using Western blotting. ( H ) RT-qPCR was used to detect the expression of miR-200a-3p. ** P <0.01, compared with the NC group; ## P <0.01, compared with the DR group; ▲▲ P <0.01, compared with the HG+agomir group.

Article Snippet: Briefly, ARPE-19 cells transfected with miR-200a-3p mimic, pcDNA3.1-pcDNA-3.1-TGF-β2, TGF-β2+miR-200a-3p mimic or pretreatment with 10 nM SRI-011381 (SRI; TGF-β2/Smad pathway activator, MedChem Express, Monmouth Junction, U.S.A.) were seeded in 96-well plates and then exposed to HG treatment.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Expressing, Western Blot, Quantitative RT-PCR

Journal: Genome biology

Article Title: Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer.

doi: 10.1186/s13059-014-0428-9

Figure Lengend Snippet: Figure 3 Genetic divergence of the ovarian metastasis from the primary gastric cancer for critical candidate drivers. The genomic position of the mutation, copy number variations (CNV) regions or loss-of-heterozygosity (LOH) intervals are shown from the cancer genomes. For the chromosome plots, the Y axis designates position with the respective chromosome, its length in megabases (MB) and ideogram designation shown to the left of the copy number profile. Deleterious mutations are shown as boxed arrows with the gene symbol. (a) The genome wide distribution of cancer-specific CNVs and LOH intervals are summarized across all chromosomes for the primary tumor and metastasis. (b) On Chromosome 3, the metastasis had unique biallelic events involving a deleterious TGFBR2 mutation and a genomic deletion affecting the other allele as seen most clearly with LOH intervals. Secondary to genomic deletions, LOH is demonstrated as a shift in the minor allelic frequency ratio value of -1 and correlates with a genomic deletion. (c) On chromosome 10, the FGFR2 gene was located in a genomic amplification region seen only in the primary and not the metastasis. The amplification is noted in a red circle.

Article Snippet: Tgfbr2 shRNAs were obtained from Origene (catalog TG516186).

Techniques: Mutagenesis, Genome Wide, Amplification

Journal: Genome biology

Article Title: Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer.

doi: 10.1186/s13059-014-0428-9

Figure Lengend Snippet: Figure 5 Dysplastic epithelium in gastric organoids. (a) Gastric organoid cultures were made from gastric tissue of neonatal mice harboring Cdh1 and Trp53 floxed alleles then subsequently infected with Fc-expressing adenovirus, or CreGFP-expressing adenovirus +/- retrovirus expressing shRNA against Tgfbr2. Images indicate immunofluorescence with nuclear DAPI staining and antibodies against CDH1, TGFBR2, or PCNA. Intrinsic GFP fluorescence from adenovirus CreGFP was abrogated by tissue passaging and subsequent formaldehyde fixation, and is not visible in immunofixation experiments. (b) GSM-06 murine gastric epithelial cells were infected with scrambled shRNA or an shRNA against murine Tgfbr2 and lysates probed by western blotting with antibodies against TGFBR2 or β-actin. (c) Gastric organoid cultures were made from gastric tissue of neonatal mice harboring Cdh1 and Trp53 floxed alleles and subsequently infected with retrovirus expressing shRNA against Tgfbr2. Images are of cultured spheres at days 2 and 20. (d) Images represent H&E stained gastric organoids with the indicated genotypes taken at low (40×) or high power (400×).

Article Snippet: Tgfbr2 shRNAs were obtained from Origene (catalog TG516186).

Techniques: Infection, Expressing, shRNA, Immunofluorescence, Staining, Fluorescence, Passaging, Western Blot, Cell Culture

Journal: Genome biology

Article Title: Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer.

doi: 10.1186/s13059-014-0428-9

Figure Lengend Snippet: Figure 6 Gastric organoid tumor explants. Gastric organoids with the indicated genotypes are shown. (a) Tumor volumes were measured over time post-injection and plotted according to genotype of the driver combinations being tested. This includes Cdh1-/-;Trp53-/- as shown in blue and Cdh1-/-;Trp53-/-;Tgfbr2 shRNA as shown in red. Error bars represent SEM. Asterisk (*) indicates P <0.01 for Cdh1-/-;Trp53-/-;Tgfbr2 shRNA compared to Cdh1-/-;Trp53-/- tumor volumes. (b, c) With different driver combinations, transformed gastric organoids were dissociated and subcutaneously (s.c.) injected into the flanks of immunodeficient NOG mice. Images indicated tumor growth at 30 days post injection. (d, e) Histological analysis of tumors confirms the presence of poorly differentiated adenocarcinoma with signet ring features, as indicated by the yellow arrows, only in the Cdh1-/-;Trp53-/-; Tgfbr2 shRNA organoids. After flank injections with dissociated organoids, histological analysis of murine lungs after 30 days revealed metastatic gastric adenocarcinoma with signet ring features at low (f) and high (g) magnification.

Article Snippet: Tgfbr2 shRNAs were obtained from Origene (catalog TG516186).

Techniques: Injection, shRNA, Transformation Assay

Journal: Molecular Pain

Article Title: Bradykinin B2 receptor triggers ITPR1-Mediated ER calcium release in trigeminal ganglion neurons to drive trigeminal neuropathic pain

doi: 10.1177/17448069261468209

Figure Lengend Snippet: Bradykinin and its B2 receptor are persistently upregulated following pIONT (a) Behavioral testing (facial nocifensive scoring) revealed that pIONT model mice exhibited significant mechanical nocifensive responses on postoperative days 3, 7, and 21 (compared to sham-operated (Sham), *P < 0.05, **P < 0.01, ***P < 0.001, Two-way ANOVA followed by the Bonferroni test). (b) Immunofluorescence staining showed that the nerve injury marker ATF3 was specifically expressed in TG neurons on postoperative day 7 in the pIONT group. Scale bar: 200 μm. (c) Quantitative analysis indicates a significant increase in the proportion of ATF3-positive cells (***P<0.001, Student’s t-test). (d–f) qPCR analysis of mRNA levels in the ipsilateral TG for BK (d), B1 receptor (e), and B2 receptor (f) (compared to sham-operated groups: *P < 0.05, **P < 0.01,***P < 0.001, one-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-B2 receptor antibody (1:1000, MCE) and anti-GAPDH antibody (1:5000, Proteintech).

Techniques: Immunofluorescence, Staining, Marker

Journal: Molecular Pain

Article Title: Bradykinin B2 receptor triggers ITPR1-Mediated ER calcium release in trigeminal ganglion neurons to drive trigeminal neuropathic pain

doi: 10.1177/17448069261468209

Figure Lengend Snippet: B2 Receptor was predominantly expressed in TG neurons following pIONT (a) Western blot analysis shows that the protein levels of the B2 receptor in the TG were significantly upregulated after pIONT compared to the sham-operated group (Sham). (b) Quantitative analysis of BDKRB2 (normalized to GAPDH) further confirmed the significant increase in B2 receptor protein levels in the pIONT group (***P<0.001, Student’s t-test). (c) Immunofluorescence staining shows that the expression of the B2 receptor (red) in the TG was significantly increased on postoperative day 7 in the pIONT group compared to the sham-operated group (Sham 7d). Scale bar:100 μm. (d) Quantitative analysis indicates a significant increase in the proportion of B2 receptor-positive cells (**P<0.01, Student’s t-test, scale bar: 100 μm). (e–g) Immunofluorescence co-localization analysis demonstrates that the B2 receptor (red) is primarily expressed on myelinated neurons (NF200+, green, e), peptidergic neurons (CGRP+, green, f), and non-peptidergic neurons (IB4+, green, g). The bar charts on the right show the quantitative co-localization ratios of the B2 receptor with each cell marker. Scale bar:100 μm.

Article Snippet: After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-B2 receptor antibody (1:1000, MCE) and anti-GAPDH antibody (1:5000, Proteintech).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Marker

Journal: Molecular Pain

Article Title: Bradykinin B2 receptor triggers ITPR1-Mediated ER calcium release in trigeminal ganglion neurons to drive trigeminal neuropathic pain

doi: 10.1177/17448069261468209

Figure Lengend Snippet: Blocking the B2 receptor effectively alleviates pIONT-induced neuropathic pain and reverses neuronal hyperexcitability. (a–b) Local injection of Icatibant significantly alleviated mechanical allodynia on postoperative day 1 (pIONT 1d) in pIONT mice (*P < 0.05, pIONT 1d + Vehicle vs. pIONT 1d + Icatibant; Two-way ANOVA followed by the Bonferroni test). (c–d) On postoperative day 7 (pIONT 7d), mechanical allodynia was similarly attenuated by Icatibant treatment (**P < 0.01, pIONT 7d + Vehicle vs. pIONT 7d + Icatibant; two-way ANOVA followed by Bonferroni test). (e-f) Representative traces of action potentials evoked by step-current injection in TG neurons. (e) Rheobase current responses in Sham (0 pA), pIONT + Vehicle (20 pA), and pIONT + Icatibant (30 pA) neurons (Δ = 10 pA, 1000 ms). (f) Firing patterns at 100 pA in Sham, pIONT + Vehicle, and pIONT + Icatibant neurons. Scale bar: 0.2s, 20 mV. (g) pIONT reduced the rheobase of the first action potential in Vehicle-treated mice, whereas Icatibant administration preserved rheobase values comparable to sham controls (*P < 0.05, one-way ANOVA followed by Tukey’s post-hoc test). (h) pIONT increased the firing frequency elicited by step-current injection in Vehicle-treated mice, an effect abolished by Icatibant(**P < 0.01, pIONT + Vehicle vs. pIONT + Icatibant). (i) pIONT enhanced the firing frequency in response to ramp-current stimulation in vehicle-treated mice, which was prevented by Icatibant co-treatment (*P < 0.05, **P < 0.01, ***P < 0.001, pIONT + vehicle vs. pIONT + Icatibant; two-way ANOVA followed by Bonferroni test). (j) The discharge RMP of action potentials was not altered by pIONT or Icatibant treatment. (P > 0.05, pIONT + vehicle vs. pIONT + Icatibant; one-way ANOVA followed by Tukey’s post-hoc test). (K) pIONT decreased action potential half-width in vehicle-treated mice. (l-m) Neither the peak amplitude (l) nor the discharge threshold (m) of action potentials was altered by pIONT or Icatibant treatment (P > 0.05, pIONT + vehicle vs. pIONT + Icatibant; one-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-B2 receptor antibody (1:1000, MCE) and anti-GAPDH antibody (1:5000, Proteintech).

Techniques: Blocking Assay, Injection