Journal: Theranostics

Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

doi: 10.7150/thno.43198

Figure Lengend Snippet: miR-222-3p directly suppresses PDCD10 expression by binding to its 3' -UTR and inhibits EOC cell migration in vitro . ( A ) A Venn diagram was used to look for candidate genes targeted by miR-222-3p. ( B ) Levels of 15 candidate genes in HO 8910 PM cells transfected with miR-222-3p mimic. ( C ) miR-222-3p inhibitor assay in SKOV3 cells showing four candidate genes with upregulation of two genes, the most significant being PDCD10. ( D ) Western blot analysis of PDCD10 levels in two different EOC cell lines after transfection with miR-222-3p mimic or inhibitor. ( E ) Schematic depiction of the double-strand formation by miR-222-3p with the 3' -UTR of PDCD10. ( F ) Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/inhibitor and PDCD10 WT/MUT. “++” stands for the double concentration. ( G and H ) Transwell and wound healing assays revealed inhibition of migration when HO 8910 PM cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed migration of EOC cell lines due to its inhibitory effect on PDCD10 (Left). Cell counting and wound healing were quantified (Right). ( I ) qPCR and ( J ) Western blot analyses of PDCD10 expression levels in HO 8910 PM cells, and Image J calculated the relative expression rate. Data are means ± SEM. Data are from six (B) experiments and representative of three (C and F-J) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

Techniques: Expressing, Binding Assay, Migration, In Vitro, Transfection, Western Blot, Luciferase, Concentration Assay, Inhibition, Cell Counting, Two Tailed Test

Journal: Theranostics

Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

doi: 10.7150/thno.43198

Figure Lengend Snippet: miR-222-3p suppresses EOC tumor metastasis in vivo by targeting PDCD10. ( A ) Schematic presentation of in vivo adhesion for equivalent numbers of GFP-labeled LV-miR-ctrl/LV-miR-222-3p and GFP-labeled ctrl vector/PDCD10 transfected stably in HO 8910 PM cells. Bar, 100 µm. ( B and C ) Representative images and quantification of intraperitoneal metastases in mice implanted intraperitoneally with the same number of HO 8910 PM cells (n= 4 mice per group). Bar, 1 cm. ( D ) Western blot analysis of PDCD10 levels in representative EOC metastatic nodules. ( E ) Representative images and bioluminescence mice images of lung tissue metastatic nodules 5 weeks (wk) after implantation. Bar, 0.5 cm. ( F ) Quantification of metastatic nodules in lung tissues of mice. ( G ) Representative images (Down) and metastatic nodule plots (Up) of mice stomach tissues formed in the restoration group. Bar, 0.5 cm. ( H ) IHC staining for PDCD10 in the stomach tissues of mice 5 weeks after implantation. Bar, 100 µm. ( I ) Representative images (Down) and metastatic nodule plots (Up) of mice liver tissues formed in the restoration group. Bar, 1 cm. ( J ) Representative HE staining of the mice lung tissues obtained from 5 weeks after implantation. Bar, 50 µm (Left) and 100 µm (Right). Data are means ± SEM and are representative of three (C, F, G, I) independent experiments. *, P< 0.05. **; P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

Techniques: In Vivo, Labeling, Plasmid Preparation, Transfection, Stable Transfection, Western Blot, Immunohistochemistry, Staining, Two Tailed Test

Journal: Theranostics

Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

doi: 10.7150/thno.43198

Figure Lengend Snippet: miR-222-3p targets PDCD10 to inhibit cell migration via EMT pathway. ( A ) Meta-analysis describing forest plots of PDCD10 expression as a univariate predictor of overall survival. ( B ) Kaplan-Meier curves for overall survival probability in 1656 OC patients with low (n=640) and high (n=1016) PDCD10 expression (analyzed with log-rank test) from KMplot ( http://kmplot.com/analysis/ ). ( C ) qPCR and Western blot analyses of PDCD10 levels in FE25 and 4 different EOC cell lines. ( D ) Representative images of PDCD10 expression in normal and tumor ovary tissues. Bar, 100 µm (Left) and 30 µm (Right). ( E ) Enrichment evaluation within the PDCD10-expressing levels for predicted GSEA results of TCGA reference gene sets for high and low PDCD10 expression groups. Genes expressed in the profile datasets were ranked by fold changes (high-expression/low-expression). GSEA correlation pathways were determined by the given algorithm. Vertical bars along the x-axis of the GSEA diagram represent the positions within the ranked list of genes set in the given sets. Positive and negative GSEA curves mean positive and negative enrichments, respectively. ( F and G ) qPCR and Western blot analyses of PDCD10, E-cad (CDH1) and VIM expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( H ) HO 8910 PM cells transduced with control vector or PDCD10 (GFP-labeled ctrl vector and PDCD10 all in green) were subjected to immunofluorescence with human-specific E-cad and VIM antibodies (in red). Bar, 10 µm. ( I ) Western blot analysis of PDCD10, E-cad, Vim translation levels in HO 8910 PM cells after treatment with miR-ctrl mimic or miR-222-3p mimic, in the presence or absence of PDCD10. Data are means ± SEM and are representative of three (C and F) independent experiments. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001, determined by unpaired two-tailed t-test.

Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

Techniques: Migration, Expressing, Western Blot, Transduction, Plasmid Preparation, Labeling, Immunofluorescence, Two Tailed Test

Journal: Theranostics

Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

doi: 10.7150/thno.43198

Figure Lengend Snippet: miR-222-3p targets PDCD10 to repress cell migration by downregulating the Wnt/ β -catenin signaling pathway. (A) qPCR analysis of β -catenin and TCF4 expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( B ) Activity of the Wnt/ β -catenin signaling pathway in HEK-293T cells detected by the TOP flash/FOP flash dual-luciferase reporting system. HEK-293T cells were transfected with TOP flash/FOP flash plasmid and pRL-SV40 vector followed by transfection with or without PDCD10, and siRNA-NC/PDCD10, siRNA-03/PDCD10, or siRNA-04. Firefly luciferase activities were tested by a luminometer, and the activity of the Wnt/ β -catenin signaling pathway was recorded as TOP/FOP. ( C ) Western blot analysis of PDCD10 and β -catenin protein expression following overexpression of PDCD10 in HO 8910 PM cell fractions. β -actin was utilized as a marker for the cytosolic fractions. ( D ) HO 8910 PM cells transduced with or without PDCD10 were subjected to immunofluorescence with human-specific β -catenin antibodies. ( β -catenin in red). Bar, 10 µm. ( E ) β -catenin nuclei-intensity (detected by Operetta High-Content Imaging System (Perkin Elmer) in HO 8910 PM cell). ( F ) Western blot analysis of PDCD10, β -catenin translation levels in HO 8910 PM cells, or after coculturing with miR-ctrl mimic or miR-222-3p mimic in the presence or absence of PDCD10 (Left). The relative expression rate was calculated by Image J (Right). Data (A-B and E-F) represent the mean ± SD in different assays (n=3), *, P< 0.05; **, P< 0.01, determined by unpaired two-tailed t-test.

Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

Techniques: Migration, Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Over Expression, Marker, Transduction, Immunofluorescence, Imaging, Two Tailed Test

Journal: Theranostics

Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10

doi: 10.7150/thno.43198

Figure Lengend Snippet: SNAI2 increases cell migration via the SNAI2/miR-222-3p/PDCD10 axis and PDCD10-mediated promotion of EMT and Wnt/ β- catenin signaling. ( A ) Pearson's correlation scatter plots showed the fold changes of PDCD10 mRNA, miR-222-3p miRNA and SNAI2 mRNA levels in EOC tissues (n=38). ( B ) SNAI2 could enhance PDCD10 expression. PDCD10 mRNA expression levels were up-regulated after SNAI2 was overexpressed, but were down-regulated after SNAI2 was knocked down. ( C ) After transfection of SNAI2-overexpressing vectors and SNAI2 knockdown vectors (SNAI2 shRNA-01 and SNAI2 shRNA-02), the expression of PDCD10 was tested by Western blot in HO 8910 PM and SKOV3 cells. ( D ) Spearman correlation analysis of PDCD10, CDH1, VIM, SNAI2 and CTNNB1 levels in OC tissues (n=597) from the TCGA datasets. ( E ) A working model describing the interaction between SNAI2/miR-222-3p/PDCD10 during cancer metastasis. PDCD10 was critical for EOC cell mesenchymal movement and cell survival by maintaining low cell adhesion and high Wnt signaling. SNAI2 regulated PDCD10 by inhibiting pri-miR-222-3p expression and subsequent targeting of genes. SNAI2 regulated PDCD10 by inhibiting miR-222-3p expression, and further activated the downstream EMT signaling and Wnt/ β- catenin signaling to promote the expression of VIM, and β- catenin. These changes contributed to the EOC cell formation and migration, ultimately increasing tumor formation and metastasis. The data (B) represent the mean ± SD in different assays (n=3), **, P< 0.01; ***, P< 0.001, determined by unpaired two-tailed t-test.

Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.

Techniques: Migration, Expressing, Transfection, shRNA, Western Blot, Two Tailed Test

Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: Expression of PDCD10 in human pituitary adenomas. ( A ) A dataset (GSE26966) was obtained from the GEO database to compare the mRNA expression level of PDCD10 between PA tumor tissues (N=14) and normal pituitary tissue (N=9). ( B ) Relative mRNA expression levels of PDCD10 by RT-qPCR in invasive (n=15) and non-invasive (n=15) pituitary adenomas. Non-invasive values were set to 1. ( C ) Representative Western blots showed the expression level of PCDC10 protein (25 kDa) in invasive and noninvasive pituitary adenomas. Band intensities were quantified and normalized to GAPDH. ( D ) Representative images of immunohistochemistry evaluated the expression of PDCD10 in invasive and non-invasive (n=15) pituitary adenomas. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: PDCD10 silencing suppresses the proliferation, migration, invasion and EMT of PA cells. ( A ) Western blotting was performed to detect the impact of PDCD10 silencing on the expression of EMT markers in Att-20 cells and TtT/GF cells. ( B ) Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation capacity after PDCD10 silencing in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was used to examine the relative migration rates of Att-20 and TtT/GF cells (magnification:100x). ( G , H ) Transwell invasion assay was used to analyze the invasion potential of Att-20 and TtT/GF cells (magnification: 200x). * P < 0.05.

Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

Techniques: Migration, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: Overexpression of PDCD10 promotes the proliferation, migration, invasion and EMT of PA cells. ( A , B ) Western blotting was performed to detect the impact of PDCD10 overexpression on the expression levels of EMT markers in Att-20 cells and TtT/GF cells. Band intensities were quantified and normalized to GAPDH. ( C , D ) CCK-8 assay was used to assess cell proliferation potential after PDCD10 overexpression in Att-20 and TtT/GF cells. ( E , F ) Scratch assay was employed to examine the relative migration rates of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification:100x). ( G , H ) Transwell invasion assay was used to detect the invasion potential of Att-20 and TtT/GF cells after PDCD10 overexpression (magnification: 200x). * P < 0.05.

Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

Techniques: Over Expression, Migration, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: PDCD10 alters the protein expression level of CXCR2 and regulates the activation of downstream AKT/ERK signal pathways. ( A , B ) Western blotting was used to detect the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 in Att-20 cells after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05. ( C , D ) In TtT/GF cells, western blotting was performed to examine the expression level of CXCR2 and phosphorylation level of AKT, ERK1/2 and STAT3 after PDCD10 silencing or overexpression. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

Techniques: Expressing, Activation Assay, Western Blot, Over Expression

Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: Activation of CXCR2 rescues the inactivation of AKT/ERK signaling and the tumor-suppressive effects induced by PDCD10 silencing. ( A , B ) Western blotting was performed to examine the phosphorylation levels of AKT and ERK1/2 after recombinant CXCL2 administration (200ng/ml) in Att-20 and TtT/GF cells with PDCD10 silencing. ( C , D ) CCK-8 assay was used to evaluate cell proliferation potential after CXCL2 administration in Att-20 and TtT/GF cells with PDCD10 silencing. ( E , F ) Relative migration rate of ATT-20 and TtT/GF cells with PDCD10 silencing was analyzed by scratch assay after CXCL2 administration (magnification: 100x). ( G , H ) Transwell invasion assay was employed to assess the invasion capacity of Att-20 and TtT/GF cells with PDCD10 silencing after CXCL2 administration (magnification: 200x). * P < 0.05.

Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

Techniques: Activation Assay, Western Blot, Recombinant, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Invasion Assay

Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: PDCD10 silencing impairs the tumorigenesis and reduces CXCR2 expression of PA cells in vivo . ( A , B ) Images for Att-20 and TtT/GF xenografts from nude mice (Left). Statistical analysis of xenograft tumor weights (Right). ( C , D ) Representative IHC images of Att-20 and TtT/GF xenograft tumor tissues for PDCD10, CXCR2 and Ki-67 staining (200x). Scale bars: 100μm. ( E , F ) Intensity of PDCD10, CXCR2 and Ki-67 staining were analyzed by IHC-Profiler. ( G , H ) Western blotting was performed to examine the expression of CXCR2 in Att-20 and TtT/GF xenograft tumor samples. Band intensities were quantified and normalized to GAPDH. * P < 0.05.

Article Snippet: Antibodies to PDCD10, E-cadherin, N-cadherin and Vimentin (VIM) were purchased from Proteintech Group.

Techniques: Expressing, In Vivo, Staining, Western Blot