Journal: PLoS ONE

Article Title: A genome-wide enrichment screen identifies NUMA1-loss as a resistance mechanism against mitotic cell-death induced by BMI1 inhibition

doi: 10.1371/journal.pone.0227592

Figure Lengend Snippet: (A) Relative mRNA expression levels of BMI1 in HAP1 cells transduced with doxycycline-inducible shRNA constructs FH1t-Random (shRandom) or FH1t-BMI1 (shBMI1) upon 96 hours of doxycycline treatment. Error bars represent SD (n = 3). A two-tailed Student’s t-test was performed for statistical testing. (B) Protein expression levels of BMI1 in HAP1 cells transduced with shBMI1 after 0–96 hours of doxycycline treatment. Vinculin was used as a loading control. (C) Cell survival upon BMI1 knockdown was confirmed in HAP1 cells transduced with shBMI1 through a colony formation assay one week after plating with or without doxycycline treatment or (D) through relative cell counts 96 hours after plating with or without doxycycline. (E) Protein expression levels of HAP1 cells treated with increasing concentrations of PTC-318. Vinculin was used as a loading control. Values below the BMI1 blot represent the Vinculin-adjusted densities (relative to the DMSO treated cells). (F) Cell survival of HAP1 cells treated with PTC-318 through colony formation assay one week after treatment with DMSO (0.1%) or PTC-318 (40 nM) and (G) relative cell counts 48 hours after treatment with DMSO (0.1%) or PTC-318 (40 nM). Error bars represent SD. Student’s t-test was performed for statistical testing.

Article Snippet: For ectopic expression of BMI1, we used the BMI1-overexpression vector with FUGW vector backbone (FUGW-BMI1; Addgene 21577) Cells in 10-cm plates were transduced using 10 μg of FH1-tUTG-RNAi vector FUGW-BMI1, 3.5 μg VSV-G, 2.5 μg REV, and 5 μg pRRE in CaCl 2 (2.5M).

Techniques: Expressing, Transduction, shRNA, Construct, Two Tailed Test, Colony Assay

Journal: PLoS ONE

Article Title: A genome-wide enrichment screen identifies NUMA1-loss as a resistance mechanism against mitotic cell-death induced by BMI1 inhibition

doi: 10.1371/journal.pone.0227592

Figure Lengend Snippet: (A) BMI1 and GFP protein expression of wildtype HAP1 cells and HAP1 cells ectopically expressing BMI1 in the absence of 3’UTR through the transduced FUGW-BMI1 construct. (B) HAP1 cell survival relative to corresponding DMSO-control 48 hours after treatment with 20 nM PTC-318. Error bars represent SD (n = 3). A two-tailed Student’s t-test was performed for statistical testing. (C) Schematic overview showing insertional mutagenesis of HAP1 cells using a retroviral gene trap (left) and the enrichment screening process (right). (D) Bubble plot depicting genes enriched for unique gene-trap insertions in HAP1 cells treated with 40 nM PTC-318. The y-axis shows the significance and the x-axis the genes for which the gene-trap insertions were mapped in alphabetical order. The size of the bubbles correspond to the number of unique inactivating gene-trap insertions.

Article Snippet: For ectopic expression of BMI1, we used the BMI1-overexpression vector with FUGW vector backbone (FUGW-BMI1; Addgene 21577) Cells in 10-cm plates were transduced using 10 μg of FH1-tUTG-RNAi vector FUGW-BMI1, 3.5 μg VSV-G, 2.5 μg REV, and 5 μg pRRE in CaCl 2 (2.5M).

Techniques: Expressing, Construct, Two Tailed Test, Mutagenesis

Journal: PLoS ONE

Article Title: A genome-wide enrichment screen identifies NUMA1-loss as a resistance mechanism against mitotic cell-death induced by BMI1 inhibition

doi: 10.1371/journal.pone.0227592

Figure Lengend Snippet: (A) Protein expression of NUMA1 in populations of HAP1 cells transduced with a doxycycline-inducible shBMI1 construct in NUMA1-WT or NUMA1-KO backgrounds (NUMA1-KO1 and NUMA1-KO2 represent two separate clones). (B and C) Survival of the HAP1 NUMA1-WT and NUMA1-KO clones transduced with the inducible shBMI1 construct and maintained with or without doxycycline (B) colony formation and (C) cell viability. (D) Expression of NUMA1 protein in HAP1 NUMA1-WT and NUMA1-KO clones treated either with 0.1% DMSO or 40 nM of PTC-318. The survival of these cells was analyzed by (E) colony formation assay and (F) cell viability. (G) Protein expression of NUMA1 and BMI1 in NUMA1-WT cells transfected with CRISPR-Cas9 targeting BMI1, single-cell sorted, and expanded (left). (H) Percentages of each genetic background based on the respective protein expression (right). Error bars represent SD (n = 3). A two-tailed Student’s t-test was performed for statistical testing.

Article Snippet: For ectopic expression of BMI1, we used the BMI1-overexpression vector with FUGW vector backbone (FUGW-BMI1; Addgene 21577) Cells in 10-cm plates were transduced using 10 μg of FH1-tUTG-RNAi vector FUGW-BMI1, 3.5 μg VSV-G, 2.5 μg REV, and 5 μg pRRE in CaCl 2 (2.5M).

Techniques: Expressing, Transduction, Construct, Clone Assay, Colony Assay, Transfection, CRISPR, Two Tailed Test

Journal: PLoS ONE

Article Title: A genome-wide enrichment screen identifies NUMA1-loss as a resistance mechanism against mitotic cell-death induced by BMI1 inhibition

doi: 10.1371/journal.pone.0227592

Figure Lengend Snippet: (A) IC50 measurements of NSCLC cell lines h520, SK-MES-1, and SWI-1573. The x-axis shows the PTC-318 concentration in μM and the y-axis the percentage of surviving cells relative to non-treated control counterparts. (B) Colony formation of h520 and SK-MES-1 cells treated with either 0.1% DMSO or 40 nM (h520) or 80 nM (SK-MES-1) of PTC-318. (C) Percentages of h520 phospho-histone H3 populations after treatment with either 0.1% DMSO or 40 nM PTC-318 determined by flow cytometry quantification. (D) Percentages of h520 phospho-histone H3 populations after treatment with either 0.1% DMSO or 80 nM PTC-318 determined by flow cytometry quantification. Error bars represent SD (n = 3). A two-tailed Student’s t-test was performed for statistical testing. (E) Protein expression of NUMA1 and BMI1 in NUMA1-WT clones of an h520 population transfected with Cas9 targeting BMI1, single-cell sorted, and expanded.

Article Snippet: For ectopic expression of BMI1, we used the BMI1-overexpression vector with FUGW vector backbone (FUGW-BMI1; Addgene 21577) Cells in 10-cm plates were transduced using 10 μg of FH1-tUTG-RNAi vector FUGW-BMI1, 3.5 μg VSV-G, 2.5 μg REV, and 5 μg pRRE in CaCl 2 (2.5M).

Techniques: Concentration Assay, Flow Cytometry, Two Tailed Test, Expressing, Clone Assay, Transfection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: SIRT6 overexpression inhibits HIF1α expression and its impact on tumor angiogenesis in lung cancer

doi:

Figure Lengend Snippet: Effect of SIRT6 overexpression on HIF-1α and HIF-2α expression in cell line A549.

Article Snippet: Cell line A549 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, RPMI 1640 medium and fetal bovine serum (FBS) from Gibco, USA, Ad-null and Ad-SIRT6 from Suzhou GenePharma, Lipofectamine ™ 2000, Trizol from Invitrogen Corporation, penicillin/streptomycin from Hyclone USA, PrimeScript ™ RT-PCR kit, and SYBR ® Premix ExTaq ™ II kit from Japan TaKaRa company, HIF-1α, HIF-2α primers from Shanghai Bioengineering Co., Ltd., VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 detection kits from Shenzhen Jingmei Biotech Co., Ltd., PHD 1-3 detection kits from R&D Systems, USA, Fluorescence labeled CD34 mAb from Miltenyi Biotech, Germany, HIF-1α and VEGF monoclonal antibodies from Santa Cruz Biotech, and immunohistochemical and DAB reagent from Beijing Zhongshan Company.

Techniques: Over Expression, Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: SIRT6 overexpression inhibits HIF1α expression and its impact on tumor angiogenesis in lung cancer

doi:

Figure Lengend Snippet: Effect of SIRT6 overexpression on PHD1-3 expression in cell line A549. 2 Effect of SIRT6 overexpression on VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 expression in lung cancer cell line A549.

Article Snippet: Cell line A549 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, RPMI 1640 medium and fetal bovine serum (FBS) from Gibco, USA, Ad-null and Ad-SIRT6 from Suzhou GenePharma, Lipofectamine ™ 2000, Trizol from Invitrogen Corporation, penicillin/streptomycin from Hyclone USA, PrimeScript ™ RT-PCR kit, and SYBR ® Premix ExTaq ™ II kit from Japan TaKaRa company, HIF-1α, HIF-2α primers from Shanghai Bioengineering Co., Ltd., VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 detection kits from Shenzhen Jingmei Biotech Co., Ltd., PHD 1-3 detection kits from R&D Systems, USA, Fluorescence labeled CD34 mAb from Miltenyi Biotech, Germany, HIF-1α and VEGF monoclonal antibodies from Santa Cruz Biotech, and immunohistochemical and DAB reagent from Beijing Zhongshan Company.

Techniques: Over Expression, Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: SIRT6 overexpression inhibits HIF1α expression and its impact on tumor angiogenesis in lung cancer

doi:

Figure Lengend Snippet: Effect of SIRT6 overexpression on VEGF expression in lung cancer cell line A549.

Article Snippet: Cell line A549 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, RPMI 1640 medium and fetal bovine serum (FBS) from Gibco, USA, Ad-null and Ad-SIRT6 from Suzhou GenePharma, Lipofectamine ™ 2000, Trizol from Invitrogen Corporation, penicillin/streptomycin from Hyclone USA, PrimeScript ™ RT-PCR kit, and SYBR ® Premix ExTaq ™ II kit from Japan TaKaRa company, HIF-1α, HIF-2α primers from Shanghai Bioengineering Co., Ltd., VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 detection kits from Shenzhen Jingmei Biotech Co., Ltd., PHD 1-3 detection kits from R&D Systems, USA, Fluorescence labeled CD34 mAb from Miltenyi Biotech, Germany, HIF-1α and VEGF monoclonal antibodies from Santa Cruz Biotech, and immunohistochemical and DAB reagent from Beijing Zhongshan Company.

Techniques: Over Expression, Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: SIRT6 overexpression inhibits HIF1α expression and its impact on tumor angiogenesis in lung cancer

doi:

Figure Lengend Snippet: Effect of SIRT6 overexpression on the growth of xenografts in nude mice.

Article Snippet: Cell line A549 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, RPMI 1640 medium and fetal bovine serum (FBS) from Gibco, USA, Ad-null and Ad-SIRT6 from Suzhou GenePharma, Lipofectamine ™ 2000, Trizol from Invitrogen Corporation, penicillin/streptomycin from Hyclone USA, PrimeScript ™ RT-PCR kit, and SYBR ® Premix ExTaq ™ II kit from Japan TaKaRa company, HIF-1α, HIF-2α primers from Shanghai Bioengineering Co., Ltd., VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 detection kits from Shenzhen Jingmei Biotech Co., Ltd., PHD 1-3 detection kits from R&D Systems, USA, Fluorescence labeled CD34 mAb from Miltenyi Biotech, Germany, HIF-1α and VEGF monoclonal antibodies from Santa Cruz Biotech, and immunohistochemical and DAB reagent from Beijing Zhongshan Company.

Techniques: Over Expression

Journal: International Journal of Clinical and Experimental Pathology

Article Title: SIRT6 overexpression inhibits HIF1α expression and its impact on tumor angiogenesis in lung cancer

doi:

Figure Lengend Snippet: Effect of SIRT6 overexpression on MVD level of xenografts in nude mice.

Article Snippet: Cell line A549 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, RPMI 1640 medium and fetal bovine serum (FBS) from Gibco, USA, Ad-null and Ad-SIRT6 from Suzhou GenePharma, Lipofectamine ™ 2000, Trizol from Invitrogen Corporation, penicillin/streptomycin from Hyclone USA, PrimeScript ™ RT-PCR kit, and SYBR ® Premix ExTaq ™ II kit from Japan TaKaRa company, HIF-1α, HIF-2α primers from Shanghai Bioengineering Co., Ltd., VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 detection kits from Shenzhen Jingmei Biotech Co., Ltd., PHD 1-3 detection kits from R&D Systems, USA, Fluorescence labeled CD34 mAb from Miltenyi Biotech, Germany, HIF-1α and VEGF monoclonal antibodies from Santa Cruz Biotech, and immunohistochemical and DAB reagent from Beijing Zhongshan Company.

Techniques: Over Expression

Journal: International Journal of Clinical and Experimental Pathology

Article Title: SIRT6 overexpression inhibits HIF1α expression and its impact on tumor angiogenesis in lung cancer

doi:

Figure Lengend Snippet: SIRT6 overexpression inhibits xenograft angiogenesis-related factors in A549 nude mice.

Article Snippet: Cell line A549 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, RPMI 1640 medium and fetal bovine serum (FBS) from Gibco, USA, Ad-null and Ad-SIRT6 from Suzhou GenePharma, Lipofectamine ™ 2000, Trizol from Invitrogen Corporation, penicillin/streptomycin from Hyclone USA, PrimeScript ™ RT-PCR kit, and SYBR ® Premix ExTaq ™ II kit from Japan TaKaRa company, HIF-1α, HIF-2α primers from Shanghai Bioengineering Co., Ltd., VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 detection kits from Shenzhen Jingmei Biotech Co., Ltd., PHD 1-3 detection kits from R&D Systems, USA, Fluorescence labeled CD34 mAb from Miltenyi Biotech, Germany, HIF-1α and VEGF monoclonal antibodies from Santa Cruz Biotech, and immunohistochemical and DAB reagent from Beijing Zhongshan Company.

Techniques: Over Expression

Journal: International Journal of Clinical and Experimental Pathology

Article Title: SIRT6 overexpression inhibits HIF1α expression and its impact on tumor angiogenesis in lung cancer

doi:

Figure Lengend Snippet: Immunohistochemical staining of xenograft HIF-1α and VEGF in nude mice with SIRT6 overexpression.

Article Snippet: Cell line A549 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, RPMI 1640 medium and fetal bovine serum (FBS) from Gibco, USA, Ad-null and Ad-SIRT6 from Suzhou GenePharma, Lipofectamine ™ 2000, Trizol from Invitrogen Corporation, penicillin/streptomycin from Hyclone USA, PrimeScript ™ RT-PCR kit, and SYBR ® Premix ExTaq ™ II kit from Japan TaKaRa company, HIF-1α, HIF-2α primers from Shanghai Bioengineering Co., Ltd., VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 detection kits from Shenzhen Jingmei Biotech Co., Ltd., PHD 1-3 detection kits from R&D Systems, USA, Fluorescence labeled CD34 mAb from Miltenyi Biotech, Germany, HIF-1α and VEGF monoclonal antibodies from Santa Cruz Biotech, and immunohistochemical and DAB reagent from Beijing Zhongshan Company.

Techniques: Immunohistochemical staining, Staining, Over Expression

Journal: Oncogenesis

Article Title: Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells

doi: 10.1038/oncsis.2016.6

Figure Lengend Snippet: Transfection of Bmi1-siRNA suppresses the proliferation, colony formation, invasion and cell cycle progression of WB-F344 cells in vitro . The qRT–PCR analysis revealed that the mRNA expression levels of Bmi1 in Bmi1-siRNA WB-F344 cells were significantly downregulated ( a ). Similar results were obtained through western blot analysis ( b ). The expression of H2AK119 ub was downregulated in WB-F344 cells by Bmi1-siRNA ( b ). Downregulation of Bmi1 expression significantly inhibited the proliferation of WB-F344 cells ( c ). The number of colonies was lower in the Bmi1-siRNA WB-F344 cells group than in the control group ( d and e ). Downregulation of Bmi1 expression significantly impaired the invasion of WB-F344 cells ( f ). The number of WB-F344 cells that migrated through the filter was markedly lower in the Bmi1-siRNA group than in the control group ( g ). Downregulation of Bmi1 reduced number of WB-F344 cells in the S phase of cell cycle ( h ). * P <0.05, ** P <0.01.

Article Snippet: A validated siRNA targeting Bmi1 and the negative control (listed in ) were purchased from GenePharma (Shanghai, China).

Techniques: Transfection, In Vitro, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Oncogenesis

Article Title: Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells

doi: 10.1038/oncsis.2016.6

Figure Lengend Snippet: Stable forced Bmi1 expression enhanced the proliferation, colony formation, invasion and cell cycle progression of WB-F344 cells in vitro . The qRT–PCR analysis revealed that the mRNA expression levels of Bmi1 were increased in forced Bmi1 expression WB-F344 cells compared with control cells ( a ). Similar results were obtained through western blot analysis (passages 15 and 30) ( b ). Upregulation of Bmi1 enhanced the proliferation ( c ), colony-formation ability ( d , e ) and invasion ( f ) of WB-F344 cells. The number of WB-F344 cells that migrated through the filter was markedly higher in the forced Bmi1 expression group than in the control group ( g ). Forced expression of Bmi1 increased the number of WB-F344 cells in the S phase of cell cycle ( h ). * P <0.05, ** P <0.01.

Article Snippet: A validated siRNA targeting Bmi1 and the negative control (listed in ) were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Control, Western Blot

Journal: Oncogenesis

Article Title: Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells

doi: 10.1038/oncsis.2016.6

Figure Lengend Snippet: Forced Bmi1 expression WB-F344 cells possess properties of CSCs. Immunofluorescence staining revealed that the expression of c-Myc was much higher in forced Bmi1 expression WB-F344 cells than in controls, and the presence of OV6 was verified in forced Bmi1 expression WB-F344 cells ( a ). The activity of ALDH in forced Bmi1 expression WB-F344 cells and the control cells were measured with an AldeflourTM assay with and without DEAB (the specific inhibitor of ALDH) ( b ). ** P <0.01.

Article Snippet: A validated siRNA targeting Bmi1 and the negative control (listed in ) were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Immunofluorescence, Staining, Activity Assay, Control

Journal: Oncogenesis

Article Title: Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells

doi: 10.1038/oncsis.2016.6

Figure Lengend Snippet: Bmi1 drives malignant transformation of WB-F344 cells in vivo . Forced Bmi1 expression WB-F344 cells showed a piled-up appearance in culture ( a ). Forced Bmi1 expression WB-F344 cells generated tumors on the right flanks of every tested nude mouse ( b , black arrow), whereas no tumors were observed on the left flanks injected with negative control cells. The tumor volume of the recipient mice is presented ( c ). The histological features of the tumors corresponded to poorly differentiated HCC ( d ). The expression levels of Bmi1, AFP and albumin were much higher in subcutaneous tumors, whereas CK19 was not expressed in subcutaneous tumors ( d ). The expression level of p16Ink4a in Bmi1-siRNA WB-F344 cells and forced Bmi1 expression WB-F344 cells were analyzed by western blot ( e ). The expression of p16Ink4a in subcutaneous tumors was detected by immunohistochemistry ( f ). Magnification: × 200. * P <0.05, ** P <0.01.

Article Snippet: A validated siRNA targeting Bmi1 and the negative control (listed in ) were purchased from GenePharma (Shanghai, China).

Techniques: Transformation Assay, In Vivo, Expressing, Generated, Injection, Negative Control, Western Blot, Immunohistochemistry

Journal: Oncogenesis

Article Title: Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells

doi: 10.1038/oncsis.2016.6

Figure Lengend Snippet: Sequences of siRNAs for Bmi1

Article Snippet: A validated siRNA targeting Bmi1 and the negative control (listed in ) were purchased from GenePharma (Shanghai, China).

Techniques: Sequencing, Control

Journal: Oncogenesis

Article Title: Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells

doi: 10.1038/oncsis.2016.6

Figure Lengend Snippet: Primers for Bmi1 and reference genes

Article Snippet: A validated siRNA targeting Bmi1 and the negative control (listed in ) were purchased from GenePharma (Shanghai, China).

Techniques: Sequencing

Journal: Animal Cells and Systems

Article Title: miR-122 promotes diabetic retinopathy through targeting TIMP3

doi: 10.1080/19768354.2020.1816580

Figure Lengend Snippet: TIMP3 was a direct target of miR-122 (A) The miR-122 wild-type and mutant binding sites in the 3′-UTR of TIMP3 were shown ( B) Luciferase assay was conducted to verify the relationship between miR-122 and TIMP3 ( C) The level of TIMP3 in ARPE-19 cells treated with high glucose and transfected with miR-122 inhibitor was determined by western blot. **: p < 0.01. ##: p < 0.01.

Article Snippet: The miR-122 mimic, negative control mimic (NC mimic), negative control inhibitor (NC inh), miR-122 inhibitor (miR-122 inh), and TIMP3 overexpression plasmid were obtained from Ribobio (Ribobio, Guangzhou, China).

Techniques: Mutagenesis, Binding Assay, Luciferase, Transfection, Western Blot

Journal: Animal Cells and Systems

Article Title: miR-122 promotes diabetic retinopathy through targeting TIMP3

doi: 10.1080/19768354.2020.1816580

Figure Lengend Snippet: miR-122 promoted cell apoptosis of high glucose-induced ARPE-19 cells by regulating TIMP3 (A) The cell viability of ARPE-19 cells treated with high glucose and transfected with miR-122 mimic or TIMP3 overexpression plasmid was measured by MTT assay ( B) The levels of TIMP3, Bcl-2, and cleaved caspase-3 in ARPE-19 cells treated with high glucose and transfected with the miR-122 mimic, or TIMP3 overexpression plasmid were determined using western blot. *: p < 0.05. **: p < 0.01. #: p < 0.05. ##: p < 0.01.

Article Snippet: The miR-122 mimic, negative control mimic (NC mimic), negative control inhibitor (NC inh), miR-122 inhibitor (miR-122 inh), and TIMP3 overexpression plasmid were obtained from Ribobio (Ribobio, Guangzhou, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, MTT Assay, Western Blot

Journal: American Journal of Cancer Research

Article Title: MicroRNA-337 regulates the PI3K/AKT and Wnt/β-catenin signaling pathways to inhibit hepatocellular carcinoma progression by targeting high-mobility group AT-hook 2

doi:

Figure Lengend Snippet: Ectopic expression of miR-337 inhibits cell proliferation and induces apoptosis in HCC. A. HepG2 and Hep3B cells transfected with miR-337 mimics or miR-NC were subjected to RT-qPCR for the detection of miR-337 expression. *P<0.05 compared with miR-NC. B. Proliferative activity of HepG2 and Hep3B cells was determined by CCK-8 assay following transfection of miR-337 mimics or miR-NC. *P<0.05 compared with miR-NC. C. The effect of miR-337 overexpression on the colony formation of HepG2 and Hep3B cells was evaluated using colony formation assay. *P<0.05 compared with miR-NC. D. Cell apoptosis rate was detected in HepG2 and Hep3B cells transfected with miR-337 mimics or miR-NC with an Annexin V-FITC apoptosis detection kit. *P<0.05 compared with miR-NC.

Article Snippet: MiR-337 mimics, miRNA mimics negative control (miR-NC), small interfering RNA (siRNA) against the expression of HMGA2 (HMGA2 siRNA), and negative control siRNA (NC siRNA) were chemically synthesized by RiboBio (Guangzhou China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Activity Assay, CCK-8 Assay, Over Expression, Colony Assay

Journal: American Journal of Cancer Research

Article Title: MicroRNA-337 regulates the PI3K/AKT and Wnt/β-catenin signaling pathways to inhibit hepatocellular carcinoma progression by targeting high-mobility group AT-hook 2

doi:

Figure Lengend Snippet: MiR-337 overexpression decreases HCC cell migration and invasion. A, B. Migration and invasion of HepG2 and Hep3B cells were investigated using Transwell migration and invasion assays following transfection of miR-337 mimics or miR-NC. *P<0.05 compared with miR-NC.

Article Snippet: MiR-337 mimics, miRNA mimics negative control (miR-NC), small interfering RNA (siRNA) against the expression of HMGA2 (HMGA2 siRNA), and negative control siRNA (NC siRNA) were chemically synthesized by RiboBio (Guangzhou China).

Techniques: Over Expression, Migration, Transfection

Journal: American Journal of Cancer Research

Article Title: MicroRNA-337 regulates the PI3K/AKT and Wnt/β-catenin signaling pathways to inhibit hepatocellular carcinoma progression by targeting high-mobility group AT-hook 2

doi:

Figure Lengend Snippet: HMGA2 is a direct target of miR-337 in HCC. A. MiR-337 and its putative binding sequences in the 3’-UTR of HMGA2. Mutation was generated on the 3’-UTR of HMGA2 in the complementary sites for the seed region of miR-337. B. HepG2 and Hep3B cells were cotransfected with miR-337 mimics or miR-NC and wild-type or mutant HMGA2 3’-UTR. The relative firefly luciferase activity was determined at 48 h posttransfection. *P<0.05 compared with miR-NC. C, D. HepG2 and Hep3B cells that were transfected with miR-337 mimics or miR-NC were subjected to RT-qPCR and Western blot analysis for detection of HMGA2 mRNA and protein expression, respectively. *P<0.05 compared with miR-NC.

Article Snippet: MiR-337 mimics, miRNA mimics negative control (miR-NC), small interfering RNA (siRNA) against the expression of HMGA2 (HMGA2 siRNA), and negative control siRNA (NC siRNA) were chemically synthesized by RiboBio (Guangzhou China).

Techniques: Binding Assay, Mutagenesis, Generated, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: MicroRNA-337 regulates the PI3K/AKT and Wnt/β-catenin signaling pathways to inhibit hepatocellular carcinoma progression by targeting high-mobility group AT-hook 2

doi:

Figure Lengend Snippet: Re-expression of HMGA2 reverses the tumor-suppressing effects of miR-337 on HCC cells. HepG2 and Hep3B cells were transfected with miR-337 mimics along with pcDNA3.1 or pcDNA3.1-HMGA2 lacking the respective 3’-UTR. A. Expression level of HMGA2 protein was analyzed by Western blot analysis in differently treated cells. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. B. CCK-8 assay was carried out to analyze cell proliferation in indicated cells. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. C. Representative images and quantification of colony formation assay in indicated cells. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. D, E. Transwell migration and invasion assays were employed to assess cell migration and invasion in indicated cells, respectively. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. F. Cell apoptosis in differently treated cells was quantified by flow cytometry analysis. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2.

Article Snippet: MiR-337 mimics, miRNA mimics negative control (miR-NC), small interfering RNA (siRNA) against the expression of HMGA2 (HMGA2 siRNA), and negative control siRNA (NC siRNA) were chemically synthesized by RiboBio (Guangzhou China).

Techniques: Expressing, Transfection, Western Blot, CCK-8 Assay, Colony Assay, Migration, Flow Cytometry

Journal: American Journal of Cancer Research

Article Title: MicroRNA-337 regulates the PI3K/AKT and Wnt/β-catenin signaling pathways to inhibit hepatocellular carcinoma progression by targeting high-mobility group AT-hook 2

doi:

Figure Lengend Snippet: MiR-337 reduces HCC tumor growth in vivo. A. MiR-337 expression was determined in tumor xenografts using RT-qPCR. *P<0.05 compared with miR-NC. B. Mice were sacrificed, and representative images of the miR-337 mimics and miR-NC xenograft tumors were obtained. C. Tumor volume was calculated every 2 days after inoculation for 2 weeks. *P<0.05 compared with miR-NC. D. Tumor weight in the different groups. *P<0.05 compared with miR-NC. E. Western blot analysis was performed to detect HMGA2, AKT, p-AKT, β-catenin, and p-β-catenin expression in xenograft tumor tissues of the miR-337 and miR-NC groups.

Article Snippet: MiR-337 mimics, miRNA mimics negative control (miR-NC), small interfering RNA (siRNA) against the expression of HMGA2 (HMGA2 siRNA), and negative control siRNA (NC siRNA) were chemically synthesized by RiboBio (Guangzhou China).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-19a-3p inhibits the cellular proliferation and invasion of non-small cell lung cancer by downregulating UBAP2L

doi: 10.3892/etm.2020.8926

Figure Lengend Snippet: UBAP2L is a direct target of miR-19a-3p. (A) Schematic representation of the miR-19a-3p target sequence with the 3'-UTR of UBAP2L. The mutant 3'-UTR of UBAP2L is also presented. (B) A dual-luciferase reporter assay was performed using A549 and H1299 cells. (C) The expression of UBAP2L mRNA in A549 and H1299 cells transfected with miR-19a-3p mimics or miR-NC was detected using a reverse transcription-quantitative polymerase chain reaction assay. (D) UBAP2L protein levels were detected using Western blotting. Data are presented as the mean ± standard deviation from triplicate experiments. **P<0.01 and ***P<0.001 vs. miR-NC. UBAP2L, ubiquitin associated protein 2 like; UTR, untranslated region; miR, miRNA; NC, negative control; WT, wild type; MUT, mutant.

Article Snippet: All cell lines were maintained in 90% media supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO 2 humidified environment at 37 ̊C. miR-19a-3p mimics, small interfering (si)RNA for UBAP2L (siUBAP2L, cat. no. 3624), the UBAP2L overexpression plasmid (pcDNA3.1-UBAP2L; cat. no. 1302) and their corresponding negative controls (NC; cat. no. 4126; miR-NC; cat. no. 3106, siNC; cat. no. 2036 and pcDNA3.1; cat. no. 1987, respectively) were purchased from Shanghai GenePharma Co., Ltd., (Shanghai, China).

Techniques: Sequencing, Mutagenesis, Luciferase, Reporter Assay, Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Ubiquitin Proteomics, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-19a-3p inhibits the cellular proliferation and invasion of non-small cell lung cancer by downregulating UBAP2L

doi: 10.3892/etm.2020.8926

Figure Lengend Snippet: UBAP2L knockdown suppressed NSCLC cell proliferation, migration and invasion. (A) Pearson's correlation analysis was used to determine the association between miR-19a-3p and UBAP2L mRNA expression levels in NSCLC tissues. A549 cells were transfected with siUBAP2L or siNC for 48 h and (B) UBAP2L protein expression was determined by western blot analysis. (C) A549 cell proliferation was determined using an MTT assay. (D) Wound healing and (E) Transwell Matrigel invasion assays were performed to assess A549 cell migration and invasion. Data are presented as the mean ± standard deviation from triplicate experiments. **P<0.01 and ***P<0.001 vs. siNC. UBAP2L, ubiquitin associated protein 2 like; NSCLC, non-small cell lung cancer; miR, miRNA; si, small interfering; NC, negative control; OD, optical density.

Article Snippet: All cell lines were maintained in 90% media supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO 2 humidified environment at 37 ̊C. miR-19a-3p mimics, small interfering (si)RNA for UBAP2L (siUBAP2L, cat. no. 3624), the UBAP2L overexpression plasmid (pcDNA3.1-UBAP2L; cat. no. 1302) and their corresponding negative controls (NC; cat. no. 4126; miR-NC; cat. no. 3106, siNC; cat. no. 2036 and pcDNA3.1; cat. no. 1987, respectively) were purchased from Shanghai GenePharma Co., Ltd., (Shanghai, China).

Techniques: Knockdown, Migration, Expressing, Transfection, Western Blot, MTT Assay, Standard Deviation, Ubiquitin Proteomics, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-19a-3p inhibits the cellular proliferation and invasion of non-small cell lung cancer by downregulating UBAP2L

doi: 10.3892/etm.2020.8926

Figure Lengend Snippet: UBAP2L upregulation partially reverses the effects of miR-19a-3p on NSCLC cell proliferation, migration and invasion. A549 cells were transfected with pcDNA3.1 or pcDNA3.1 + UBAP2L expression plasmids, which in turn were co-transfected with the miR-19a-3p expression plasmid. (A) Western blotting was performed to assess UBAP2L protein expression. GAPDH was used as a loading control. (B) MTT, (C) wound healing and (D) Transwell Matrigel invasion assays were performed to determine A549 cell proliferation, migration and invasion. Data are presented as the mean ± standard deviation from triplicate experiments. *P<0.05 and ***P<0.001 vs. miR-19a-3p mimics + pcDNA3.1. UBAP2L, ubiquitin associated protein 2 like; miR, miRNA; NSCLC, non-small cell lung cancer; optical density.

Article Snippet: All cell lines were maintained in 90% media supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO 2 humidified environment at 37 ̊C. miR-19a-3p mimics, small interfering (si)RNA for UBAP2L (siUBAP2L, cat. no. 3624), the UBAP2L overexpression plasmid (pcDNA3.1-UBAP2L; cat. no. 1302) and their corresponding negative controls (NC; cat. no. 4126; miR-NC; cat. no. 3106, siNC; cat. no. 2036 and pcDNA3.1; cat. no. 1987, respectively) were purchased from Shanghai GenePharma Co., Ltd., (Shanghai, China).

Techniques: Migration, Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Standard Deviation, Ubiquitin Proteomics