shrna pgpu6 plasmid negative control sh-nc  (Shanghai GenePharma)

Journal: Heliyon

Article Title: DNMT3A-mediated DNA methylation and transcription inhibition of FZD5 suppresses lung carcinogenesis

doi: 10.1016/j.heliyon.2024.e29733

Figure Lengend Snippet: Inhibition of FZD5 inhibits urethane-induced lung tumorigenesis in mice. C57BL/6 mice were challenged with urethane to induce lung tumorigenesis, followed by tail vein injection of lentiviral vectors-carried sh-FZD5 or sh-NC. A, FZD5 mRNA in the mouse lung tissues examined by RT-qPCR (n = 8); B, tumorigenesis rate in the mouse lung (n = 8); C, representative images of the mouse lung in each group; D, number of tumor nodules in the mouse lung (n = 8); E, pathological changes in the mouse lung examined by HE staining; F-G, expression of PCNA (F) and FZD5 (G) in the mouse lung tissues examined by the IHC assay (n = 8). Differences are compared by the unpaired t -test. * p < 0.05.

Article Snippet: After two months of urethane administration, artificial gene alteration was induced in the mice through tail vein injection of lentiviral vectors featuring the negative control (NC) plasmids (oe-NC or sh-NC), the overexpression DNMT3A or FZD plasmids (oe-DNMT3A/oe-FZD), or the short hairpin (sh) RNA of FZD5 (each one supplied by the VectorBuilder Inc., Guangzhou, Guangdong, China).

Techniques: Inhibition, Injection, Quantitative RT-PCR, Staining, Expressing

Journal: Heliyon

Article Title: DNMT3A-mediated DNA methylation and transcription inhibition of FZD5 suppresses lung carcinogenesis

doi: 10.1016/j.heliyon.2024.e29733

Figure Lengend Snippet: DNMT3A suppresses lung tumorigenesis in urethane-treated mice. C57BL/6 mice were challenged with urethane to induce lung tumorigenesis, followed by tail vein injection of lentiviral vectors-carried oe-DNMT3A/oe-NC and oe-FZD5. A-B, mRNA expression of DNMT3A (A) and FZD5 (B) in the mouse lung tissues examined by RT-qPCR (n = 8); C, tumorigenesis rate in the mouse lung (n = 8); D, representative images of the mouse lung in each group; E, number of tumor nodules in the mouse lung (n = 8); F, pathological changes in the mouse lung examined by HE staining; G, expression of FZD5 and PCNA in the mouse lung tissues examined by the IHC assay (n = 8). Differences are compared by the one-way ANOVA. * p < 0.05.

Article Snippet: After two months of urethane administration, artificial gene alteration was induced in the mice through tail vein injection of lentiviral vectors featuring the negative control (NC) plasmids (oe-NC or sh-NC), the overexpression DNMT3A or FZD plasmids (oe-DNMT3A/oe-FZD), or the short hairpin (sh) RNA of FZD5 (each one supplied by the VectorBuilder Inc., Guangzhou, Guangdong, China).

Techniques: Injection, Expressing, Quantitative RT-PCR, Staining

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 is upregulated in LUAD. GEPIA database exhibited (A) the MNX1 expression level in tumor tissues of LUAD and (B) the overall survival of LUAD patients with high or low expression of MNX1. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines (PC-9, A549, H1975 and H3255) were used to examine the (C) mRNA level and (D) protein expression of MNX1 using reverse transcription-quantitative PCR and western blotting, respectively. *P<0.05, **P<0.01, ***P<0.001 vs. BEAS-2B. MNX1, Motor neuron and pancreas homeobox 1; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: Short hairpin (sh) RNA (pGPU6 plasmid) targeting MNX1 (sh-MNX1-1, forward, 5′-CCGGGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGC-3′; sh-MNX1-2, forward, 5′-CCGGCGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCGTTTTTG-3′ and reverse, 5′-AATTCAAAAACGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCG-3′), pcDNA3.1 vector expressing MNX1 (oe-MNX1) or CCDC34 (oe-CCDC34) and the negative control (NC) of shRNA (sh-NC, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) and pcDNA3.1 (oe-NC) were obtained from Shanghai GenePharma Co., Ltd. A549 cells (1×10 5 ) were aliquoted into 6-well plates.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Gene Expression

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown represses proliferation, migration and invasion of A549 cells. Following transfection with sh-NC or sh-MNX1-1/sh-MNX1-2 in A549 cells, (A) the mRNA level and (B) protein expression of MNX1 were examined using reverse transcription-quantitative PCR and western blotting, respectively. (C) CCK-8 assay was used to examine cell viability. (D) Cell colony formation assay was conducted to assay formed colonies. (E-G) Wound-healing and Transwell assays were performed to examine cell migration and invasion, respectively. Magnification, ×100. (H) Protein expression of Ki67, PCNA, MMP2 and MMP9 was examined using western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; NC, negative control (empty vector); CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen.

Article Snippet: Short hairpin (sh) RNA (pGPU6 plasmid) targeting MNX1 (sh-MNX1-1, forward, 5′-CCGGGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGC-3′; sh-MNX1-2, forward, 5′-CCGGCGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCGTTTTTG-3′ and reverse, 5′-AATTCAAAAACGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCG-3′), pcDNA3.1 vector expressing MNX1 (oe-MNX1) or CCDC34 (oe-CCDC34) and the negative control (NC) of shRNA (sh-NC, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) and pcDNA3.1 (oe-NC) were obtained from Shanghai GenePharma Co., Ltd. A549 cells (1×10 5 ) were aliquoted into 6-well plates.

Techniques: Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Colony Assay, shRNA, Negative Control, Plasmid Preparation, Cell Counting

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown hinders cell cycle progression and promotes apoptosis of A549 cells. Following transfection with sh-NC or sh-MNX1 in A549 cells, (A) Cell cycle distribution was assessed using flow cytometry analysis. (B) Protein expression of cyclin B1 and p-Histone H2A.X was detected using western blotting. (C) Cell apoptosis rate was detected using flow cytometry analysis. (D) Protein expression of Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9 was detected using western blotting. **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; NC, negative control (empty vector); p-, phosphorylated.

Article Snippet: Short hairpin (sh) RNA (pGPU6 plasmid) targeting MNX1 (sh-MNX1-1, forward, 5′-CCGGGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGC-3′; sh-MNX1-2, forward, 5′-CCGGCGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCGTTTTTG-3′ and reverse, 5′-AATTCAAAAACGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCG-3′), pcDNA3.1 vector expressing MNX1 (oe-MNX1) or CCDC34 (oe-CCDC34) and the negative control (NC) of shRNA (sh-NC, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) and pcDNA3.1 (oe-NC) were obtained from Shanghai GenePharma Co., Ltd. A549 cells (1×10 5 ) were aliquoted into 6-well plates.

Techniques: Knockdown, Transfection, Flow Cytometry, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown inhibits tumor growth in vivo. An in vivo LUAD animal model was established and the (A) body weight and (B) tumor volume were monitored every 3 days. (C) After sacrifice, the tumors were taken out and images captured. (D) Tumor weight was recorded. (E) Protein expression of Ki67 and PCNA in tumor tissues was detected using western blotting. **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; LUAD, Lung adenocarcinoma; sh, short hairpin RNA; NC, negative control (empty vector); PCNA, proliferating cell nuclear antigen.

Article Snippet: Short hairpin (sh) RNA (pGPU6 plasmid) targeting MNX1 (sh-MNX1-1, forward, 5′-CCGGGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGC-3′; sh-MNX1-2, forward, 5′-CCGGCGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCGTTTTTG-3′ and reverse, 5′-AATTCAAAAACGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCG-3′), pcDNA3.1 vector expressing MNX1 (oe-MNX1) or CCDC34 (oe-CCDC34) and the negative control (NC) of shRNA (sh-NC, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) and pcDNA3.1 (oe-NC) were obtained from Shanghai GenePharma Co., Ltd. A549 cells (1×10 5 ) were aliquoted into 6-well plates.

Techniques: Knockdown, In Vivo, Animal Model, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 binds to CCDC34 promoter and transcriptionally regulates CCDC34 expression. (A) GEPIA database exhibited the CCDC34 expression level in tumor tissues of LUAD. *P<0.05. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines A549 were applied to examine the (B) mRNA level and (C) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. BEAS-2B. A549 cells were transfected with oe-NC or oe-MNX1 and the (D) mRNA level and (E) protein expression of MNX1 using reverse RT-qPCR and western blotting, respectively. A549 cells were transfected with oe-MNX1 or sh-MNX1 or their negative controls and the (F) mRNA level and (G) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. oe-NC; ## P<0.01, ### P<0.001 vs. sh-NC. (H) As predicted from JASPAR website, MNX1 might bind to CCDC34 promoter. (I) Luciferase reporter assay was performed to verify the interaction between MNX1 and CCDC34 promoter and the luciferase activity was examined using dual-luciferase reporter system. ***P<0.001 vs. oe-NC. (J) ChIP assay was used to verify the interaction between MNX1 and CCDC34 promoter. ***P<0.001 vs. anti-IgG. MNX1, Motor neuron and pancreas homeobox 1; CCDC34, coiled-coil domain-containing 34; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector); RT-qPCR, Reverse transcription-quantitative PCR; ChIP, chromatin immunoprecipitation.

Article Snippet: Short hairpin (sh) RNA (pGPU6 plasmid) targeting MNX1 (sh-MNX1-1, forward, 5′-CCGGGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGC-3′; sh-MNX1-2, forward, 5′-CCGGCGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCGTTTTTG-3′ and reverse, 5′-AATTCAAAAACGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCG-3′), pcDNA3.1 vector expressing MNX1 (oe-MNX1) or CCDC34 (oe-CCDC34) and the negative control (NC) of shRNA (sh-NC, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) and pcDNA3.1 (oe-NC) were obtained from Shanghai GenePharma Co., Ltd. A549 cells (1×10 5 ) were aliquoted into 6-well plates.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Reporter Assay, Activity Assay, Gene Expression, shRNA, Over Expression, Negative Control, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: CCDC34 overexpression abolishes the inhibitory effect of MNX1 knockdown on proliferation, migration and invasion in A549 cells. Following transfection with oe-NC or oe-CCDC34 in A549 cells, (A) the mRNA level and (B) protein expression of CCDC34 were examined using reverse transcription-quantitative PCR and western blotting, respectively. ***P<0.001 vs. oe-NC. (C) CCK-8 assay was used to examine cell viability. (D) Cell colony formation assay was conducted to assay formed colonies. (E-G) Wound-healing and Transwell assays were performed to examine cell migration and invasion, respectively. Magnification, ×100. (H) Protein expression of Ki67, PCNA, MMP2 and MMP9 was examined using western blot. *P<0.05, ***P<0.001 vs. sh-NC. ## P<0.01, ### P<0.001 vs. sh-MNX1+oe-NC. CCDC34, coiled-coil domain-containing 34; MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector); PCNA, proliferating cell nuclear antigen.

Article Snippet: Short hairpin (sh) RNA (pGPU6 plasmid) targeting MNX1 (sh-MNX1-1, forward, 5′-CCGGGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGC-3′; sh-MNX1-2, forward, 5′-CCGGCGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCGTTTTTG-3′ and reverse, 5′-AATTCAAAAACGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCG-3′), pcDNA3.1 vector expressing MNX1 (oe-MNX1) or CCDC34 (oe-CCDC34) and the negative control (NC) of shRNA (sh-NC, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) and pcDNA3.1 (oe-NC) were obtained from Shanghai GenePharma Co., Ltd. A549 cells (1×10 5 ) were aliquoted into 6-well plates.

Techniques: Over Expression, Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Colony Assay, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: CCDC34 overexpression abolishes the function of MNX1 knockdown on cell cycle and apoptosis in A549 cells. (A) Cell cycle distribution was assessed using flow cytometry analysis. (B) Protein expression of cyclin B1 and p-Histone H2A.X was detected using western blotting. (C) Cell apoptosis rate was detected using flow cytometry analysis. (D) Protein expression of Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9 was detected using western blotting. ***P<0.001 vs. sh-NC; # P<0.05, ### P<0.001 vs. sh-MNX1+oe-NC. CCDC34, coiled-coil domain-containing 34; MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector).

Article Snippet: Short hairpin (sh) RNA (pGPU6 plasmid) targeting MNX1 (sh-MNX1-1, forward, 5′-CCGGGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAGGAAGCGGAGAAACAGAACTCGAGTTCTGTTTCTCCGCTTCCTGC-3′; sh-MNX1-2, forward, 5′-CCGGCGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCGTTTTTG-3′ and reverse, 5′-AATTCAAAAACGAGGACGACGAGGACCATTTCTCGAGAAATGGTCCTCGTCGTCCTCG-3′), pcDNA3.1 vector expressing MNX1 (oe-MNX1) or CCDC34 (oe-CCDC34) and the negative control (NC) of shRNA (sh-NC, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) and pcDNA3.1 (oe-NC) were obtained from Shanghai GenePharma Co., Ltd. A549 cells (1×10 5 ) were aliquoted into 6-well plates.

Techniques: Over Expression, Knockdown, Flow Cytometry, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation