Journal: Cancers

Article Title: MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion

doi: 10.3390/cancers12123518

Figure Lengend Snippet: Colony formation assay: ( A – C ), number of colonies—overexpression of miR-215-5p significantly reduced the number of colonies in HCT-116 and RKO cells and increased the number of colonies in HCT-15 cells. ( D – F ), the colonies’ diameter—overexpression of miR-215-5p significantly reduced the diameter of colonies in the case of HCT-116, RKO, and HCT-15 cells. ( G – I ), representative pictures of colonies stained with crystal violet. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For functional studies, we stably transfected cell lines HCT-116, RKO, and HCT-15 using TurboFectin 8.0 and pCMV-miR-215 vector (OriGene Technologies, Rockville, MD, USA, CAT#: SC400278) and empty pCMV-MIR vector (CAT#: PCMVMIR).

Techniques: Colony Assay, Over Expression, Staining

Journal: Cancers

Article Title: MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion

doi: 10.3390/cancers12123518

Figure Lengend Snippet: Transwell migration/invasion assay: ( A – C ) graphs of migrating cells per view, and representative pictures of migrated cells stained with Hoechst 33342. Overexpression of miR-215-5p significantly reduced the number of migrating HCT-15 cells. ( D – F ), and graphs of invading cells per view and representative pictures of invaded cells stained with Hoechst 33342. Overexpression of miR-215-5p significantly reduced the number of invading HCT-15 cells and visibly reduced the number of invading HCT-116 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For functional studies, we stably transfected cell lines HCT-116, RKO, and HCT-15 using TurboFectin 8.0 and pCMV-miR-215 vector (OriGene Technologies, Rockville, MD, USA, CAT#: SC400278) and empty pCMV-MIR vector (CAT#: PCMVMIR).

Techniques: Migration, Invasion Assay, Staining, Over Expression

Journal: Cancers

Article Title: MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion

doi: 10.3390/cancers12123518

Figure Lengend Snippet: Tumorigenicity assay: ( A – C ), graphs of tumor weights—overexpression of miR-215-5p significantly reduced tumor weight in the case of HCT-116 and HCT-15 cells, and a similar trend was observed in the case of RKO cells. ( D – F ), graphs of tumor volumes—overexpression of miR-215-5p significantly reduced tumor volume in the case of RKO and HCT-15 cells, cell line HCT-116 showed a similar trend. ( G – I ), graphs of normalized (RNU48) expression of miR-215-5p in tumors. Expression of miR-215-5p remained significantly increased in HCT-116, RKO, and HCT-15 tumors. ( J – L ), pictures of removed control/miR-215-5p tumors and representative pictures of subcutaneous control/miR-215-5p tumors in the animal model. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For functional studies, we stably transfected cell lines HCT-116, RKO, and HCT-15 using TurboFectin 8.0 and pCMV-miR-215 vector (OriGene Technologies, Rockville, MD, USA, CAT#: SC400278) and empty pCMV-MIR vector (CAT#: PCMVMIR).

Techniques: Tumorigenicity Assay, Over Expression, Expressing, Animal Model

Journal: Cancers

Article Title: MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion

doi: 10.3390/cancers12123518

Figure Lengend Snippet: Transcriptome profiling: ( A ) Vulcano plot with the top ten most significantly deregulated mRNAs in HCT-15-miR-215-5p cells in comparison to control HCT-15 cells. ( B ) hierarchical clustrogram and heatmap of the top fifty deregulated mRNAs between HCT-15-miR-215-5p cells (red: miR-215-5p) and control HCT-15 cells (blue: mock). Genes clustered using unsupervised clustering. ( C ) KEGG pathway analysis—the top ten signaling pathways regulated by miR-215-5p in HCT-15 cells. ( D ) Gene Ontology overrepresentation analysis—top 10 overrepresented Gene Ontology terms (across all three sub-ontologies) regulated by miR-215-5p in HCT-15 cells.

Article Snippet: For functional studies, we stably transfected cell lines HCT-116, RKO, and HCT-15 using TurboFectin 8.0 and pCMV-miR-215 vector (OriGene Technologies, Rockville, MD, USA, CAT#: SC400278) and empty pCMV-MIR vector (CAT#: PCMVMIR).

Techniques:

Journal: Cancers

Article Title: MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion

doi: 10.3390/cancers12123518

Figure Lengend Snippet: Validation of sequencing data by RT-qPCR. ( A ) Expression analyses of subcutaneous tumors generated in NSG mice using HCT-15 cells stably transfected with miR-215-5p and control cells. ( B ) Expression analyses of healthy colon tissue, primary tumor, and metastatic liver tissue of CRC patients. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For functional studies, we stably transfected cell lines HCT-116, RKO, and HCT-15 using TurboFectin 8.0 and pCMV-miR-215 vector (OriGene Technologies, Rockville, MD, USA, CAT#: SC400278) and empty pCMV-MIR vector (CAT#: PCMVMIR).

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Generated, Stable Transfection, Transfection

Journal: British Journal of Cancer

Article Title: FAT1: a potential target for monoclonal antibody therapy in colon cancer

doi: 10.1038/bjc.2016.145

Figure Lengend Snippet: mAb198.3 efficacy in colon cancer xenografts. ( A ) Preventive model. Athymic mice were engrafted subcutaneously with HCT15 cells and treated intravenously two times a week with 12 mg mAb per kg of mAb198.3 or isotype control mAb, or untreated. ( B ) Therapeutic model. mAb198.3 was administered repeatedly in athymic mice bearing either HCT15 or HT29 xenograft of ∼100 mm 3 . Median tumour volumes of each mAb-treated and -untreated mouse group are shown.

Article Snippet: HCT15 or HT29 human colon carcinoma cells (5 × 10 6 ) were injected subcutaneously into the right flank of nude athymic mice (Harlan Laboratories; 8 mice per group).

Techniques: Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B was downregulated in colorectal cancer (CRC) tissues and cells. (A,B) A microarray profile revealed that circFAM120B was a differently expressed circRNA that was significantly downregulated in CRC tissues ( n = 10). (C) The expression of circFAM120B in tumor tissues ( n = 50) and normal tissues ( n = 50) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). (D) The expression of circFAM120B in LoVo, HCT15, and NCM460 cells was detected by qRT-PCR. (E,F) LoVo and HCT15 cells were exposed to actinomycin D to test the stability of circFAM120B. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

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

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B overexpression weakened cell proliferation, migration/invasion and glycolysis. In circFAM120B-overexpressed LoVo and HCT15 cells, (A–C) cell proliferation was assessed by CCK-8 assay and colony formation assay. (D,E) Cell migration and invasion were determined by transwell assay (magnification: 100×). (F,G) Extracellular acidification rate (ECAR) was measured by glycolysis stress test to evaluate glycolysis metabolism. (H,I) Lactate production and glucose consumption were quantified to assess glycolysis progression using corresponding kits. (J,K) Glycolysis-related markers, HK2 and LDHA, were quantified by western blot to observe glycolysis. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Over Expression, Migration, CCK-8 Assay, Colony Assay, Transwell Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: MiR-645 was a target of circFAM120B. (A,B) Biotinylated-circFAM120B probe could significantly enrich the level of miR-645. (C) Wild-type circFAM120B sequence containing miR-645 binding sites was mutated to generate mutant-type circFAM120B sequence for dual-luciferase reporter assay. (D,E) Dual-luciferase reporter assay was performed to further validate the relationship between miR-645 and circFAM120B. (F) RIP assay was performed to validate the relationship between miR-645 and circFAM120B. (G) The expression of miR-645 in tumor tissues ( n = 50) and normal tissues ( n = 50) was detected by qRT-PCR. (H) The expression of miR-645 in LoVo, HCT15, and NCM460 cells was detected by qRT-PCR. (I) The expression of miR-645 in LoVo and HCT15 cells with circFAM120B overexpression was detected by qRT-PCR. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B blocked CRC progression in vitro by targeting miR-645. LoVo and HCT15 cells were transfected with oe-circFAM120B alone or oe-circFAM120B + miR-645, with vector or oe-circFAM120B + miR-NC as a control. (A) The expression of miR-645 in these transfected cells was detected by qRT-PCR. (B,C) Cell proliferation was assessed by CCK-8 assay. (D) Cell proliferation was assessed by colony formation assay. (E,F) Cell migration and invasion were monitored by transwell assay (magnification: 100×). (G,H) Glycolysis was evaluated by ECAR using glycolysis stress test. (I,J) Lactate production and glucose consumption were quantified to monitor glycolysis. (K,L) The expression of HK2 and LDHA was quantified by western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: In Vitro, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Migration, Transwell Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: TGFBR2 was a target of miR-645. (A) Wild-type TGFBR2 3′UTR sequence containing miR-645 binding sites was mutated to generate mutant-type TGFBR2 3′UTR sequence for dual-luciferase reporter assay. (B,C) Dual-luciferase reporter assay was conducted to verify the interaction between miR-645 and TGFBR2. (D) RIP assay was conducted to verify the interaction between miR-645 and TGFBR2. (E,F) The expression of TGFBR2 suppressed by miR-645 restoration was detected by qRT-PCR and western blot. (G,H) The expression of TGFBR2 in tumor tissues ( n = 50) and normal tissues ( n = 50) was detected by qRT-PCR and western blot. (I,J) The expression of TGFBR2 in LoVo, HCT15 and NCM460 cells was detected by qRT-PCR and western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: miR-645 promoted CRC progression in vitro by mediating TGFBR2. LoVo and HCT15 cells were transfected with anti-miR-645 or anti-miR-645 + si-TGFBR2, with anti-miR-NC or anti-miR-645 + si-NC as the corresponding control. (A,B) The expression of TGFBR2 was examined using qRT-PCR and western blot. (C–E) Cell proliferation was investigated using CCK-8 assay and colony formation assay. (F,G) Cell migration and invasion were monitored by transwell assay (magnification: 100×). (H,I) ECAR was measured by glycolysis stress test. (J,K) Lactate production and glucose consumption were detected using the corresponding kits. (L,M) The expression of HK2 and LDHA was measured by western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: In Vitro, Transfection, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Colony Assay, Migration, Transwell Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircFAM120B Blocks the Development of Colorectal Cancer by Activating TGF-Beta Receptor II Expression via Targeting miR-645

doi: 10.3389/fcell.2021.682543

Figure Lengend Snippet: CircFAM120B activated the expression of TGFBR2 by targeting miR-645. In LoVo and HCT15 cells transfected with oe-circFAM120B, vector, oe-circFAM120B + miR-645 or oe-circFAM120B + miR-NC, the expression of TGFBR2 was detected by (A,B) qRT-PCR and (C) western blot. * P < 0.05.

Article Snippet: Colorectal cancer cell lines, including LoVo and HCT15, were purchased from ProCell Co., Ltd. (Wuhan, China) and maintained in matched special medium (ProCell Co., Ltd.).

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot