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: Cell Proliferation

Article Title: SATB 2 targeted by methylated miR‐34c‐5p suppresses proliferation and metastasis attenuating the epithelial‐mesenchymal transition in colorectal cancer

doi: 10.1111/cpr.12455

Figure Lengend Snippet: SATB2 prevents metastasis of colorectal cancer (CRC) cells in vivo. A, Bioluminescent images of in vivo incidence of lung metastasis and primary tumour in nude mice subjected to tail vein inoculation with HT‐29 cells whose endogenous SATB2 was stably knocked down (labelled as HT‐29 Lv‐sh‐SATB2) or not (HT‐29 Lv‐control). Fourteen nude mice were grouped into 2, with each having 7 mice. The incidence rate of lung metastasis in group where mice inoculated with HT‐29 Lv‐sh‐SATB2 cells was 6 of 7. In contrast, the incidence of lung metastasis in group where mice inoculated with HT‐29 Lv‐control cells were 1 of 7; there was significant difference with P value being .029 using Cross‐table analysis. B, Haematoxylin‐eosin (HE) section analysis of lung metastasis nodes and its quantitative counts of visible nodes per lung; ***P < .001 compared with Lv‐control group using the independent sample t‐test analysis. C, Similarly and parallel to HT‐29 cells, the evaluation of incidence of lung metastasis was verified in another kind of CRC cell line HCT‐15 whose endogenous SATB2 was stably knocked down (HCT‐15 Lv‐sh‐SATB2) or not (HCT‐15 Lv‐control). As presented, there was also significant difference in the case of incidence of lung metastasis, with P value being .021 using cross‐table analysis. D, HE section analysis of lung metastasis nodes and its quantitative counts of visible nodes per lung, ***P < .001 relative to Lv‐control group using the independent sample t‐test analysis. In terms of bioluminescent image presented, shown were the representative figures selected among the 7 candidates

Article Snippet: The human CRC cell lines HT‐29, Colo‐320, SW480, SW620 and HCT‐15 were obtained from China Center for Type Culture Collection.

Techniques: In Vivo, Stable Transfection

Journal: Cell Proliferation

Article Title: SATB 2 targeted by methylated miR‐34c‐5p suppresses proliferation and metastasis attenuating the epithelial‐mesenchymal transition in colorectal cancer

doi: 10.1111/cpr.12455

Figure Lengend Snippet: Repressed miR‐34c‐5p in CRC resulted from methylation in its promoter sequence. A, Basal level of miR‐34c‐5p in the same panel of CRC cell lines as SATB2 was detected using immunoblotting. B, Detection of miR‐34c‐5p level in the 85 paired cases of CRC and its normal control using qRT‐PCR. U6, the internal loading control. Independent sample t test was used to analyse the statistical difference between CRC and its paired normal control. C, Spearman correlation was carried out to analyse the correlation between miR‐34c‐5p and SATB2 expression on mRNA level. As displayed, the R 2 coefficient was .131, the sample size was 85 cases and P value was .0007. D, Detection of methylated miR‐34c‐5p level in CRC and paired normal control using methylated qRT‐PCR. E, Similarly, Spearman correlation was carried out to analyse the correlation between methylated miR‐34c‐5p and miR‐34c‐5p expression on mRNA level, as shown, the R 2 coefficient was −.401, the sample size was 85 cases, and P value was .003. F, the miR‐34c‐5p level was assayed before and after treatment with 5‐aza‐2′‐deoxycytidine (abbreviated as 5‐Aza‐dC), an inhibitor for DNA methyltransferase, for 48 h in CRC cell lines HT‐29 and HCT‐15. *P < .05, **P < .01, ***P < .001 in comparison with their control group, that is, HCoEpiC using independent sample t test. G: expression variation of SATB2 as well as E‐cadherin and N‐cadherin in the presence and absence of 5‐Aza‐dC treatment for 48 h, as exemplified by western blot

Article Snippet: The human CRC cell lines HT‐29, Colo‐320, SW480, SW620 and HCT‐15 were obtained from China Center for Type Culture Collection.

Techniques: Methylation, Sequencing, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cell Proliferation

Article Title: SATB 2 targeted by methylated miR‐34c‐5p suppresses proliferation and metastasis attenuating the epithelial‐mesenchymal transition in colorectal cancer

doi: 10.1111/cpr.12455

Figure Lengend Snippet: MiR‐34c‐5p was identified to be able to directly and negatively modulate SATB2. A, Bioinformatic prediction of the potential binding sites of SATB2 in the mature sequence of miR‐34c‐5p, which was highly conserved in mammalians. The bold blue words were the highly conserved binding site of miR‐34c‐5p binding to SATB2. B, Verification of the potential binding of miR‐34c‐5p with SATB2 using Luciferase reporter assay. Independent sample t test was used to analyse the statistical difference relative to control groups. Experiment was performed in triplicate and shown was the representative. C, Expression variation of SATB2 as well as the typical EMT relevant biomarkers, including E‐cadherin and N‐cadherin, after transfection with miR‐34c‐5p mimics and inhibitor sequence into HT‐29 and SW480 cells, as exemplified by immunoblot. The experiment was performed independently in triplicate and shown were the representative figures selected among the candidates. D, In vivo evaluation of metastatic variation of CRC cells whose endogenous miR‐34c‐5p was stably re‐expressed using lentivirus (Lv) vector fused with bioluminescent tag. For HT‐29 cells, 14 nude mice were grouped into 2, with each 7. In control group, mice were subjected to the tail vein inoculation of HT‐29 cells transfected with Lv‐miR‐34c‐5p‐scramble (hereafter referred to as Lv‐control), whereas in experimental group, mice subjected to the tail vein inoculation of HT‐29 cells transfected with Lv‐miR‐34c‐5p. The same holds true for HCT‐15 cells. Presented were the representative bioluminescent images picked up among the candidates from at least 3 different times of repeat. *P<0.05 compared with control group using independent sample t test

Article Snippet: The human CRC cell lines HT‐29, Colo‐320, SW480, SW620 and HCT‐15 were obtained from China Center for Type Culture Collection.

Techniques: Binding Assay, Sequencing, Luciferase, Reporter Assay, Expressing, Transfection, Western Blot, In Vivo, Stable Transfection, Plasmid Preparation

Journal: Cell Proliferation

Article Title: SATB 2 targeted by methylated miR‐34c‐5p suppresses proliferation and metastasis attenuating the epithelial‐mesenchymal transition in colorectal cancer

doi: 10.1111/cpr.12455

Figure Lengend Snippet: SATB2 suppresses the epithelial‐mesenchymal transition (EMT) in colorectal cancer (CRC) cell lines. A, Endogenous basal level of SATB2 in a panel of CRC cell lines available to us; the observed band size of SATB2 and β‐actin band was 83 and 42 kilodalton (kDa), respectively; B, transient knock‐down of SATB2 using siRNA technique in HT‐29 and HCT‐15 cells. Scramble‐siRNA was used as negative control of siRNA sequences transfected. The observed band size of E‐cadherin, N‐cadherin and vimentin band was 110, 125 and 54 kDa, respectively; C, Likewise, exogenous SATB2 fused with green fluorescent protein (GFP) was re‐expressed in CRC cell lines HT‐29 and HCT‐15. The observed band size of SATB2 fused with GFP was approximately 110 kDa, with GFP tag being observed at 27 kDa. Shown were the representative figures picked among the candidates from at least independently 3 different times of repeat

Article Snippet: The human CRC cell lines HT‐29, Colo‐320, SW480, SW620 and HCT‐15 were obtained from China Center for Type Culture Collection.

Techniques: Negative Control, Transfection

Journal: Cell Division

Article Title: Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression

doi: 10.1186/s13008-024-00129-7

Figure Lengend Snippet: BHLHE40 mediates the transcription of NEAT1 in CRC cells. A Genes co-expressed with NEAT1 in CRC were obtained from UALCAN. B The intersection of genes co-expressed with NEAT1 in COAD and READ and the transcription factors targeting NEAT1 downloaded from hTFtarget. C The expression of these eight transcription factors in CRC was analyzed in the UALCAN. D The expression of BHLHE40 in both COAD and READ. E The binding fragment of BHLHE40 on the NEAT1 promoter with the highest score. F RT-qPCR detection of BHLHE40 expression in CRC tissues and adjacent tissues by RT-qPCR (n = 54). G Expression of NEAT1 in CRC cell lines and HCoEpiC cells by RT-qPCR. H Knockdown efficiency of BHLHE40 by RT-qPCR. I RT-qPCR detection of NEAT1 expression after knockdown of BHLHE40 in LoVo and HCT-15 cells. J Changes in luciferase activity after knockdown of BHLHE40 using luciferase activity assay. K BHLHE40 binding to the NEAT1 promoter in CRC cell using ChIP assay. Results were expressed as magnitude of relative expression (means ± SD) from three independent experiments. F Paired t-test; G one-way ANOVA; H – K two-way ANOVA. * p < 0.05

Article Snippet: Human colon epithelium cell HCoEpiC (YS1700C, YaJi Biological, Shanghai, China), and human CRC cell lines LoVo (CL-0144), HCT-15 (CL-0097) and Caco-2 (CL-0050, all from Procell, Wuhan, Hubei, China) were used in this study.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Knockdown, Luciferase, Activity Assay

Journal: Cell Division

Article Title: Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression

doi: 10.1186/s13008-024-00129-7

Figure Lengend Snippet: NEAT1 reverses the anti-tumor effects of sh-BHLHE40 in CRC cells. A RT-qPCR detection of NEAT1 overexpression efficiency. B MTT assay for LoVo and HCT-15 cell viability. C Transwell assay for LoVo and HCT-15 cell migration ability. D Transwell assay for LoVo and HCT-15 cell invasion ability. E Western blot for E-cadherin, N-cadherin, and Vimentin expression in LoVo and HCT-15 cells. F Flow cytometry for LoVo and HCT-15 cell apoptosis. Results were expressed as magnitude of relative expression (means ± SD) from three independent experiments. A – F two-way ANOVA. * p < 0.05

Article Snippet: Human colon epithelium cell HCoEpiC (YS1700C, YaJi Biological, Shanghai, China), and human CRC cell lines LoVo (CL-0144), HCT-15 (CL-0097) and Caco-2 (CL-0050, all from Procell, Wuhan, Hubei, China) were used in this study.

Techniques: Quantitative RT-PCR, Over Expression, MTT Assay, Transwell Assay, Migration, Western Blot, Expressing, Flow Cytometry

Journal: Cell Division

Article Title: Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression

doi: 10.1186/s13008-024-00129-7

Figure Lengend Snippet: NEAT1 reverses the metastasis-suppressing and tumor-suppressing properties of sh-BHLHE40. HCT-15 cells after infection were injected subcutaneously into nude mice to observe tumor growth. A Tumor growth curve. B Immunohistochemical analysis of BHLHE40 and Ki67 expression in xenograft tumor tissues of nude mice. C HCT-15 cells after infection were injected into nude mice via the tail vein, and lung metastasis formation was observed using HE staining. D Western blot for BHLHE40 and EMT-related protein expression in metastatic tissues with lung infiltration. Results were expressed as magnitude of relative expression (means ± SD) from six nude mice in each group. Two-way ANOVA. * p < 0.05

Article Snippet: Human colon epithelium cell HCoEpiC (YS1700C, YaJi Biological, Shanghai, China), and human CRC cell lines LoVo (CL-0144), HCT-15 (CL-0097) and Caco-2 (CL-0050, all from Procell, Wuhan, Hubei, China) were used in this study.

Techniques: Infection, Injection, Immunohistochemical staining, Expressing, Staining, Western Blot

Journal: Cell Division

Article Title: Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression

doi: 10.1186/s13008-024-00129-7

Figure Lengend Snippet: BHLHE40 mediates NEAT1 transcription to activate Wnt/β-catenin signaling in CRC cells. A Western blot for the E-cadherin, N-cadherin, Vimentin, Wnt, β-catenin, c-myc, and cyclin D1 protein expression in CRC cells in response to oe-BHLHE40 (oe-NC as control) or oe-BHLHE40 + sh-NEAT1 (oe-BHLHE40 + sh-NC as control). B Effects of oe-BHLHE40 and iCRT3 combined treatment on Wnt/β-catenin pathway activity in CRC cells was measured using TOP/FOP flash assay. C MTT assay for LoVo and HCT-15 cell viability. D Transwell assay for LoVo and HCT-15 cell migration ability. E Transwell assay for LoVo and HCT-15 cell invasion ability. F Flow cytometry for LoVo and HCT-15 cell apoptosis. Results were expressed as magnitude of relative expression (means ± SD) from three independent experiments. Two-way ANOVA. * p < 0.05

Article Snippet: Human colon epithelium cell HCoEpiC (YS1700C, YaJi Biological, Shanghai, China), and human CRC cell lines LoVo (CL-0144), HCT-15 (CL-0097) and Caco-2 (CL-0050, all from Procell, Wuhan, Hubei, China) were used in this study.

Techniques: Western Blot, Expressing, Control, Activity Assay, MTT Assay, Transwell Assay, Migration, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Podocalyxin-Like Protein 1 Regulates TAZ Signaling and Stemness Properties in Colon Cancer

doi: 10.3390/ijms18102047

Figure Lengend Snippet: Suppression of PODXL decreases TAZ downstream gene expressions. ( A ) Real-time PCR analysis of the PODXL mRNA level in PODXL-knockdown HCT15 cells. ( B ) Knockdown of PODXL-downregulated TAZ downstream gene expressions, as measured by real-time PCR assay. * p < 0.05; ** p < 0.01, as assessed by an unpaired t -test. ( C ) Western blot analysis of protein levels of the Hippo cascade in PODXL knockdown HCT15 cells. GAPDH, glyceraldehyde 3 phosphate dehydrogenase; RFP, Red Fluorescent Protein.

Article Snippet: The human colon cancer cell lines of HCT116, LoVo, HT29, and HCT15 were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Podocalyxin-Like Protein 1 Regulates TAZ Signaling and Stemness Properties in Colon Cancer

doi: 10.3390/ijms18102047

Figure Lengend Snippet: Expressions of PODXL and TAZ are associated with the stem cell signature in colorectal cancer. ( A ) Real-time PCR analysis of pluripotent stem cell markers and cancer stem-like gene expressions in PODXL- (upper panel) and TAZ-knockdown (lower panel) HCT15 cells. * p < 0.05; ** p < 0.01, as assessed by an unpaired t -test. ( B ) Knockdown of PODXL and TAZ-suppressed tumorsphere formation in HCT15 cells. Pictures were taken under a reverse microscope at 40× (upper panel) or 100× (lower panel) magnification. * p < 0.05 as assessed by an unpaired t -test. ( C ) Correlations of PODXL and TAZ with stem cell-related gene signatures. Data were retrieved from gene expression omnibus (GSE68468 and GSE40967). NES, normalized enrichment score.

Article Snippet: The human colon cancer cell lines of HCT116, LoVo, HT29, and HCT15 were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Microscopy, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Podocalyxin-Like Protein 1 Regulates TAZ Signaling and Stemness Properties in Colon Cancer

doi: 10.3390/ijms18102047

Figure Lengend Snippet: Expression of PODXL confers resistance to conventional chemotherapies in colorectal cancer. ( A ) Western blot analysis of the PODXL protein level in CRC cell lines. ( B ) Determination of 50% inhibitory concentration (IC 50 ) values of 5-fluorouracil (5-FU) and irinotecan (CPT11) toward CRC cells. CRC cells were treated with various concentrations of 5-FU or CPT11 for 48 h, and cell viability was measured by MTT assay. Results are shown as the mean ± standard error of the mean ( n = 3). The IC 50 value was obtained using the GraphPad Prism5 program. ( C ) Knockdown of PODXL increased sensitivity in response to chemotherapies. 5-FU and CPT11 (0–40 μM) were added in mock and PODXL knockdown HCT15 cells for 48 h, and cell viability was measured by MTT assay. ( D ) HCT15 cells were treated with verteporfin (VP) for 24 h, and protein expression was analyzed by Western blotting. ( E ) HCT15 cells were treated with 5-FU or CPT11 in the presence or absence of VP for 24 (left panel) or 48 h (right panel), and cell viability was measured by MTT assay. Results are shown as the mean ± standard error of the mean ( n = 3). ( F ) HCT15 cells were treated with 5-FU or CPT11 combined with VP, and formation of tumorsphere was assessed. A two-tailed Student’s t -test was applied to determine the statistical significance of the indicated groups. * p < 0.05, ** p < 0.01.

Article Snippet: The human colon cancer cell lines of HCT116, LoVo, HT29, and HCT15 were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Expressing, Western Blot, Concentration Assay, MTT Assay, Knockdown, Two Tailed Test