Journal: Journal of Cancer

Article Title: Inhibition of the cell migration, invasion and chemoresistance of colorectal cancer cells through targeting KLF3 by miR-365a-3p

doi: 10.7150/jca.61967

Figure Lengend Snippet: Expression levels of miR-365a-3p are downregulated in CRC tissues and cell lines. (A) RT-qPCR was used to analyze miR-365a-3p expression levels in 162 CRC and adjacent normal tissues. (B) Log2 transformed value of miR-365a-3p expression level ratio between CRC and adjacent normal tissues. (C) RT-qPCR was used to determine miR-365a-3p expression levels in different CRC cell lines (HCT8, DLD1, LoVo, SW48, HT29, SW480, RKO and HCT116) and a human normal fetal colonic mucosa cell line (FHC). RT-qPCR was performed to analyze miR-365a-3p expression levels in CRC samples with or without (D) lymph node metastasis or (E) distant organ metastasis. (F) Association between relative miR-365a-3p expression levels in CRC tumor tissue and tumor volume of surgically resected clinical samples. RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; CRC, colorectal cancer. **P<0.05 compared with group FHC, *** P<0.01 compared with group FHC.

Article Snippet: CRC cell lines (HCT8, DLD1, LoVo, SW48, HT29, SW480, RKO and HCT116) and a human normal fetal colonic mucosa cell line (FHC) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transformation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cancer

Article Title: Inhibition of the cell migration, invasion and chemoresistance of colorectal cancer cells through targeting KLF3 by miR-365a-3p

doi: 10.7150/jca.61967

Figure Lengend Snippet: Effect of miR-365a-3p on the migration and invasion of CRC cell lines. (A) RT-qPCR was used to analyze miR-365a-3p expression levels in SW480 and LOVO cells transfected with three different miR-365a-3p-specific inhibitors or mimics, respectively. (B) RT-qPCR was used to determine miR-365a-3p expression levels in SW480 and LOVO cells transfected with lentiviral vectors carrying a miR-365a-3p-specific inhibitor or mimic, respectively. (C-F) Migratory and invasive abilities of SW480 and LOVO cells transfected with lentiviral vectors carrying a miR-365a-3p-specific inhibitor or mimic, respectively, were determined. RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA. **P<0.05 compared with group INC, mNC, LV-INC, or LV-mNC. *** P<0.01 compared with group INC, mNC, LV-INC, or LV-mNC.

Article Snippet: CRC cell lines (HCT8, DLD1, LoVo, SW48, HT29, SW480, RKO and HCT116) and a human normal fetal colonic mucosa cell line (FHC) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cancer

Article Title: Inhibition of the cell migration, invasion and chemoresistance of colorectal cancer cells through targeting KLF3 by miR-365a-3p

doi: 10.7150/jca.61967

Figure Lengend Snippet: Interference of miR-365a-3p expression alters the viability of CRC cells following treatment with chemotherapy agents. Viability of LOVO cells transfected with a lentiviral vector carrying a miR-365a-3p-specific mimic and treated with increasing doses of (A) doxorubicin, (B) fluorouracil or (C) cisplatin. Viability of SW480 cells transfected with a lentiviral vector carrying a miR-365a-3p-specific inhibitor and treated with increasing doses of (D) doxorubicin, (E) fluorouracil or (F) cisplatin. miR, microRNA. **P<0.05 compared with group LV-INC or LV-mNC. *** P<0.01 compared with group LV-INC or LV-mNCFigure 3. Interference of miR-365a-3p expression alters the viability of CRC cells following treatment with chemotherapy agents. Viability of LOVO cells transfected with a lentiviral vector carrying a miR-365a-3p-specific mimic and treated with increasing doses of (A) doxorubicin, (B) fluorouracil or (C) cisplatin. Viability of SW480 cells transfected with a lentiviral vector carrying a miR-365a-3p-specific inhibitor and treated with increasing doses of (D) doxorubicin, (E) fluorouracil or (F) cisplatin. miR, microRNA. **P<0.05 compared with group LV-INC or LV-mNC. *** P<0.01 compared with group LV-INC or LV-mNC.

Article Snippet: CRC cell lines (HCT8, DLD1, LoVo, SW48, HT29, SW480, RKO and HCT116) and a human normal fetal colonic mucosa cell line (FHC) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Journal of Cancer

Article Title: Inhibition of the cell migration, invasion and chemoresistance of colorectal cancer cells through targeting KLF3 by miR-365a-3p

doi: 10.7150/jca.61967

Figure Lengend Snippet: KLF3 is a target gene of miR-365a-3p and modulates chemoresistance in CRC cell lines. (A) miR-365a-3p binding site in the WT-KLF3 3'-UTR region was predicted and vectors carrying WT- and MUT-KLF3 3'-UTR region were synthesized. (B and C) Dual luciferase reporter gene assay was conducted to investigate the effect on KLF3 expression in LOVO and SW480 cells co-transfected with the lentiviral vectors carrying a miR-365a-3p-specific inhibitor or mimic, respectively, and WT- or MUT-KLF3 3'-UTR vectors. (D) Western blotting and reverse transcription-quantitative PCR were used to determine the regulatory effects of KLF3-specific siRNAs on KLF3 protein and mRNA expression levels in LOVO cells, respectively. (E-G) Viability of SW480 cells transfected with a lentiviral vector carrying a miR-365a-3p-specific inhibitor, with or without the co-transfection with KLF3-specific siRNAs. Each group of cells was treated with increasing doses of fluorouracil, cisplatin or doxorubicin. (H and I) Cell migration and invasion were measured following transfection with a lentiviral vector carrying a miR-365a-3p-specific inhibitor, with or without co-transfection with KLF3-specific siRNAs. miR, microRNA; WT, wild-type; MUT, mutant; KLF3, Kruppel-like factor; UTR, untranslated region; siRNA, small interfering RNA. **P<0.05 compared with group NC, LV-mNC, or LV-INC. *** P<0.01 compared with group NC, LV-mNC, or LV-INC.

Article Snippet: CRC cell lines (HCT8, DLD1, LoVo, SW48, HT29, SW480, RKO and HCT116) and a human normal fetal colonic mucosa cell line (FHC) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Synthesized, Luciferase, Reporter Gene Assay, Expressing, Transfection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Plasmid Preparation, Cotransfection, Migration, Mutagenesis, Small Interfering RNA

Journal:

Article Title: Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

doi: 10.1128/IAI.69.1.52-57.2001

Figure Lengend Snippet: Binding of purified and biotinylated TibA to HCT8 monolayers. Bound TibA was detected by peroxidase-coupled streptavidin. Color development in each well was measured at 405 nm. Symbols: ♦, saturation curve performed with 0.0, 1.0, 2.4, 4.8, 9.6, 19.2, and 48.0 pmol of TibA per well; ▴, competition curve performed with 4.8 pmol of labeled TibA plus 0.0, 1.0, 4.8, 9.6, or 48.0 pmol of unlabeled TibA. Every experiment was performed at least three times. The extent of biotin labeling varied from day to day, resulting in significant differences in color development. Therefore, both curves shown here represent the average of two experiments performed in duplicate.

Article Snippet: The human ileocecal epithelial cell line HCT8 (ATCC CCL 244) was maintained in RPMI 1640 medium containing 10% fetal bovine serum, 1 mM l -glutamine, and 1 mM sodium pyruvate (Life Technologies).

Techniques: Binding Assay, Purification, Labeling

Journal:

Article Title: Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

doi: 10.1128/IAI.69.1.52-57.2001

Figure Lengend Snippet: Inhibition of TibA-mediated invasion by anti-TibA antiserum. Invasion assays were performed in the absence of antibodies (▪) or in the presence of preabsorbed and affinity-purified IgG from rabbit preimmune serum or polyclonal anti-TibA antiserum. (A) Invasion of HCT8 cells relative to E. coli DH5α(pET109), representing 100% (actual invasion of this strain in the absence of antibody was 1.30% ± 0.05%). Statistically significant effects of antibody treatment on the invasion efficiency of DH5α(pET109) are indicated by ∗ (P < 0.01) or ∗∗ (P < 0.005) as determined by analysis of variance of three experiments, each performed in triplicate. (B) Invasion of HCT8 cells relative to ETEC strain H10407, representing 100% (actual invasion of this strain in the absence of antibody was 0.24% ± 0.02%). TIB3 is a tib locus deletion mutant of H10407. Data are shown as averages for three replicates. Statistically significant effects of antibody treatment on the invasion efficiency of H10407 are indicated by ∗ (P < 0.01) as determined by analysis of variance of three experiments, each performed in triplicate.

Article Snippet: The human ileocecal epithelial cell line HCT8 (ATCC CCL 244) was maintained in RPMI 1640 medium containing 10% fetal bovine serum, 1 mM l -glutamine, and 1 mM sodium pyruvate (Life Technologies).

Techniques: Inhibition, Affinity Purification, Mutagenesis

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: Effects of ribosomal inactivation on intracellular fat and gene profiles. (A-D) HCT-8 (A and B) and HepG2 (C and D) cells were treated with the vehicle, 1000 ng/mL RIS-1, or 2 mM RIS-2 for 24 h. (A and B) Lipid droplets were detected using a transmission electron microscope (TEM) at the original magnification x1000 (scale bar(s), 2 mm) with white arrows indicating lipid droplets in each cell. (C and D) Total lipids from the whole cell lysate were analyzed for lipidomic profiling using mass spectrometry (performed by Lipotype GmbH, Dresden, Germany). (E) Pearson 's correlation analysis of relationships between transcription levels of key components of the ribotoxic stress responses and lipid metabolism in patients with IBD (gse117993 ( Denson 's, n = 190)). Correlation matrix visualization was generated using the corrplot function of the R-package (R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/ ). Correlations of transcriptional expression among genes were interpreted according to a general guideline for Pearson's coefficient value: r > 0.7, high (+); 0.5 < r < 0.7, moderate (+); 0.3 < r < 0.5, moderate (+) or low (+); 0.1 < r < 0.3, low (+); -0.1 < r < 0.1, negligible; -0.3 < r < -0.1, low (-). (F) HCT-8 cells were treated with the vehicle or 500 ng/mL RIS-1 for 1 h. Functional gene ontology using RNA sequencing analysis of the cellular mRNA was evaluated based on p values for each category. (G-H) HCT-8 (G) and HepG2 (H) cells were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 24 h. Each mRNA was measured using reverse transcription-quantitative PCR. (I-K) LDL uptake into human intestinal epithelial cells (upper panels) and cellular LDLR expression (lower pannels) were measured using LDL-Dylight TM 550 (red) and DyLight TM -488-conjugated anti-LDLR antibody (green), respectively. The microscopic analysis was performed at the original magnification of 200× (scale bar(s), 50 μm) with each quantitative graph (J and K, * p < 0.05, ** p < 0.01, and *** p < 0.001). LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; CE, cholesterol esters; DAG, diacylglycerol; PA, phosphatidylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine (-ether); PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TAG, triacylglycerol.

Article Snippet: Human epithelial cell lines including intestinal HCT-8 and hepatic HepG2 were supplied by the American Type Culture Collection (ATCC; Rockville, MD, USA).

Techniques: Transmission Assay, Microscopy, Mass Spectrometry, Generated, Expressing, Functional Assay, RNA Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SNHG1 expression is up-regulated in colorectal cancer and is correlated with prognosis. a Hierarchical cluster heat map of differentially expressed lncRNAs in colorectal cancer and corresponding normal tissues generated from RNA sequencing data from the TCGA database. Red in the heat map denotes upregulation; blue denotes downregulation. The red arrow indicates SNHG1. b Expression of SNHG1 in the TCGA, GSE9348 and GSE8671 cohorts. c qRT-PCR analysis of SNHG1 expression in 80 pairs of colorectal cancer and corresponding normal tissues. d Kaplan-Meier survival analysis of CRC patients’ overall survival based on SNHG1 expression in our cohort ( n = 130, P < 0.001). e Kaplan-Meier survival analysis of CRC patients’ overall survival based on SNHG1 expression in GSE29621 ( n = 65, P = 0.035). f SNHG1 expression in colorectal cancer cell lines (DLD-1, HCT-116, HT-29, SW-620, HCT-8 and SW-480) compared with normal colorectal epithelial cells FHC detected by qRT-PCR. ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: Expressing, Generated, RNA Sequencing, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SP1 activates SNHG1 transcription in colorectal cancer cells. a Analysis of SP1 ChIP-seq, H3K4me3 ChIP-seq and DnaseI-seq data of HCT-116 cells in the SNHG1 locus. b SNHG1 expression was detected by qRT-PCR in HCT-116 and HCT-8 cells transfected with SP siRNAs or the SP1 vector. c The correlation between SP1 and SNHG1 expression analyzed in 30 paired colorectal cancer samples ( n = 30, r = 0.38, P = 0.03). d ChIP assays were performed to detect SP1 occupancy at the SNHG1 promoter region, α-Satellite and DHFR were employed as negative and positive control respectively for SP1 ChIP assays. e Dual luciferase reporter assays were used to determine the SP1 binding sites on the SNHG1 promoter region. The upper left corner of the picture was SP1 binding motif provided by the JASPAR CORE database. * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: ChIP-sequencing, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Positive Control, Luciferase, Binding Assay

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SNHG1 affects colorectal cancer cells growth. a SNHG1 expression was detected by qRT-PCR in HCT-116 and HCT-8 cells transfected with two SNHG1 siRNAs. b HCT-116 and HCT-8 cells transfected with SNHG1 siRNAs were subjected to the CCK-8 assay after transfection. c HCT-116 and HCT-8 cells transfected with SNHG1 siRNAs were seeded onto 6-well plates. The number of colonies was counted on the 14th day after seeding. d Representative images of mice bearing tumors from empty vector, sh-SNHG1#1 vector and SNHG1 vector groups, and the tumor volume growth curves after injections in different groups. e SNHG1 expression was detected in tumors from different groups of mice using qRT-PCR. f Representative images of hematoxylin and eosin (HE) staining and Ki67 immunostaining of tumor samples from different groups. Scale bar = 50 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Plasmid Preparation, Staining, Immunostaining

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SNHG1 regulates colorectal cancer cell proliferation and apoptosis. a Results of gene set enrichment analysis (GSEA) were plotted to visualize the correlation between the expression of SNHG1 and cell cycle and DNA repair gene signatures in TCGA cohort. b EdU assays were used to determine the cell proliferation ability of si-SNHG1 transfected cells. c Flow cytometric cell cycle distribution assays to detect the proportion of colorectal cancer cell cells in G1, S, and G2/M phases after transfection with SNHG1 siRNAs. d The cell cycle related proteins CyclinD1, CDK4, CDK6, and CyclinD2 were detected by western blot following SNHG1 silencing. e The effect of SNHG1 knockdown on cell apoptosis was analyzed by flow cytometric cell apoptosis assays. f Apoptosis related proteins Caspase-3, cleaved Caspase-3, PARP, cleaved PARP and Bax were detected by western blot after SNHG1 knockdown. Scale bar = 50 μm. ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: Expressing, Transfection, Western Blot, Knockdown

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SNHG1 acts as a sponge for miR-154-5p in the cytoplasm. a Representative FISH images indicated subcellular location of SNHG1 in HCT-116 and HCT-8 cells (red). Nuclei were stained by DAPI (blue). SNHG1 sense probe was employed as a negative control. b Relative SNHG1 expression levels in nuclear and cytosolic fractions of HCT-116 and HCT-8 cells. Nuclear controls: U6; Cytosolic controls: GAPDH. c Dual luciferase reporter assays were used to determine miRNAs that directly interacted with SNHG1. Luciferase activity is presented as relative luciferase activity normalized to activity of their respective negative control. d Dual luciferase reporter assays were conducted with wild type and mutant type (putative binding sites for miR-154-5p were mutated) luciferase reporter vectors. Right panel, sequence alignment of miR-154-5p and its predicted binding sites (green) in SNHG1. Predicted miR-154-5p target sequence (blue) in SNHG1 (Luc-SNHG1-wt) and position of mutated nucleotides (red) in SNHG1 (Luc-SNHG1-mut). e RNA immunoprecipitation with an anti-Ago2 antibody was used to assess endogenous Ago2 binding to RNA in HCT-116 cells, IgG was used as the control. SNHG1 and miR-154-5p levels were determined by qRT–PCR and presented as fold enrichment in Ago2 relative to input. RIP efficiency of Ago2 protein was detected by western blot. f RNA pull-down assays were used to examine the interaction of SNHG1 and Ago2 in HCT-116 cells. g CCK-8 assays demonstrated that SNHG1 silencing inhibited HCT-116 cell growth. MiR-154-5p down-regulation rescued growth inhibition caused by SNHG1 knockdown. h EdU assays revealed that SNHG1 overexpression promotes HCT-116 cell proliferation. Co-transfecting miR-154-5p mimics with the SNHG1 plasmid abolished the increased proliferation rates. i The correlation between miR-154-5p and SNHG1 expression analyzed in 30 paired colorectal cancer samples (n = 30, r = − 0.48, P = 0.008). Scale bar = 50 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: Staining, Negative Control, Expressing, Luciferase, Activity Assay, Mutagenesis, Binding Assay, Sequencing, RNA Immunoprecipitation, Control, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Inhibition, Knockdown, Over Expression, Plasmid Preparation

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SNHG1 regulates expression of the miR-154-5p target gene, CCND2. a CCND2 expression was detected by qRT-PCR in SNHG1 siRNAs transfected or SNHG1 siRNAs and miR-154-5p inhibitors co-transfected HCT-116 cells. b CCND2 expression was detected by qRT-PCR in SNHG1 vector transfected or SNHG1 vector and miR-154-5p mimics co-transfected HCT-116 cells. c Western blot analyses of CCND2 expression after knockdown of SNHG1, overexpression of miR-154-5p or knockdown of SNHG1 + inhibition of miR-154-5p in HCT-116 cells. d Dual luciferase reporter assays demonstrated that miR-154-5p overexpression reduced Luc-CCND2 luciferase activity and SNHG1 overexpression abolished miR-154-5p induced reductions in luciferase activity in HCT-116 cells. e CCND2 expression was measured by western blot after silencing of endogenous SNHG1 and transfection with either SNHG1-mut vector, which contains mutations at the putative miR-154-5p binding site, or SNHG1 vector in HCT-116 cells. f The correlation between CCND2 and SNHG1 expression analyzed in 30 paired colorectal cancer samples ( n = 30, r = 0.38, P = 0.036). g EdU assays demonstrated HCT-116 cells proliferation rates after knockdown of SNHG1, knockdown of CCND2 or both knockdown of SNHG1and CCND2. h CCK-8 assays demonstrated that CCND2 knockdown could reverse growth promotion caused by SNHG1 overexpression in HCT-116 cells. i Immunohistochemistry analysis of CCND2 protein levels in tumor tissues formed from SNHG1 knockdown or control cells. j Detection of CCND2 protein levels in colorectal cancer and normal tissues by IHC. Scale bar = 50 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Knockdown, Over Expression, Inhibition, Luciferase, Activity Assay, Binding Assay, CCK-8 Assay, Immunohistochemistry, Control

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SNHG1 participates in epigenetic repression of KLF2 and CDKN2B by interacting with PRC2. a GSEA showed a significant correlation between the SNHG1 and genes in PRC2 related pathway. b Scatter plot showing the expression relationship among SNHG1, EZH2, SUZ12 and EED in colorectal tumor tissues from TCGA database. The upper right squares show the Pearson correlation between each other. c RIPs experiments for EZH2, SUZ12 and EED were performed and the coprecipitated RNA was subjected to qRT-PCR for SNHG1. GAPDH was employed as a negative control. d RNA pull-down was used to examine the association of SNHG1 and EZH2. AR binding to HuR was used as a positive control. e PRC2 target genes expression was detected by qRT-PCR in SNHG1 siRNAs transfected HCT-116 cells. f CDKN2B and KLF2 expression was detected by qRT-PCR in SNHG1 vector transfected or SNHG1 vector and EZH2 siRNAs co-transfected CRC cells. g CDKN2B and KLF2 protein levels were detected by western blot in indicated conditions. h ChIP assays were performed to detect EZH2 and H3K27me3 occupancy in the CDKN2B promoter region. i ChIP assays were performed to detect EZH2 and H3K27me3 occupancy in the KLF2 promoter region. * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Negative Control, Binding Assay, Positive Control, Transfection, Plasmid Preparation, Western Blot

Journal: Molecular Cancer

Article Title: The long noncoding RNA SNHG1 regulates colorectal cancer cell growth through interactions with EZH2 and miR-154-5p

doi: 10.1186/s12943-018-0894-x

Figure Lengend Snippet: SNHG 1 promotes colorectal cancer progression partly by regulating KLF2 and CDKN2B expression. a Left panel, CCK-8 assays demonstrated that silence of SNHG1 inhibited cancer cell growth. KLF2 knockdown could rescue growth inhibition caused by SNHG1 knockdown in HCT-116 cells. Right panel, CCK-8 assays demonstrated that silence of SNHG1 inhibited cancer cell growth. CDKN2B (P15) knockdown could rescue growth inhibition caused by SNHG1 knockdown in HCT-116 cells. b EdU assays showed that SNHG1 knockdown inhibited cancer cell proliferation. Co-transfecting KLF2 or CDKN2B siRNAs with SNHG1 siRNAs reversed the decreased proliferation rates in HCT-116 cells. c EdU assays showed that EZH2 knockdown could inhibit proliferation promotion caused by SNHG1 overexpression in HCT-116 cells. d Immunohistochemistry analysis of EZH2, KLF2 and CDKN2B protein levels in colorectal cancer and normal tissues. e Immunohistochemistry analysis of KLF2 and CDKN2B protein levels in tumor tissues formed from SNHG1 knockdown or control cells. f Schematic of the proposed mechanism of SNHG1 in colorectal cancer cells. In the cytoplasm, SNHG1 acts as a ceRNA to sponge miR-154-5p and upregulated the expression of CCND2 (CyclinD2). In the nucleus, SNHG1 is involved in PRC2 mediated epigenetic repression of KLF2 and CDKN2B (P15). KLF2 is also an upstream regulatory factor of CDKN2B. Besides, CDKN2B is a well-studied inhibitor of CCND2. Downstream genes of SNHG1 formed a regulatory network to regulate growth of colorectal cancer. Scale bar = 50 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1 and HT-29) and the human normal colorectal epithelial cell (FHC) were obtained from American Type Culture Collection.

Techniques: Expressing, CCK-8 Assay, Knockdown, Inhibition, Over Expression, Immunohistochemistry, Control