Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 is upregulated in colorectal cancer. (A) GEPIA ( http://gepia.cancer‐pku.cn/ ) prediction of the expression profile of CERS6‐AS1 in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). (B) The starBase database ( http://starbase.sysu.edu.cn/ ) shows the expression of CERS6‐AS1 in COAD. (C) CERS6‐AS1 expression in colorectal cancer cell lines (Caco‐2, HCT‐15, HCT‐15) and human normal colorectal mucosa epithelial cells (FHC). (D) A subcellular fractionation assay was used to detect the cellular location of CERS6‐AS1 in Caco‐2 and HCT‐15 cells. * p <0.05, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Expressing, Fractionation

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 deficiency suppresses malignant phenotypes of colorectal cancer cells. (A) The CERS6‐AS1 knockdown efficiency in colorectal cancer cells was measured by RT‐qPCR. (B,C) The viability and proliferation ability of Caco‐2 and HCT‐15 cells post‐transfection with sh‐CERS6‐AS1#1/#2 was assessed with CCK‐8 and colony formation assays. (D–F) The migration and invasion of Caco‐2 and HCT‐15 cells were assessed by Transwell assays. (G) The levels of EMT‐associated proteins (E‐cadherin, N‐cadherin, Slug, Twist) were measured using western blot after transfection of sh‐CERS6‐AS1#1/#2. (H) Cell stemness was measured using a sphere‐formation assay in cells after transfection with CERS6‐AS1#1/#2. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Knockdown, Quantitative RT-PCR, Transfection, CCK-8 Assay, Migration, Western Blot, Tube Formation Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 targets miR‐15b‐5p in colorectal cancer cells. (A) The miRNAs with binding sites for CERS6‐AS1 were searched in starBase. (B) The RNA pull‐down assay was used to explore the binding between the candidate miRNAs and CERS6‐AS1 in Caco‐2 and HCT‐15 cells. (C) The miR‐15b‐5p level in colon adenocarcinoma (COAD) was predicted by starBase. (D) MiR‐15b‐5p level in colorectal cancer cells. (E) The binding sequence of CERS6‐AS1 and miR‐15b‐5p was obtained from starBase. (F) The overexpression efficiency of miR‐15b‐5p in Caco‐2 and HCT‐15 cells using miR‐15b‐5p mimics. (G) A luciferase reporter assay was used to examine the binding between CERS6‐AS1 and miR‐15b‐5p after the indicated transfection. (H) RIP assay was used to detect whether CERS6‐AS1 and miR‐15b‐5p were enriched in the anti‐Ago2 precipitates. (I) The influence of CERS6‐AS1 silencing on miR‐15b‐5p expression was assessed using RT‐qPCR in Caco‐2 and HCT‐15 cells post‐transfection with CERS6‐AS1#1/#2. ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Binding Assay, Pull Down Assay, Sequencing, Over Expression, Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: SPTBN2 is directly targeted by miR‐15b‐5p. (A) The downstream target genes of miR‐15b‐5p were searched in starBase under the following screening conditions: CLIP‐data: strict stringency (≥5), degradome‐data: high stringency (≥3), pan‐cancer: 8 cancer types. (B) The expression of 10 candidate mRNAs in colorectal cancer cells posttransfection with miR‐15b‐5p mimics. (C) Survival analysis in patients with COAD with high or low levels of SPTBN2 based on starBase. (D,E) GEPIA showed SPTBN2 expression in COAD and READ, and starBase showed SPTBN2 expression in COAD. (F) SPTBN2 mRNA and protein levels in colorectal cancer cells were assessed using RT‐qPCR and western blot. (G) The potential binding sequences between SPTBN2 and miR‐15b‐5p from starBase. (H) SPTBN2 mRNA and protein expression was measured using RT‐qPCR and western blot in Caco‐2 and HCT‐15 cells transfected with sh‐CERS6‐AS1#1/#2. (I) The SPTBN2 mRNA level was assessed by RT‐qPCR in Caco‐2 and HCT‐15 cells transfected with miR‐15b‐5p mimics. (J) CERS6‐AS1 expression posttransfection with pcDNA3.1/CERS6‐AS1 in colorectal cancer cells. (K) A RIP assay was used to explore the relationship of CERS6‐AS1, miR‐15b‐5p and SPTBN2 in colorectal cancer cells. (L) A luciferase reporter assay was used to validate the interaction of CERS6‐AS1, miR‐15b‐5p, and SPTBN2 in colorectal cancer cells. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

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

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: CERS6‐AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR ‐15b‐5p to regulate SPTBN2

doi: 10.1002/kjm2.12503

Figure Lengend Snippet: CERS6‐AS1 promotes malignant behaviors in cells by upregulating SPTBN2 in colorectal cancer. (A) The SPTBN2 overexpression efficiency was examined using RT‐qPCR in colorectal cancer cells. (B,C) The viability and proliferation of Caco‐2 and HCT‐15 cells after transfection with sh‐CERS6‐AS1#2 or sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2 were measured by CCK‐8 and colony formation assays. (D–F) The migration and invasion capacities of Caco‐2 and HCT‐15 cells were assessed using Transwell assays in the sh‐CERS6‐AS1#2 and sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2 groups. (G) Western blot analysis showed the levels of proteins linked to the EMT process in Caco‐2 and HCT‐15 cells posttransfection with sh‐CERS6‐AS1#2 or sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2. (H) A sphere‐formation assay was used to examine cell stemness in the sh‐CERS6‐AS1#2 and sh‐CERS6‐AS1#2+pcDNA3.1/SPTBN2 groups. * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: A human normal colorectal mucosa epithelial cell line (FHC, catalog number: CRL‐1831) and colorectal cancer cell lines (Caco‐2, catalog number: HTB‐37; T84, catalog number: CCL‐248; HCT‐15, catalog number: CCL‐225) were provided by the American Type Culture Collection (ATCC, Manassas, VA) and then incubated in Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Shanghai, China) with 10% fetal bovine serum (FBS; OriCell, Suzhou, Jiangsu, China) and 1/100 penicillin/streptomycin (Biochrom, Cambridge, UK) at 37°C in 5% CO 2 .

Techniques: Over Expression, Quantitative RT-PCR, Transfection, CCK-8 Assay, Migration, Western Blot, Tube Formation Assay