Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A Silencing of circUBAP2 was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting circUBAP2 (si-circUBAP2-1 or si-circUBAP2-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-circUBAP2-1 was chosen for further experiments for its better transfection efficiency. Then, MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for B Cell migration by Transwell assay; C Cell migration by wound healing assay; D The protein levels of Vimentin and Twist was examined by Immunoblotting; E The concentrations of IL-1β and IL-17 in the culture medium by ELISA. F , G The Volcano plot and hierarchical clustering heatmap showing differentially expressed mRNAs in CXCL11-treated MHCC-97H based on RNA sequencing. Upregulated genes were applied for H Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling enrichment analysis and I Gene Ontology (GO) of biological process enrichment analysis. J The expression of IFIT1/IFIT3 in CXCL11-treated and untreated HCC cells based on RNA sequencing data. * P < 0.05, ** P < 0.01, compared with the control group; # P < 0.05, ## P < 0.01, compared with the si-NC group; $ P < 0.05, $$ P < 0.01, compared with the CXCL11 + si-circUBAP2 group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Small Interfering RNA, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Migration, Transwell Assay, Wound Healing Assay, Western Blot, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Expressing, Control

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A The mRNA expression of IFIT1 and IFIT3 was examined in 12 cases of nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by real-time PCR. B The protein contents and distribution of IFIT1 and IFIT3 were examined in nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by IHC staining. C MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for the mRNA expression of IFIT1 and IFIT3 by real-time PCR. D IFIT1 or IFIT3 silencing was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting IFIT1 or IFIT3 (si-IFIT1-1 or si-IFIT1-2; si-IFIT3-1 or si-IFIT3-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-IFIT3-1 and si-IFIT1-2 were chosen for further experiments due to better transfection efficiency. Next, MHCC-97H and Huh-7 cells were transfected with si-NC, si-IFIT1, or si-IFIT3, and examined for E Cell migration by Transwell assay; F Cell migration by wound healing assay; G Cellular protein content and distribution of Vimentin was examined by IF staining; H The protein levels of Vimentin and Twist was examined by Immunoblotting; I The concentrations of IL-1β and IL-17 in the culture medium by ELISA. * P < 0.05, ** P < 0.01, compared with the si-NC group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Transfection, Small Interfering RNA, Negative Control, Migration, Transwell Assay, Wound Healing Assay, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A ENCORI was used to predict miRNAs that circUBAP2 might target, and TargetScan was used to predict miRNAs that might target IFIT1/IFIT3. These two sets of miRNAs intersected at hsa-miR-4756-5p, hsa-miR-1321, hsa-miR-552-3p, hsa-miR-3611, and hsa-miR-24-3p. The expression of miR-4756-5p, miR-1321, miR-552-3p, miR-3611, and miR-24-3p was examined in MHCC-97H and Huh-7 cells with or without CXCL11 treatment. miR-4756 was chosen for further experiments due to its downregulation in both HCC cell lines in response to CXCL11 stimulation. B MHCC-97H and Huh-7 were transfected with si-circUBAP2 and examined for the expression of miR-4756 by real-time PCR. C The expression of miR-4756 was examined in 12 cases of nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by real-time PCR. D miR-4756 overexpression or inhibition was achieved in MHCC-97H and Huh-7 cells by transfecting miR-4756 mimics or miR-4756 inhibitor; the transfection efficiency was confirmed by real-time PCR. E MHCC-97H and Huh-7 cells were transfected with miR-4756 mimics or miR-4756 inhibitor and examined for the protein levels of IFIT1 and IFIT3 by Immunoblotting. F – H Wild- and mutant-type circUBAP2, IFIT1 3′-UTR, or IFIT3 3′-UTR luciferase reporter plasmids were constructed as described and named as wt-circUBAP2/mut-circUBAP2, wt-IFIT1/mut-IFIT1, and wt-IFIT3/mut-IFIT3. These plasmids were co-transfected in 293T cells with miR-4756 mimics or miR-4756 inhibitor and the luciferase activity was determined. * P < 0.05, ** P < 0.01, compared to si-NC + inhibitor NC group. ## P < 0.01, compared to si-cirUBAP2+miR-4756 inhibitor group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Over Expression, Inhibition, Western Blot, Mutagenesis, Luciferase, Construct, Activity Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: MHCC-97H and Huh-7 cells were co-transfected with si-circUBAP2 and miR-4756 inhibitor and examined for A The protein levels of IFIT1 and IFIT3 by Immunoblotting; B The concentrations of IL-1β, and IL-17 in the culture medium by ELISA; C Cell migration by Transwell assay; D Cell migration by wound healing assay; E The cellular protein content and distribution of Vimentin was examined by IF staining; F The protein levels of Vimentin and Twist was examined by Immunoblotting. * P < 0.05, ** P < 0.01, compared with the si-NC + inhibitor NC group; ## P < 0.01, compared with the si-circUBAP2 + miR-4756 inhibitor group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, Wound Healing Assay, Staining