Additional file 1 of Hsa_circ_0009092/miR-665/NLK signaling axis suppresses colorectal cancer progression via recruiting TAMs in the tumor microenvironment
Creators: name: Song, Jialin
name: Liu, Qing
name: Han, Lei
name: Song, Tiantian
name: Huang, Sihao
name: Zhang, Xinyao
name: He, Qiuming
name: Liang, Chenxi
name: Zhu, Shuai
name: Xiong, Bin
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Statistics
subject: FOS: Mathematics

description: Additional file 1:Figure S1.Circ_0009092 was a single-stranded covalently closed RNA molecules. (A) The expression of circ_0009092 was detected by qRT-PCR in RNA extracts from several CRC cell lines as well as normal cell line. GAPDH was used for qRT-PCR normalization. (B) qRT-PCR analysis of the expression of circ_0009092 and OGDH mRNA after treatment with RNase R in Lovo cells. (C) The presence of circ_0009092 was validated in CRC cell lines by qRT-PCR. Divergent primers amplified circ_0009092 in cDNA but not in genomic DNA. GAPDH was used for negative control. (D) ROC curves showing predictability using circ_0009092 (AUC=0.7944). Statistical analysis between two groups was conducted using two-tailed t-test. Error bars, SD.Figure S2.Identification analysis of miR-665 in CRC samples. (A) FISH detection of circ_0009092 and miR-665 in CRC tumor and ANT. Nuclei were stained with DAPI. Scale bar, 50μm.Figure S3.Circ_0009092 inhibited CRC cell progression by targeting miR-665. (A) EdU assays were utilized to detect the viability and proliferation of Lovo and HCT-116 cells. (B) The colony formation activity of HCT116 and Lovo cells evaluated. (C) Wound healing assay were used to detect the migration of Lovo and HCT-116 cells. The migration (D) and invasion (E) ability of CRC cells were detected by transwell assays. Statistical analysis between two groups was performed using two-tailed t-test. One-way ANOVA statistical tests were adopted for more than two groups. Data are the means ± SD of three experiments. *P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.001.Figure S4.Circ_0009092/miR-665 regulated NLK expression in CRC cells. (A) qRT-PCR analysis of the predicted target gene expression in CRC cells. GAPDH was used for qRT-PCR normalization. (B) The correlation between NLK and circ_0009092 (n= 80). (C) Dual-luciferase reporter assays of HCT-116 cells transfected with miR-665 mimic, NLK-WT, and NLK-MUT. Data are pooled from three independent experiments. Statistical analysis between two groups was conducted using two-tailed t-test. *P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.001. ns: no significance. Error bars, SD.Figure S5.The expression distribution of immune score in tumor tissues and normal tissues. The abscissa represents immune cell types, and the ordinate represents the expression distribution of immune score in different groups. (A) Immune cell score heatmap, different colors represent different expression distribution in different samples. (B) The percentage abundance of tumor infiltrating immune cells in each sample. Different colors represent different types of immune cells. The abscissa represents the sample, and the ordinate represents the percentage of immune cell content in a single sample. The statistical difference of two groups was compared through the Wilcox test, significance difference of three groups was tested with Kruskal-Wallis test. *P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.001.Figure S6.Circ_0009092/miR-665/NLK regulated CCL2 expression to affect the recruitment of macrophages. (A) Chemotaxis analysis of THP-1 macrophages with different supernatant. (B) Quantification analysis of chemotactic cells in five fields was counted manually. Statistical analysis between two groups was conducted using two‐tailed t‐test. One‐way ANOVA statistical tests were used for more than two groups. *P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.001.Figure S7.NLK regulated the phosphorylation and O-GlcNAcylation of STAT3 by binding to STAT3. (A) Results of immunofluorescence staining showing that NLK colocalizes with STAT3 in CRC cells. (B) Interaction between STAT3 and OGT/O-GlcNAcylation modification was evaluated by coIP assays in CRC cells transfected with miR-665 mimic. (C) Interaction between STAT3 and OGT/O-GlcNAcylation modification was evaluated by coIP assays in CRC cells transfected with miR-665 inhibitor. (D) The relative CCL2 promoter transcriptional activity was detected by dual luciferase assay and normalized to Renilla luciferase activity in CRC cells transfected with oe- circ_0009092 or miR-665 mimic. (E) The relative STAT3 transcriptional activity was detected by dual luciferase assay and normalized to Renilla luciferase activity in CRC cells transfected with sh- circ_0009092 or miR-665 inhibitor. Statistical analysis between two groups was conducted using two‐tailed t‐test. One‐way ANOVA statistical tests were used for more than two groups. *P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.001.Table S1.Clinicopathologic parameters of colorectal cancer patients (n= 30).Table S2.The sequences of the primers for qRT-PCR.Table S3.Antibodies used in this study.