Fig. 8

LINC00662 regulated CLDN8/IL22 co-expression and the activation of ERK signaling pathway by competitively binding with miR-340-5p (a) TargetScan database showed that binding site of CLDN8/or IL22 and miR-340-5p; (b) Luciferase reporter assays were used to prove that miR-340-5p can target CLDN8/or IL22; (c) Western blot was used to detect the expressions of CLDN8, IL22, p-ERK and ERK in protein levels in miR-340-5p inhibitors transfected HCT29 and LS174T cells and miR-340-5p mimics transfected LOVO and CT26 cells; (d) Statistical graph of CLDN8 protein level; (e) Statistical graph of IL22 protein level; (f) Statistical graph of p-ERK/ERK level; (g) cBioPrortal database showed co-expression relationship of CLDN8 and IL22 genes; (h) Co-immunoprecipitation experiments showed that CLDN8 directly interact with IL22 in colon cell lines; After LINC00662 overexpression plasmids and miR-340-5p mimics were transfected into HCT29 and LS174T cells, (i and j) Western blot was used to detect the expressions of CLDN8, IL22, p-ERK and ERK in protein levels; After LINC00662 knockdown plasmids and miR-340-5p mimics were transfected into LOVO and CT26 cells, (k and l) Western blot was used to detect the expressions of CLDN8, IL22, p-ERK and ERK in protein levels; (m-p) Luciferase activity of vector containing different region of IL22 was measured in cells with LINC00662 overexpression in colon cancer cell lines (HCT29, LS174T, LOVO and CT26 cells). GAPDH was used as a load control. Data are presented as the mean ± standard deviation. *P < 0.01 vs. NC inhibitors and #P < 0.01 vs. NC mimics