Upregulated circTMEM59 Inhibits Cell Growth and Metastasis by miR-668-3p/ID4 Axis in Colorectal Cancer

Figure 6

ID4, a target gene of miR-668-3p, is regulated by circTMEM59. (a) Venn diagram of predicted miR-668-3p targets by three programs (miRWalk, TargetScan, and miRDB). (b) The seed regions of miR-668-3p, the seed-recognizing sites in the ID4 3 UTR, and the nucleotides mutated in ID4 mutant 3 UTR are shown. (c) Luciferase reporter assay was conducted to verify that miR-668-3p bound to the 3-UTR region of ID4 directly. miR-668-3p overexpression significantly suppressed, while miR-668-3p loss increased the luciferase activity that carried wild-type (WT) but not mutant (MUT) 3-UTR of ID4. (d) RIP assays confirmed the binding status between miR-668-3p and ID4 in CRC cell lines, respectively. (e) Relative expression of ID4 in CRC tissues, adjacent tissues, and different tumor size of CRC. (f) Correlation analysis of the expression of circTMEM59 and ID4, miR-668-3p and ID4 in 100 CRC samples. (g) miR-668-3p overexpression decreased the level of ID4 protein in HT29 cells, while miR-668-3p reduction increased the level of ID4 protein in DLD1 cells. (h) CircTMEM59 reduction decreased the level of ID4 protein in HT29 cells, and circTMEM59 overexpression increased the level of ID4 protein in DLD1 cells. (i) Relative protein levels of ID4 in HT29 cells cotransfected with NC, sh-circTMEM59 + Ctl, and sh-circTMEM59 + inhibitor. (j) Relative protein levels of ID4 in DLD1 cells cotransfected with Ctl, circTMEM59 + NC, and circTMEM59 + mimics. (k) The IHC of ID4, Bcl-2, caspase-3, CDK4, cyclin D1, E-cadherin, Vimentin, and ki-67 in the low expression ID4 of tumors and high expression ID4 of tumors (scale bar: 100 μm). Data represent the . Student’s -test was used to determine statistical significance: , , and .

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)