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. 2019 Apr 8;234(11):19895–19910. doi: 10.1002/jcp.28588

Figure 6.

Figure 6

CircFNDC3B increased CD44 levels by binding to IGF2BP3. (a) The level of CD44 mRNA in MGC‐803 and BGC‐823 cells was evaluated by qRT‐PCR. MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) for 48 hr. BGC‐823 cells were transfected with siRNA (S1, S2) or negative control siRNA (NC) for 48 hr. The results suggested that ectopic expression of circFNDC3B increased CD44 mRNA, whereas silencing of circFNDC3B reduced CD44 mRNA (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (b,c) Western blot was performed to evaluate CD44 protein in MGC‐803 and BGC‐823 cells. The gray scale quantitative results suggested ectopic expression of circFNDC3B remarkably upregulated CD44 levels, and inversely, silencing of circFNDC3B downregulated CD44 levels. (d) We also overexpressed circFNDC3B on BGC‐823 cells, and the results showed that overexpression of circFNDC3B increased CD44 expression (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B). (e) We detected the role of IGF2BP3 between circFNDC3B and CD44 mRNA. MGC‐803 cells were transfected with siRNA against IGF2BP3 (S1, S2, S3) to detect the level of silencing IGF2BP3 and CD44 expression by Western blot. The results showed that silencing IGF2BP3 obviously reduced CD44 expression (WT: wild‐type; NC‐IGF2BP3: negative control; S1‐IGF2BP3, S2‐IGF2BP3, S3‐IGF2BP3: siRNA targeting IGF2BP3). (f) Then, MGC‐803 cells were transfected with siRNA against IGF2BP3 and transfected with the circFNDC3B vector again after 48 hr. Western blotting was performed to detect CD44 levels. The gray scale quantitative results suggested that when IGF2BP3 was silenced in MGC‐803 cells, overexpression of circFNDC3B did not truly increase the expression of CD44 (WT: wild‐type; NC+EV: negative control siRNA and empty vector; S1+OVER, S2+OVER, S3+OVER: siRNA against IGF2BP3 and circFNDC3B vector). (g) qRT‐PCR results showed that IGF2BP3 mRNA and CD44 mRNA and circFNDC3B levels were regulated (WT: wild‐type; NC+EV: negative control siRNA and empty vector; S1+OVER, S2+OVER, S3+OVER: siRNA against IGF2BP3 and circFNDC3B vector). Data were expressed as the mean ± SEM and were analyzed by independent samples t test, n = 3. *p < 0.05, ** p < 0.01, *** p < 0.0001. FNDC3B: fibronectin type III domain‐containing protein 3B; qRT‐PCR: quantitative reverse transcription‐PCR; SEM: standard error of the mean; siRNA: small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]