Figures & data
Figure 1. FGD5-AS1 expression is abnormal high in PC cells and plays carcinogenic effect
(A) The expression of FGD5-AS1 in PAAD tissues and the normal tissues was obtained from the TCGA database via searching GEPIA (http://gepia.cancer-pku.cn/). (B) The expression of FGD5-AS1 in PC cells and the normal cell was detected by RT-qPCR. (C) The knockdown efficiency of FGD5-AS1 in PANC-1 and SW1990 cells transfected with sh-FGD5-AS1 was detected via RT-qPCR. (D–E) Colony formation and EdU assay were applied for estimating cell proliferative capability in cells transfected with inhibited FGD5-AS1. (F) Flow cytometry analysis was conducted to evaluate cell apoptosis when FGD5-AS1 was knocked down in cells. (G) Transwell assay was applied for measuring cell migratory capability after silencing FGD5-AS1. (H) Western blot assay tested the protein level of Bax, Bcl2, MMP2 and MMP9. *P < .05, **P < .01.
Figure 2. FGD5-AS1 acts as a sponge of miR-520a-3p
(A–B) The location of FGD5-AS1 in cells was tested through FISH and subcellular fractionation assays. (C) RNA pull down detected the binding situation between FGD5-AS1 and the possible miRNAs. (D) MiR-520a-3p expression was estimated via RT-qPCR in PC cells. (E) RIP assay was carried out to detect the relationship between miR-520a-3p and FGD5-AS1. (F) RNA pull down assay was performed to verify the combination between miR-520a-3p and FGD5-AS1. (G) The binding sites of miR-520a-3p and FGD5-AS1. (H) RT-qPCR of the transfection efficiency of miR-520a-3p. (I) The interaction of miR-520a-3p and FGD5-AS1 was further proven through luciferase reporter assay. **P < .01.
Figure 3. KIAA1522 is targeted by miR-520a-3p in PC cells
(A) Through the prediction of PITA, RNA22, miRmap, microT and miRanda database under the strict condition (CLIP-Data ≥5), there were 14 mRNAs that might bind with miR-520a-3p. (B) RT-qPCR analysis was utilized to test the expression level of the candidate mRNAs when miR-520a-3p was overexpressed in PANC-1 and SW1990 cells. (C) RT-qPCR analysis was utilized to test KIAA1522 expression when FGD5-AS1 was silenced. (D–E) Western blot assay detected KIAA1522 protein level in cells transfected miR-520a-3p mimics or sh-FGD5-AS1. (F) RIP assay was applied for measuring the correlation of FGD5-AS1, miR-520a-3p and KIAA1522. (G) RNA pull down assay further proved the combination of miR-520a-3p and KIAA1522. (H) The binding sites of miR-520a-3p and KIAA1522 were predicted by starBase. (I) Luciferase reporter assay was applied for verifying the binding sites of miR-520a-3p and KIAA1522. **P < .01.
Figure 4. FGD5-AS1 expedites cell proliferation and migration of PC via upregulation of KIAA1522
(A) Transfection efficiency of KIAA1522 was detected via RT-PCR as well as western blot assay. (B–C) The cell proliferative ability was measured by colony formation and EdU assay in different groups. (D) Flow cytometry analysis was conducted to estimate cell apoptosis in different groups. (E) Transwell assay was performed to evaluate cell migratory capability in different transfection groups. (F) Western blot assay was utilized to access protein levels of Bax, Bcl2, MMP2 and MMP9 in different transfection groups. **P < .01.
