Fig. 4: PCAT1 acts as a sponge of miR-326. | Cell Death & Disease

Fig. 4: PCAT1 acts as a sponge of miR-326.

From: Long noncoding RNA PCAT1, a novel serum-based biomarker, enhances cell growth by sponging miR-326 in oesophageal squamous cell carcinoma

Fig. 4

a Fractionation of KYSE30 cells followed by RT-qPCR was used to determine the localization of PCAT1. GAPDH served as the control for cytoplasmic expression, and NEAT1 was the control for nuclear expression. b Schematic diagram of binding sites between PCAT1 and miR-326, as well as the mutation of binding sites in PCAT1. The mutated sequences are underlined. c–e RT-qPCR analysis of miR-326 expression in KYSE30, KYSE150 and KYSE450 cells after transfection of miR-326 mimics. U6 was used as an internal reference. Statistical significance was assessed using two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. f–h Dual-luciferase reporter assays showed the effect of miR-326 mimics on the luciferase activity of wild-type and mutant PCAT1 constructs in KYSE30, KYSE150 and KYSE450 cells. Statistical significance was assessed using two-tailed Student’s t test. *P < 0.05; **P < 0.01. i RT-qPCR analysis of PCAT1 expression in KYSE30 and KYSE150 cells after transfection of miR-326 mimics. GAPDH was used as an internal reference. Statistical significance was assessed using two-tailed Student’s t test. *P < 0.05; **P < 0.01. j, k RT-qPCR analysis of PCAT1 and miR-326 expression in KYSE150 cells co-transfected with PCAT1 vector and miR-326 mimics. GAPDH or U6 was used as an internal reference. Statistical significance was assessed using two-tailed Student’s t test. *P < 0.05; **P < 0.01. l, m Colony formation assay was performed in PCAT1-overexpressed KYSE150 cells transfected with miR-326 mimics. Representative images (l) and quantification analysis (m) are shown (n = 3). Statistical significance was assessed using one-way ANOVA. *P < 0.05; **P < 0.01. n The growth curve monitored by the RTCA-MP system was performed in PCAT1-overexpressed KYSE150 cells transfected with miR-326 mimics. o Western blot analysis of KRAS level in KYSE150 cells transfected with miR-326 mimics. p Western blot analysis of AKT and phospho-AKT (p-AKT) in PCAT1-overexpressed KYSE150 cells transfected with miR-326 mimics. Representative images (left) and quantification analysis (right) are shown (n = 3). Statistical significance was assessed using one-way ANOVA. *P < 0.05

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