Fig. 3: Impact of five miRNAs on BMP signalling cascade. | Cell Death Discovery

Fig. 3: Impact of five miRNAs on BMP signalling cascade.

From: Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma

Fig. 3

Immunoblot analysis with cell lysates of Huh7 cells transfected with (A) vector (pRNAU6.1) and five miRNAs independently, and (B) scrambled oligo, ACVR2A-AS, and BMPR1B-AS oligo to verify the expression of total SMAD5, p-SMAD5, ERK1/2, and p-ERK1/2. GAPDH was an internal loading control. (C) The expression analysis of SMAD4 in the purified nuclear and cytoplasmic fraction of Huh7 cells transfected with vector, pPre-miRNA, and pPre-miRNA + anti-miRNA oligo. Histone H3 and GAPDH were used as loading control for nuclear and cytoplasmic fraction respectively. Huh7 cells were transfected with vector, pPre-miRNA, and pPre-miRNA + anti-miRNA oligo, and assessed the expression of (D) EMT markers (SNAIL and Vimentin) and (E) Stemness markers (OCT4, CD44, and NANOG) by immunoblot analysis, (F) quantified spheroid number on ultralow attachment plate, and (G) detected doxorubicin sensitivity. Huh7 cells were transfected with vector, ACVR2A-AS, and BMPR1B-AS and quantified (H) the expression of EMT markers (ZEB1, SNAIL, VIMENTIN, E-CADHERIN) and Stemness markers (CD133, OCT4, NANOG, CD44) by qRT-PCR, (I) spheroid number, and (J) doxorubicin sensitivity. p value was calculated using unpaired student’s t-test for F, G, I, J, and two-way ANOVA for H. *, **, *** indicate p value < 0.05, <0.01, and <0.001, respectively.

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