Research Paper Volume 16, Issue 19 pp 12909—12927

Figure 3. Knockdown of MIAT impairs the arecoline-induced myofibroblastic transformation in BMFs. (A) Primary nBMFs (obtained from two healthy individuals; nBMFs−1 and −2) were cultured with arecoline (0, 10, and 20 μg/mL) for 24 hours, followed by RNA-sequencing analysis to determine the levels of MIAT (p < 0.01). (B) Normal BMFs (−1 and −2) were cultured with arecoline (0, 5, 10, and 20 μg/mL) for 24 hours, followed by qRT-PCR analysis to determine the MIAT expression. (C–F) Normal BMFs (−1 and −2) were transfected with lentiviruses expressing non-targeting ShRNA (Sh-Luc.) and Sh-MIAT (Sh-MIAT−1 and −2). After 48 hours, the cells were cultured with or without arecoline (20 μg/mL) for 24 hours for the induction of myofibroblasts transdifferentiation. The expression of MIAT in each group was assessed using qRT-PCR analysis. Data are presented as mean ± SD (n=3); *p < 0.05 vs. Sh-Luc.; #p < 0.05 vs. Sh-Luc. with arecoline treatment (C). The protein expression of α-SMA in each group was determined using Western blotting analysis (D). Cells (nBMFs−1 and −2) were cultured in collagen gel for an additional 48 hours, and the resulting gel area after cell contraction was measured (E). Cells (nBMFs−1 and −2) were cultured in Transwell system for an additional 24 hours, and their migration ability was quantified (F). Data are presented as mean ± SD (n=3); *p < 0.05 vs. Sh-Luc.; #p < 0.05 vs. Sh-Luc. with arecoline treatment; Scale bar, 50 μm (E, F).