Figures & data
Figure 1. MiR-144-5p was verified to be low-expressed in thyroid carcinoma.
(a) The expression of miR-144-5p in the thyroid carcinoma tissues was analyzed by qRT-PCR, U6 was used as an internal control. (b) The expression of miR-144-5p in the thyroid carcinoma cells was analyzed by qRT-PCR, U6 was used as an internal control. (c-d) The transfection efficiency of miR-144-5p mimic and inhibitor in TPC-1 (C) and IHH4 (d) cells were detected by qRT-PCR, U6 was used as an internal control. (+++P < 0.001, vs. Nthy-ori3-1; *P < 0.05, ***P < 0.001, vs. NC). (M: miR-144-5p mimic, I: miR-144-5p inhibitor, qRT-PCR: quantitative real-time PCR, NC: negative control)
Figure 2. MiR-144-5p mimic suppressed the viability, proliferation, migration, and invasion of TPC-1 and IHH4 cells, and miR-144-5p caused the opposite results.
(a, e) The proliferation of TPC-1 cells after transfection was detected by colony formation assay. (b, f) The migration rate of TPC-1 cells after transfection was evaluated by wound-healing assay. (c, g) The invasion rate of TPC-1 cells after transfection was determined by transwell assay. (d) The viability of TPC-1 cells after transfection was measured by MTT assay. (h, l) The proliferation of IHH4 cells after transfection was determined via colony formation assay. (i, m) The migration rate of IHH4 cells after transfection was measured by wound-healing assay. (j, n) The invasion rate of IHH4 cells after transfection was detected by transwell assay. (k) The viability of IHH4 cells after transfection was evaluated using MTT assay. (**P < 0.01, ***P < 0.001, vs. NC). (M: miR-144-5p mimic, I: miR-144-5p inhibitor, NC: negative control)
Figure 3. MiR-144-5p targeted STON2 and down-regulated the expression of STON2 in TPC-1 and IHH4 cells.
(a) The binding sites between miR-144-5p and STON2 were predicted by TargetScan. (b-c) The binding relationship of miR-144-5p and STON2 in TPC-1 (B) and IHH4 (C) cells was verified by dual-luciferase reporter assay. (d-e) The transfection efficiency of STON2 overexpression plasmids in TPC-1 (D) and IHH4 (E) cells was detected by qRT-PCR, GAPDH used as the internal control. (f-g) The transfection efficiency of STON2 overexpression plasmids in TPC-1 (F) and IHH4 (G) cells was detected by Western blotting, GAPDH used as the internal control. (f-i) The expression of STON2 in TPC-1 (f-g) and IHH4 (h-i) cells was detected by qRT-PCR (F, H) and Western blotting (G, I), GAPDH was used as an internal control. (+++P < 0.001, vs. MC; **P < 0.01, ***P < 0.001, vs. Vector; ^^^P < 0.001, vs. MC+Vector; ###P < 0.001, vs. M+ Vector; &&&P < 0.001, vs. MC+STON2). (M: miR-144-5p mimic, MC: mimic control)
Figure 4. MiR-144-5p mimic weakened the promoting effect of STON2 overexpression on migration and invasion of TPC-1 and IHH4 cells.
(a, c) The migration rate of TPC-1 cells after transfection was determined by wound-healing assay. (b, d) The invasion rate of the TPC-1 cells after transfection was detected by transwell assay. (e, g) The migration of IHH4 cells after transfection was measured by wound-healing assay. (f, h) The invasion of the IHH4 cells after transfection was measured by transwell assay. (^^P < 0.01, ^^^P < 0.001, vs. MC+Vector; ###P < 0.001, vs. M+ Vector; &&&P < 0.001, vs. MC+STON2). (M: miR-144-5p mimic, MC: mimic control)
Figure 5. MiR-144-5p mimic enhanced the inhibitory effects of PTX on cell viability, proliferation, migration, and invasion of TPC-1 and IHH4 cells.
(a) The viability of TPC-1 cells after PTX treatment for 48 h was detected by MTT assays. (b) The expression of miR-144-5p in TPC-1 cells after transfection and PTX treatment was detected by qRT-PCR, U6 was used as an internal control. (c) The viability of TPC-1 cells after transfection and PTX treatment was measured by MTT assay. (d, g) The proliferation of TPC-1 cells after transfection and PTX treatment was detected by colony formation assay. (E, H) The migration of TPC-1 cells after transfection and PTX treatment was detected using wound-healing assay. (f, i) The invasion of TPC-1 cells after transfection and PTX treatment was detected by transwell assay. (j) The viability of IHH4 cells after PTX treatment for 48 h was detected by MTT assay. (k) The expression of miR-144-5p in IHH4 cells after transfection and PTX treatment was quantified by qRT-PCR, U6 was used as an internal control. (l) The viability of IHH4 cells after transfection and PTX treatment was detected by MTT assay. (m, p) The proliferation of IHH4 cells after transfection and PTX treatment was detected by colony formation assay. (n, q) The migration of IHH4 cells after transfection and PTX treatment was detected by wound-healing assay. (o, r) The invasion of IHH4 cells after transfection and PTX treatment was detected by transwell assay. (**P < 0.01, ***P < 0.001, vs. MC; ^^P < 0.01, ^^^P < 0.001, vs. PTX+MC). (PTX: paclitaxel, M: miR-144-5p mimic, MC: mimic control)
Figure 6. The effect of UTMD-mediated miR-144-5p overexpression on the miR-144-5p expression, proliferation, and invasion of PTX-induced TPC-1 and IHH4 cells was stronger than liposome-mediated miR-144-5p overexpression.
(a) Microbubbles were examined by an inverted microscope. (b-c) The expression of miR-144-5p in TPC-1 (b) and IHH4 (c) cells after transfection and PTX treatment was detected by qRT-PCR, U6 was used as an internal control. (d, g) The proliferation of TPC-1 cells after transfection and PTX treatment was evaluated by colony formation assay. (E, H) The migration of TPC-1 cells after transfection and PTX treatment was detected by wound-healing assay. (f, i) The invasion rate of TPC-1 cells after transfection and PTX treatment was measured by transwell assay. (j, m) The proliferation of IHH4 cells after transfection and PTX treatment was determined by colony formation assay. (k, n) The migration of IHH4 cells after transfection and PTX treatment was detected by wound-healing assay. (l, o) The invasion rate of IHH4 cells after transfection and PTX treatment was quantified by transwell assay. (***P < 0.001, vs. Control; ^^^P < 0.001, vs. PTX; ###P < 0.001, vs. PTX+UTMD-MC;+P < 0.05, ++P < 0.01, +++P < 0.001, vs. PTX+UTMD-M). (PTX: paclitaxel, M: miR-144-5p mimic, MC: mimic control, UTMD: ultrasound targeted microbubble destruction)
Figure 7. The effect of UTMD-mediated miR-144-5p overexpression on the miR-144-5p expression, proliferation, and invasion of PTX-induced TPC-1 and IHH4 cells was stronger than liposome-mediated miR-144-5p overexpression.
(a) The expressions of MMP-9, E-cadherin, and N-cadherin in TPC-1 cells after transfection and PTX treatment were detected by Western blot. GAPDH was used as an internal control. (b) The expressions of MMP-9, E-cadherin, and N-cadherin in IHH4 cells after transfection and PTX treatment were measured using Western blot. GAPDH was used as an internal control. (***P < 0.001, vs. Control; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, vs. PTX; ##P < 0.01, ###P < 0.001, vs. PTX+UTMD-MC;+P < 0.05, ++P < 0.01, vs. PTX+UTMD-M). (PTX: paclitaxel, M: miR-144-5p mimic, MC: mimic control, UTMD: ultrasound targeted microbubble destruction)
Figure 8. The effect of UTMD-mediated miR-144-5p overexpression on STON2-overexpressed TPC-1 and IHH4 cells was stronger than liposome-mediated miR-144-5p overexpression.
(a-c) The expression of STON2 in TPC-1 cells after overexpression STON2 and miR-144-5p was detected by qRT-PCR (a) and Western blot (b-c). GAPDH was used as an internal control. (d-e) The expressions of MMP-9, E-cadherin, and N-cadherin in TPC-1 cells after overexpression STON2 and miR-144-5p was detected by Western blot. GAPDH was used as an internal control. (f-h) The expression of STON2 in IHH4 cells after overexpression STON2 and miR-144-5p was detected by qRT-PCR (f) and Western blot (g-h), GAPDH was used as an internal control. (i-j) The expressions of MMP-9, E-cadherin, and N-cadherin in TPC-1 cells after overexpression STON2 and miR-144-5p were detected by Western blot, GAPDH was used as an internal control. (**P < 0.01, ***P < 0.001, vs. Control; ^^^P < 0.001, vs. STON2; ###P < 0.001, vs. STON2+ UTMD-MC;+P < 0.05, ++P < 0.01, +++P < 0.001, vs. PTX+UTMD-M). (M: miR-144-5p mimic, MC: mimic control, UTMD: ultrasound targeted microbubble destruction)
Figure 9. UTMD-mediated miR-144-5p overexpression enhanced the inhibitory effects of PTX on the growth of subcutaneous-xenotransplant tumor.
(a) The images of the subcutaneous-xenotransplant tumor tissues. (b) The volume of the tumor tissue was recoded. (c) The tumor weight at the day 12 after treatment was recorded. (d) The expression of Ki67 in the tumor tissue was detected by immunohistochemical assay. (**P < 0.01, ***P < 0.001, vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. PTX). (PTX: paclitaxel, M: miR-144-5p mimic, MC: mimic control, UTMD: ultrasound targeted microbubble destruction)
Supplemental material
