Figure 1.
Expression level of miR-20a in human thyroid tissue samples.
Y axis represents relative miR-20a expression level normalized to U6 (2−ΔΔCt value). Quantitative real-time PCR was performed using total RNA from 65 human tissues (8 ATCs, 22 DTCs, 24 benign, and 11 normal). Error bars represent standard error of mean (* indicates p<0.05).
Figure 2.
MiR-20a overexpression inhibits cellular proliferation.
(A–D). Thyroid cancer cell line proliferation with miR-20a overexpression. The Y axis represents the cell number. (E–F) Thyroid cancer in vivo growth, ex vivo tumor harvests and weight. Error bars represent standard error of mean (* indicates p<0.05; ** indicates p<0.01).
Figure 3.
MiR-20a overexpression decreases tumor spheroids.
(A) Representative image of spheroids in culture with miR-20a overexpression. (B) Quantification of spheroid difference with miR-20a overexpression. The total area occupied by the spheroids within an image was measured by circumscribing the perimeter of each spheroid, marking the entire area, and calculating the pixel numbers with ImageJ software (Maryland, USA). The Y axis represents the size and number of the spheroids. Error bars represent SEM (* indicates p<0.05).
Figure 4.
MiR-20a overexpression inhibits thyroid cancer cell invasion.
(A) TPC-1 thyroid cancer cell line, (B) XTC-1 thyroid cancer cell line, (C) FTC-133 thyroid cancer cell line, and (D) C643 thyroid cancer cell line. The Y axis represents the invasion index of the thyroid cancer cells. Error bars represent standard error of mean (* indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001).
Figure 5.
MiR-20a overexpression decreases LIMK1 protein expression in thyroid cancer cell lines.
(A) Immunoblots for LIMK1 protein expression in C643, XTC-1, FTC-133, and TPC-1 cell lines, which were transfected with either miR-20a or miR-NC for 72 hours. (B) Immunoblots for endogenous LIMK1 and GAPDH in FTC-133 cells transfected with either miR-20a or miR-NC for 7 days, 14 days and 21days.
Table 1.
Genes identified to be regulated by miR-20a using both microarray analysis and target scan analysis.*
Figure 6.
MiR-20a targets LIMK1 and LIMK1 regulates cellular invasion.
(A) Luciferase activity of pEZX-LIMK1-UTR in FTC-133 cells when co-transfected with miR-20a or miR-NC. All luciferase measurements were made in triplicates and readings were performed at 24 hours post-transfection. Error bars represent standard error of mean (* indicates p<0.05). (B) LIMK1 siRNA knockdown in FTC-133 thyroid cancer cell line. LIMK1 mRNA expression by quantitative RT-PCR (top panel). LIMK1 protein expression by Western blot (bottom panel). Data shown is for 72 hours after siRNA transfection. (C) Cellular invasion with LIMK1 knockdown. Transfection of LIMK1 siRNAs inhibited FTC-133 thyroid cancer cells invasion. The Y axis represents the invasion index of the thyroid cancer cells. Data shown is for 72 hours after siRNA transfection. Error bars represent standard error of mean (* indicates p<0.05; ** indicates p<0.01). (D) Transfection of LIMK1 siRNAs has no effect on the migration of FTC-133 thyroid cancer cells. The Y axis represents the wound distance. Data shown is for 72 hours after siRNA transfection. Error bars represent standard error of mean.