Figure 1.
Average expression levels of miR-26a and CKS2 in human PTC specimens (n = 40) vs. normal thyroid tissues (n = 40).
A. miR-26a down-regulation in PTC specimens (n = 40) vs. normal thyroid tissues (n = 40) (p<0.001). B. Increased CKS2 expression increased in PTC specimens results shown by RT-PCR and western blot (p = 0.003).
Table 1.
The clinical date of analyzed tissue samples.
Figure 2.
miR-26a inhibition of TPC-1 cell growth.
A. Effect of miR-26a on TPC-1 cell proliferation measured by CCK8 assay. B. Effect of stable over-expression of miR-26a on TPC-1 cell proliferation measured by CCK8 assay. C. Expression of miR-26a in miR-26a-TPC-1 cells is higher than EV-TPC-1 cell and TPC-1 cell. D. Colony formation assay of TPC-1 cells transfected with miR-26a, miR control, anti-miR-26a, anti-miR control, and miR-26a-TPC-1 cell vs EV-TPC-1 cell. E. The relative numbers of colonies obtained by counting five vision fields. Error bars correspond to 95% confidence intervals. F. Cell-cycle distribution of TPC-1 cells transfected with miR-26a mimic, anti-mir-26a and their nonspecific controls for 48 h. G. Flow cytometry and Annexin V assays showing the number of cells in apoptosis in TPC-1 cells stably over-expressing miR-26a or control TPC-1 cells with blank PLL3.7 (EV-TPC-1 cell).
Figure 3.
CKS2 is a target of miR-26a and miR-26a regulates CKS2 downstream signaling molecules.
A. Putative binding site of miR-26a on the CKS2 3′UTR along with the mutation in the predicted seed region. B. Reporter assays on HEK-293T cells transfected with the reporter vectors containing either the wildtype or mutated CKS2 3′UTR. C. Protein levels of CKS2 in TPC-1 cells transfected with miR-26a mimic, anti-miR-26a or their nonspecific controls. D. miR-26a mediated reduction of CKS2 levels is associated with decreased expression of cyclinB1 and cdk1, and increased expression of bcl-xl and Akt, shown by RT-PCR and western blot. E. siRNA for CKS2 decreases expression of cyclinB1 and cdk1, and increases expression of bcl-xl and Akt, shown by RT-PCR and western blot. F. Transfection with anti-miR-26a increases cyclin B1 and cdk1 expression, and decreases bcl-xl and Akt expression, as shown by RT-PCR and western blot.
Figure 4.
Knockdown of CKS2 inhibits TPC-1 cell growth.
A. Effect of miR-26a transfection or CKS2 knockdown on TPC-1 cell proliferation measured by CCK8 assay. B. Colony formation assay of TPC-1 cells transfected with miR-26a mimic, miR control, CKS2 siRNA and siRNA control, and the relative numbers of colonies counted under a microscope in five vision fields. Error bars correspond to 95% confidence intervals. C. Cell-cycle distribution of TPC-1 cells transfected with miR-26a mimic, miR control, CKS2 siRNA and siRNA control for 48 h. D. Flow cytometry and annexin V assays reflect the number of apoptotic TPC-1 cells after transfection with miR-26a mimic, miR control, CKS2 siRNA or siRNA control.
Figure 5.
Stable over-expressison of CKS2 and anti-miR-26a promotes TPC-1 cell growth.
A. Effects of CKS2 or anti-miR-26a over-expression on TPC-1 cell proliferation. CKS2 = TPC-1 cell transfected with CKS2-PMCB vector; EV = TPC-1 cell transfected with EV-PMCB vector. B. Colony formation assay of TPC-1 cells transfected with CKS2-PMCB, EV-PMCB, anti-miR-26a and anti-miR control, and the relative numbers of colonies counted under microscope in five vision fields. Error bars correspond to 95% confidence intervals. C. Cell-cycle distribution of TPC-1 cells transfected with CKS2-PMCB, EV-PMCB, anti-miR-26a and anti-miR control for 48 h. D. Flow cytometry and Annexin V assays reflect the number of apoptotic TPC-1 cells after transfection with CKS2-PMCB, EV-PMCB, anti-miR-26a and anti-miR control.
Figure 6.
CKS2 overexpression rescues the growth suppressive effect of miR-26a.
A. Effects of CKS2 over-expression on miR-26a-TPC-1 cell proliferation. MiR-26a+CKS2 = miR-26a-TPC-1 cell transfected with CKS2-PMCB vector; MiR-26a +EV = miR-26a-TPC-1 cell transfected with EV-PMCB vector. B. Colony formation assay of miR-26a-TPC-1 cells transfected with CKS2-PMCB or EV-PMCB, and the relative numbers of colonies counted under microscope in five vision fields. Error bars correspond to 95% confidence intervals. C. Cell-cycle distribution of miR-26a-TPC-1 cells transfected with CKS2-PMCB or EV-PMCB for 48 h. D. Flow cytometry and Annexin V assays reflect the number of apoptotic miR-26a-TPC-1 cells after transfection with CKS2-PMCB, or EV-PMCB.
Figure 7.
miR-26a suppresses tumor growth of TPC-1 cells in nude mice.
A. Growth curve of tumors volumes over 21 days of observation. Each data point represents the mean ± SEM of six mice. B. The tumors were dissected from the mice and measured. C. IHC Ki-67 and PNCA staining of sections of transplanted tumors formed by TPC-1 cells infected with miR-26a-PLL3.7 or EV-PLL3.7.