miR-26a and its target CKS2 modulate cell growth and tumorigenesis of papillary thyroid carcinoma

Abstract

Background: While many studies have shown that levels of miR-26a are lower in papillary thyroid carcinoma (PTC), the role and mechanism of miR-26a in PTC are unclear.

Method: We used database searches to select potential mRNA targets of miR-26a. Anti-miR-26a, miR-26a mimic, siRNA for CKS2 and their effects on cell growth, cell-cycle distribution and colony formation were evaluated. We also evaluate the over-expressed miR-26a in TPC-1 cells in severe combined immune-deficient mice. We used luciferase reporter assays, real-time PCR and western blot analysis to measure the expression and activity of miR-26a, CKS2, and related factors such as cyclin B1, cyclin A, cdk1, bcl-xl and Akt. Finally, we measured the relationship between the levels of miR-26a and CKS2 in PTC and normal thyroid tissues.

Results: Relative to normal thyroid tissues, miR-26a is consistently down-regulated in TPC specimens, and CKS2 was identified as a potential target. Up-regulated miR-26a expression or down-regulated CKS2 expression in TPC-1 and CGTH W3 cells lines caused G2 phase-arrest. Decreased miR-26a expression or increased CKS2 expression could have inverse function on PTC cell lines. CyclinB1, cyclinA, bcl-xl and AKt are indirectly regulated by miR-26a in a CKS2-dependent manner. Finally, CKS2 is overexpressed in PTC specimens relative to normal thyroid tissue, and a significant inverse correlation exists between miR-26a and CKS2 expression in clinical PTC specimens.

Conclusion: Our data indicate that miR-26a functions as a growth-suppressive miRNA in PTC, and that its suppressive effects are mediated mainly by repressing CKS2 expression.

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).

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.