MicroRNA-18a prevents senescence of mesenchymal stem cells by targeting CTDSPL

Abstract

Stem cell therapy requires massive-scale homogeneous stem cells under strict qualification control. However, Prolonged ex vivo expansion impairs the biological functions and results in senescence of mesenchymal stem cells (MSCs). We investigated the function of CTDSPL in the premature senescence process of MSCs and clarified that miR-18a-5p played a prominent role in preventing senescence of long-term cultured MSCs and promoting the self-renewal ability of MSCs. Over-expression of CTDSPL resulted in an enlarged morphology, up-regulation of p16 and accumulation of SA-β-gal of MSCs. The reduced phosphorylated RB suggested cell cycle arrest of MSCs. All these results implied that CTDSPL induced premature senescence of MSCs. We further demonstrated that miR-18a-5p was a putative regulator of CTDSPL by luciferase reporter assay. Inhibition of miR-18a-5p promoted the expression of CTDSPL and induced premature senescence of MSCs. Continuous overexpression of miR-18a-5p improved self-renewal of MSCs by reducing ROS level, increased expression of Oct4 and Nanog, and promoted growth rate and differentiation capability. We reported for the first time that the dynamic interaction of miR-18a-5p and CTDSPL is crucial for stem cell senescence.

Keywords: CTDSPL; mesenchymal stem cells; miR-18a-5p; senescence.

Figure 1

Late passaged UCMSCs showed senescence-associated changes and impaired biological functions. (A) Population double time (PDT) of UCMSCs during long-term expansion. (B) The cell size of early and late passaged UCMSCs was measured using forward scatter (FSC) and side scatter (SSC) by flow cytometry. (C) Immunophenotypes characterization of early and late passaged UCMSCs. (D) SA-β-gal staining of early and late passaged UCMSCs. (E) Quantification of the SA-β-gal staining positive cells (n = 3). (F) Western blot analysis of p16 expression in UCMSCs of passages 4, 8, and 11. (G) Early and late passaged UCMSCs were induced to osteogenic (Ost) or adipogenic (Ado) differentiation. Osteogenic efficiency was evaluated by alkaline phosphatase (ALP) staining; adipogenic efficiency was evaluated by oil red O staining. (H) The differentiation rate was quantized. (I) Wound healing assay was performed for early and late passaged UCMSCs and (J) migrated cell numbers were quantified in 0 hours, 6 hours, 12 hours and 24 hours (n = 3). ^***p < 0.001.

Figure 2

miR-26a/b and CTDSPL expression were up-regulated following senescence of UCMSCs. (A) Quantitative RT-PCR assay was performed to detect miR-26a and miR-26b expression in UCMSCs. (B) Gene structure of CTDSPL. (C) Western blot assay was performed to detect CTDSPL expression in UCMSCs. (D) The expression of P16 was quantized. (E) CTDSPL was also quantized. ^*p < 0.05; ^**p < 0.01.

Figure 3

Over-expression of CTDSPL induced premature senescence of UCMSCs. (A) SA-β-gal staining of control and CTDSPL over-expressed UCMSCs. (B) SA-β-gal positive cells were quantified (n = 3). (C) Morphology changes were quantified by flow cytometry. (D) Representative confocal microscopy images of GFP tagged control or CTDSPL transfected UCMSCs. F-actin was labeled with Alexa Fluor 633 conjugated phalloidin; nuclei were stained with Hoechst 33342. (E) Western blot assay of p16 expression after transfected with control or CTDSPL plasmid. (F) Western blot assay of RB and pRB expression after transfected with control or CTDSPL plasmid. ^***p < 0.001.

Figure 4

Inhibition of miR-18a-5p induced premature senescence of UCMSCs. (A) Relative expression of miR-18a-5p following passaging was analyzed by quantitate RT-PCR. (B) Western blot assay of CTDSPL expression after miR-NC inhibitor or miR-18a-5p inhibitor transfection. (C) SA-β-gal staining of UCMSCs transfected with miR-NC inhibitor or miR-18a-5p inhibitor. (D) SA-β-gal positive cells were quantified (n = 3). (E) Western blot assay of p16 expression in UCMSCs after miR-NC or miR-18a-5p inhibitor transfection. (F) Schematic representation of the reporter plasmids psiCHECK2-CTDSPL-3UTR-Wild, psiCHECK2-CTDSPL-3UTR-Mutation and psiCHECK2-CTDSPL-3UTR-Delation. (G) Luciferase reporter assay was performed to verify the direct repression of CTDSPL by miR-18a-5p. ^*p < 0.05; ^**p < 0.01.

Figure 5

miR-18a-5p overexpression reduced ROS levels of late passaged UCMSCs (passage 11). (A, B) Total ROS, mitochondrial mass and mitochondrial ROS levels of early and late passaged UCMSCs were detected by flow cytometry (A) and confocal microscope (B). (C) Flow cytometry analysis of the total ROS, mitochondrial mass and mitochondrial ROS levels of miR-18a-5p and control lentivirus vector transduced UCMSCs and (D) relative fluorescence intensity of miR-18a-5p overexpressing groups relative to control groups were quantified (n = 3). ^*p < 0.05; ^**p < 0.01.

Figure 6

Stable expression of miR-18a-5p improved self-renewal of UCMSCs. (A) Western blot assay of Oct4, Nanog and P16 in miR-18a-5p and control lentivirus vector transduced UCMSCs. (B, C) Competitive growth assay of miR-18a-5p and control transduced UCMSCs. GFP ratios were measured by flow cytometry before and after two passaging cultures. (D) GFP ratio of UCMSCs in competitive growth assay before and after culture. The efficiency of osteogenic differentiation (E) was increased in UCMSCs stably expressing miR-18a-5p (F), and similarly, the potential for adipogenic differentiation (G) was enhanced in the group with stable miR-18a-5p transduction. Osteogenic efficiency was evaluated by ALP staining; adipogenic efficiency was evaluated by oil red O staining. The results were quantized in Figure F and Figure H. ^*p < 0.05; ^**p < 0.05.