CDR1as regulated by hnRNPM maintains stemness of periodontal ligament stem cells via miR-7/KLF4

Abstract

CDR1as is a well-identified circular RNA with regulatory roles in a variety of physiological processes. However, the effects of CDR1as on stemness of periodontal ligament stem cells (PDLSCs) and the underlying mechanisms remain unclear. In this study, we detect CDR1as in human PDLSCs, and subsequently demonstrate that CDR1as maintains PDLSC stemness. Knockdown of CDR1as decreases the expression levels of stemness-related genes and impairs the cell's multi-differentiation and cell migration abilities, while overexpression of CDR1as increases the expression levels of stemness-related genes and enhances these abilities. Furthermore, our results indicate that the RNA-binding protein hnRNPM directly interacts with CDR1as and regulates its expression in PDLSCs. In addition, we show that CDR1as promotes the expression of stemness-related genes in PDLSCs by inhibiting miR-7-mediated suppression of KLF4 expression. Collectively, our results demonstrate that CDR1as participates in the molecular circuitry that regulates PDLSC stemness.

Keywords: KLF4; circRNA CDR1as; hnRNPM; miR-7; periodontal ligament stem cell; stemness.

FIGURE 1

Identification and validation of PDLSCs and CDR1as. A. PDLSCs were derived from PDL explants on day 7 and cultured in normal medium until passage number 3. B. Single colonies formed after PDLSCs were cultured for 10 days. C. PDLSCs were characterized by detection of mesenchymal stem cell surface markers (CD44 and CD90) through flow cytometric analysis. Leukocyte markers (CD34 and CD45) were used as a negative control. D. Alizarin Red staining showing the mineralized matrix of osteo‐induced PDLSCs. E. Oil Red O staining showing the oil deposition in adipo‐induced PDLSCs F. Divergent and convergent primers of CDR1as were designed to specifically target the circular junction site and the linear region of CDR1as, respectively, for qRT‐PCR. The circular structure of CDR1as was validated by agarose gel electrophoresis. G. The head‐to‐tail splicing of the CDR1as RT‐PCR product was confirmed by Sanger sequencing. Scale bar (A, B, D, E), 100 μm

FIGURE 2

Knockdown of CDR1as down‐regulates the expression of stemness markers and PDLSC osteogenic differentiation. A. Approximately 80% of PDLSCs were green fluorescent, and thus successfully transfected by lentivirus, in the sh‐NC, sh‐CDR1as#1 and sh‐CDR1as#2 groups. The CDR1as expression levels in these groups were analysed by qRT‐PCR. The sh‐CDR1as#1 group was selected for subsequent experiments. B. mRNA and protein expression levels of stemness‐associated genes (SOX2, OCT4 and Nanog) as measured by qRT‐PCR and Western blot in the sh‐CDR1as#1 and sh‐NC groups. C. Mineralized matrix deposition by PDLSCs cultured with osteogenic inductive medium for 21 days, as demonstrated by Alizarin Red staining and quantified by CPC assay. D. Protein expression levels of ALP and Runx2 after culturing with osteogenic inductive medium for 7 days, as demonstrated by ALP staining and Western blot. E. Oil deposition of PDLSCs after culturing with adipogenic inductive medium for 21 days, as demonstrated by Oil Red O staining and quantified by optical absorbance at 510 nm after adding isopropyl alcohol. Scale bar (A, C, D, E), 100 μm. Quantitative qRT‐PCR data are presented as mean ± SD of three experiments. *P < .05; **P < .01; NS, not significant, by Student's t test

FIGURE 3

Knockdown of CDR1as inhibits migration and wound healing capacities of PDLSCs. A. DNA synthesis of PDLSCs was assessed by EdU assay after transfection with sh‐CDR1as#1 and sh‐NC for 48 h. B. Quantitative EdU assay data from A. C. Proliferation of PDLSCs in three groups was assessed at 24 h, 48 h and 72 h using a CCK8 kit. D. Migration ability of PDLSCs in three groups was assessed by transwell assay. Cells that migrated to the underside of the membrane were stained and counted. E. The average wound widths at 0 h and 24 h were analysed to assess the wound healing capacity of PDLSCs. Scale bar (A), 100 μm. Scale bar (D, E), 200 μm. Quantitative data are presented as mean ± SD. *P < .05; NS, not significant, by Student's t test

FIGURE 4

Overexpression of CDR1as up‐regulates the expression of stemness markers and PDLSC osteogenic differentiation. A. Approximately 80% of PDLSCs were red fluorescent, and thus successfully transfected by lentivirus, in the ov‐NC and ov‐CDR1as groups. The expression levels of CDR1as in these groups were analysed by qRT‐PCR. B. mRNA and protein expression levels of stemness‐associated genes (SOX2, OCT4 and Nanog) as measured by qRT‐PCR and Western blot in the ov‐CDR1as and ov‐NC groups. C. Mineralized matrix deposition by PDLSCs cultured with osteogenic inductive medium for 21 days, as demonstrated by Alizarin Red staining and quantified by CPC assay. D. Protein expression levels of ALP and Runx2 after culturing with osteogenic inductive medium for 7 days, as demonstrated by ALP staining and Western blot. E. Oil deposition of PDLSCs after culturing with adipogenic inductive medium for 21 days, as demonstrated by Oil Red O staining and quantified by optical absorbance at 510 nm after adding isopropyl alcohol. Scale bar (A, C, D, E), 100 μm. Quantitative qRT‐PCR data are presented as mean ± SD of three experiments. *P < .05; **P < .01; NS, not significant, by Student's t test

FIGURE 5

Overexpression of CDR1as maintains migration and wound healing capacities of PDLSCs. A. DNA synthesis of PDLSCs was assessed by EdU assay after transfection with ov‐NC and ov‐CDR1as#1 for 48 h. B. Quantitative EdU assay data from A. C. Proliferation of PDLSCs in three groups was assessed at 24 h, 48 h and 72 h using a CCK8 kit. D. Migration ability of PDLSCs in three groups was assessed by transwell assay. Cells that migrated to the underside of the membrane were stained and counted. E. The average wound widths at 0 h and 24 h were analysed using ImageJ 1.51 software to assess the wound healing capacity of PDLSCs. Scale bar (A), 100 μm. Scale bar (D, E), 200 μm. Quantitative data are presented as mean ± SD. *P < .05; **P < .01; NS, not significant, by Student's t test

FIGURE 6

hnRNPM promotes the expression of CDR1as in PDLSCs. In the RNA pull‐down assay, the biotinylated CDR1as probe (biotin‐CDR1as) was designed to pull down CDR1as and RBPs. Biotinylated random oligo (biotin‐NC) was used as a negative control. A. Normalized CDR1as levels in input and biotin‐CDR1as and biotin‐NC elutions of the pull‐down assay, as measured by qRT‐PCR. B. Silver staining of eluted samples from the PDLSC pull‐down assay. C. hnRNPM protein levels in eluted samples from the pull‐down assay, as measured by Western blot. D. hnRNPM mRNA expression levels in PDLSCs transfected with si‐NC and four siRNAs targeting different regions of hnRNPM, as analysed by qRT‐PCR. E. hnRNPM protein expression levels in PDLSCs transfected with si‐NC and four siRNAs targeting different regions of hnRNPM, as analysed by Western blot. F. CDR1as levels in PDLSCs transfected with si‐NC and four siRNAs targeting different regions of hnRNPM, as analysed by qRT‐PCR. Quantitative qRT‐PCR data are presented as mean ± SD of three experiments. **P < .01, by Student's t test

FIGURE 7

CDR1as regulates stemness of PDLSCs via miR‐7 and KLF4. A. The expression levels of miR‐7 and KLF4 in the sh‐CDR1as#1 and sh‐NC groups were analysed. B. Schematic illustration showing the differences between the two luciferase reporters, including one containing the complete KLF4 3′‐UTR sequence (KLF4‐wt) and one containing the KLF4 3′‐UTR sequence, with mutated sequences in the two miR‐7 binding sites (KLF4‐mut). The reporter assay showed the luciferase activity of KLF4‐wt and KLF4‐mut in 293T cells co‐transfected with miR‐7 mimics. C. KLF4 mRNA and protein expression levels in PDLSCs transfected with si‐NC and four siRNAs targeting different regions of KLF4, as analysed by qRT‐PCR and Western blot. D. mRNA and protein expression levels of stemness‐associated genes (SOX2, OCT4 and Nanog) in PDLSCs transfected with si‐NC and siRNA targeting KLF4, as measured by qRT‐PCR and Western blot. Quantitative qRT‐PCR data are presented as mean ± SD of three experiments. **P < .01, by Student's t test