Regulation of Srpr Expression by miR-330-5p Controls Proliferation of Mouse Epidermal Keratinocyte

Abstract

Srpr is a gene encoding α subunit of the signal recognition particle receptor which is involved in the targeting and translocation of nascent secretory and membrane proteins to the endoplasmic reticulum. Previous studies showed aberrant expression of Srpr in several cell types with abnormal growth rate. Although Srpr is expressed in various tissues including skin, the role of Srpr in keratinocytes and regulation of its expression by miRNAs have not been studied. In this study, we investigated the role of SRPR and regulation of its expression by miRNA in skin keratinocytes. We found that SRPR was highly expressed in epidermal keratinocytes and regulated keratinocyte proliferation by affecting cell cycle progression. We also demonstrated that miR-330-5p directly inhibits Srpr expression. These data suggest that miR-330-5p-mediated regulation of the SRPR level is needed for the regulation of proliferation of epidermal keratinocytes.

Fig 1. Abundant expression of SRPR in mouse epidermal keratinocyte.

(A-B) Srpr was abundantly expressed in keratinocyte (PAM212 and HaCaT cell) and mouse dorsal skin at both mRNA by RT-PCR (A) and protein level by western blot analysis (B). (C-D) SRPR expression in dorsal skin (C) and HF (D) of BALB/C mice at postnatal days P10, P14, P17, P21, P28, P35, P42 and P49 by immunohistochemistry. Brown signals indicated the SRPR-positive epidermis cells (arrowhead), HF (arrow) and hair cortex (star). Scale bar = 100 μm.

Fig 2. SRPR regulates the proliferation of keratinocyte.

(A) Western blot analysis showed that SRPR protein expression was significantly decreased at 50nM and 100nM Srpr siRNA transfected PAM212 cells. GAPDH was used as a loading control. (B) Quantitative analysis of western blot using ImageJ software. The data was normalized against GAPDH expression. (C) Proliferation assay revealed that Srpr siRNA transfection inhibited proliferation of PAM212 cells in comparison with control at both 50 nM and 100 nM in a dose-dependent manner. (D) Relative viable cells were measured at 48 h after transfection. (E, F) In contrast, relative viable PAM212 cells were increased by transfection of Srpr CDS construct. (G, H) Cell cycle assay was performed and revealed that Srpr knockdown induced cell cycle arrest at G0/G1 and number of cell at S phase were decreased. (A-H) Results are the average of three independent experiments *P<0.05; **P<0.01; ***P<0.001. NS = no significant.

Fig 3. MiR-330-5p regulates Srpr expression in mouse epidermal keratinocyte.

(A) Targetscan algorithm predicted that a conserved binding sequence of miR-330-5p was present in the 3’ UTR of Srpr mRNA. (B) Real-time quantitative PCR was performed to validate the result of previous study using the same total RNA used for microarray analysis. (C) MiR-330-5p down-regulated the Srpr expression at 25nM, 50nM and 100nM mimic transfected PAM212 cells. The data was normalized against Gapdh expression. (D) Western blot analysis also revealed that SRPR protein level was decreased at 50nM and 100nM miR-330-5p mimic transfected PAM212 cells in comparison to the control. β-actin was used as a loading control. (E) Quantitative analysis of western blot using ImageJ software. The SRPR band intensity was normalized against GAPDH expression. (F) Srpr expression was significantly increased by inhibition of miR-330-5p. (B-F) Results are the average of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Fig 4. Srpr is a direct target of miR-330-5p in mouse keratinocyte.

(A) Location of the binding site for miR-330-5p in 3’UTR of Srpr mRNA. Deletion is depicted in red hyphen. (B) Dual Luciferase assay showed that miR-330-5p directly inhibited the luciferase activity by targeting the binding site in Srpr 3’UTR. However, luciferase activity did not change significantly when cells were co-transfected with the deletion mutant lacking the miR-330-5p-binding site and the miR-330-5p. (C-E) Mir-330-5p suppressed the proliferation of PAM212 cells through inhibition of SRPR expression. (C) Real-time quantitative PCR showed that cotransfection of miR-330-5p and the full length SRPR-cDNA (Srpr CDS) was able to abolish the decreased expression of Srpr by miR-330-5p alone. (D) Proliferation of PAM212 cells was decreased by miR-330-5p. Co-transfection with the Srpr CDS construct rescued inhibition of cell proliferation by miR-330-5p. (E) Relative viable cells were measured at 72 h after transfection. (B-E) Results are the average of three independent experiments. **P<0.01; ***P<0.001. NS = no significance.