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. 2018 Sep 15;19(1):334.
doi: 10.1186/s12891-018-2253-x.

Up-regulated expression of E2F2 is necessary for p16INK4a-induced cartilage injury

Xinnan Bao  1 , Xinyu Hu  2
Affiliations

Up-regulated expression of E2F2 is necessary for p16INK4a-induced cartilage injury

Xinnan Bao et al. BMC Musculoskelet Disord. .

Abstract

Background: Cartilage degradation would result in osteoarthritis (OA). p16INK4awas found in some age-related diseases. In this study, we aimed to determine the role of p16INK4a during OA and to investigate the underlying mechanisms.

Methods: Enzyme-linked immunosorbent assay (ELISA) was performed to test the activity of senescence-associated secretory phenotype (SASP). Real-time PCR (RT-PCR) and Western blot were used to determine the expressions of target genes.

Results: The increased expressions of p16INK4a and E2F2 were accompanied with cartilage degradation induced by IL-1β. Over-expression of p16INK4a enhanced the secretion of SASP markers (TGFβ, IL-6, IL-8, IL-1α, MMP3 and MMP13), reduced the expression of type II procollagen (COL2A1).Thus, the over-expression of p16INK4a lead to cartilage injury. Moreover, we found that the expression of E2F2 was enhanced in p16INK4a over-expression group, and that cartilage injury caused by p16INK4a was alleviated by depleting E2F2.

Conclusions: p16INK4a was up-regulated during the cartilage injury in OA. p16INK4a promoted cartilage injury by increasing the expression of E2F2. Thus, this study extends the molecular regulation network for understanding pathological progression of OA, and provides potential therapeutic target for OA.

Keywords: E2F2; Osteoarthritis (OA); Senescence-associated secretory phenotype (SASP); p16INK4a.

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Conflict of interest statement

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
(a) Western blot assay for the expression of Col2A1, p16INK4a and E2F2. (b) Real-time PCR (RT-PCR) for the expression of Col2A1, p16INK4a and E2F2. (c) Western blot assay for the expression of E2F2 in the cells treated with 10 ng/ml IL-1β.*P < 0.05,**P < 0.01 vs. control. Data are shown as mean ± SD, n = 4
Fig. 2
Fig. 2
(a) Determination of the secretion of SASP markers, including TGFβ, IL-6, IL-8, IL-1α, MMP3 and MMP13, using ELISA assay. **P < 0.01 vs. control. IL-1β, cells were treated with IL-1β. Data were shown as mean ± SD, n = 5
Fig. 3
Fig. 3
(a) ELISA assay for the secretion of SASP markers after the over-expression of p16INK4a. (b) RT-PCR for detecting the expressions of Col2A1, p16INK4a and E2F2. (c) The expressions of Col2A1, p16INK4a and E2F2 by Western blot assay. *P < 0.05 and **P < 0.01 vs. empty. Empty, cells were transfected with over-expression empty vector; p16INK4a, cells were transfected with p16INK4a over-expression vector. Data were shown as mean ± SD, n = 5
Fig. 4
Fig. 4
(a) The expression of Col2A1, p16INK4a and E2F2 determined by RT-PCR. (b-c) The expressions of Col2A1, p16INK4a and E2F2 determined by Western blot assay. Empty group, cells were transfected with over-expression empty vector and siRNA negative control; p16INK4a group, cells were transfected with p16INK4a over-expression vector and siRNA negative control; E2F2 group, cells were transfected with E2F2 over-expression vector and siRNA negative control; p16INK4a + E2F2 group, cells were over-expressed with E2F2 and p16INK4a and siRNA negative control; si-E2F2 group, cells were transfected with si-E2F2 and over-expression empty vector; p16INK4a + si-E2F2 group, cells were transfected with si-E2F2 and p16INK4a. *P < 0.05 vs. empty, #P < 0.05 vs. p16INK4a. Data were shown as mean ± SD, n = 4
Fig. 5
Fig. 5
ELISA assay for SASP markers. Empty group, cells were transfected with over-expression empty vector and siRNA negative control. p16INK4a group, E2F2 indicated the over-expressions of p16INK4a and E2F2 respectively. Si-E2F2 indicated the depletion of E2F2. *P < 0.05 vs. empty. #P < 0.05, vs. p16INK4a. Data were shown as mean ± SD, n = 5
Fig. 6
Fig. 6
(a) Cell proliferation was detected by WST-1 cell counting kit. *P < 0.05 vs. control. Data were shown as mean ± SD, n = 5. (b) The expressions of CDC6, MCM6, p16INK4a and E2F2 determined by RT-PCR. Data were shown as mean ± SD, n = 4.*P < 0.05 vs. empty.&P < 0.05, vs. p16INK4a
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