Fig. 3. The effects of Circ-4099 on the expression of extracellular matrix and inflammatory cytokines in hNP.

a Plasmid map of the Circ-4099 over-expression plasmid. b The qPCR products were used to confirm the Circ-4099 sequence. c, d hNP cells were transfected with Circ-4099, NC (PLCDH-ciR) or negative control, and total RNA was collected. qPCR was used to detect the expression of Circ-4099 and its linear RNA (DENND5A) (linear + circRNA). The results showed that the Circ-4099 over-expression plasmid can extremely enhance Circ-4099 expression. These data confirm the validity and efficiency of the Circ-4099 over-expression plasmid. e, f qRT-PCR showed that Circ-4099 over-expression led to increased Collagen II and Aggrecan mRNA expression, which was suppressed by treatment with miR-616-5p mimics, while MMP3 and ADAMTS5 showed few significant differences in regulation. g, h Western blotting also showed that he Collagen II and Aggrecan protein expression levels were significantly increased by circ-4099 over-expression treatment. We next tested the effects of Circ-4099 on expression of inflammatory cytokines in hNP. i, j The IL-1β, TNF-α, and PGE2 mRNA levels were significantly suppressed in hNP cells transduced with Circ-4099 over-expression plasmid, which was inhibited by treatment with miR-616-5p mimics. k, l IL-1β, TNF-α, IL-6, and PGE2 cytokine release into medium was detected by ELISA, which showed similar results