Primary cilia are hornlike sensory organelles that play an important role in regulating the function of chondrocyte[17]. INPP5E regulates mitosis and ciliary disassembly of primary cilia[5]. However, the regulation of INPP5E on primary cilia in chondrocytes has not been reported. IFT88 is a core component of the intracflagellar transport complex B and is essential for cilia construction[18]. Downregulation or loss of IFT88 is known to impair cilia occurrence[19]. Ace-tubulin is a key protein in regulating ciliary peristalsis and controlling its stability[20]. In this study, we found that silencing INPP5E inhibited the expression of IFT88 and ace-tubulin, suggesting that inhibition of INPP5E reduced primary cilia formation in chondrocytes.
Recent studies have shown that activation of autophagy requires primary cilia, and that autophagy is involved in controlling cilia formation[21]. In this study, we treated chondrocytes with CH, the expression of beclin-1, LC3 I and LC3 II were reduced significantly. The elevated expression of beclin-1, LC3 I or LC3 II was positive associated with the level autophagy[22, 23]. These results further suggest that inhibition of primary cilia reduces chondrocytes autophagy.
MiRNAs are small non-coding RNAs that inhibit gene mRNA expression by interfering with transcription[24]. In this study, two miRNAs hsa-miR-181a-5p and hsa-miR-138-5p were found bound with INPP5E, and inhibits its expression. Hsa-miR-181a-5p inhibits autophagy in MCF-10A cells[25]. Hsa-miR-138-5p inhibits autophagy in pancreatic cancer cells[26]. Over-expression of hsa-miR-181a-5p or hsa-miR-138-5p inhibits beclin-1, LC3 I or LC3 II in chondrocytes. Over-expression of hsa-miR-181a-5p or hsa-miR-138-5p inhibits the promoting effect of INPP5E on beclin-1, LC3 I or LC3 II in chondrocytes. Therefore, hsa-miR-181a-5p or hsa-miR-138-5p inhibits autophagy of chondrocytes by inhibiting INPP5E.
The effect of hsa-miR-181a-5p or hsa-miR-138-5p on primary cilia has not been reported. In the current study, over-expression of hsa-miR-181a-5p or hsa-miR-138-5p inhibits IFT88 and ace-tubulin in chondrocytes, suggesting that hsa-miR-181a-5p or hsa-miR-138-5p inhibits autophagy of chondrocytes by inhibiting primary cilia. Moreover, over-expression of hsa-miR-181a-5p or hsa-miR-138-5p inhibits the promoting effect of INPP5E on IFT88 and ace-tubulin in chondrocytes. Therefore, hsa-miR-181a-5p or hsa-miR-138-5p down-regulates chondrocyte autophagy by inhibiting inPP5E-induced primary cilia.
CeRNAs are mutually regulated transcripts of competing shared miRNAs at the post-transcriptional level. The CeRNA network links the function of mRNAs to non-coding RNAs such as miRNAs, lncRNAs, and circRNAs[27]. In this study, SNHG12 was found bound with hsa-miR-181a-5p or hsa-miR-138-5p, and inhibits its expression. SNHG12 has been reported to promote autophagy[16]. In this study, the role of SNHG12 in promoting autophagy was also confirmed in chondrocytes. Moreover, silencing SNHG12 inhibits the promoting effect of INPP5E on beclin-1, LC3 I or LC3 II in chondrocytes. Therefore, SNHG12 promotes autophagy of chondrocytes through the hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E axis. In addition, we found for the first time that SNHG12 promotes the expression of IFT88 and ace-tubulin in chondrocytes. Silencing SNHG12 inhibits the promoting effect of INPP5E on IFT88 and ace-tubulin in chondrocytes. Therefore, SNHG12 promotes chondrocyte autophagy by promoting INPP5E-induced primary cilia formation.
Inhibition of mTORC1 signaling by primary cilia has been demonstrated[6]. In this study, we also found that after treating chondrocytes with CH, the protein expression of p-mTOR was increased. Interestingly, expression of ace-tubulin was elevated when we treated chondrocytes with rapa. Therefore, there exists a mTOR-primary cilia-mTOR loop in chondrocytes. MTOR is a highly conserved kinase that is important for autophagy regulation[28].In this study, we found that inhibition of mTOR promotes the autophagy in chondrocytes. In addition, inhibition of INPP5E reduced the promoting effect of rapa on autophagy. Therefore, INPP5E promotes chondrocyte autophagy by inhibiting mTOR-primary cilia-mTOR loop.
Collagen II is one of the cross-linked copolymers of the core fiber network during chondrogenesis[29]. Articular cartilage maintains its function based on the longevity of Collagen II [30]. Silencing INPP5E inhibits the expression of collagen II in chondrocytes. Silencing SNHG12 or over-expression of hsa-miR-181a-5p or hsa-miR-138-5p inhibits the promoting effect of INPP5E on collagen II in chondrocytes. These results suggest that SNHG12 promotes collagen II through hsa-miR-181a-5p/hsa-miR-138-5p- INPP5E axis in chondrocytes. The proliferation and maturation of chondrocytes requires attachment to a matrix rich in collagen II[31]. Thus, SNHG12 might the proliferation and maturation of chondrocytes. Dicam is reported to promote chondrocyte proliferation and maturation through hedgehog signaling pathway in primary cilia[32]. Inhibition of PI3K/AKT/mTOR signaling pathway inhibits chondrocyte proliferation[33]. Therefore, SNHG12 might promote chondrocyte proliferation and maturation by activating mTOR signal via promoting INPP5E-mediated primary cilia formation.
Cyclin D1 integrates extracellular mitotic signaling and cell cycle progression[34]. Silencing INPP5E inhibits the expression of cyclin D1 in chondrocytes. Silencing SNHG12 or over-expression of hsa-miR-181a-5p or hsa-miR-138-5p inhibits the promoting effect of INPP5E on cyclin D1 in chondrocytes. These results suggest that SNHG12 promotes collagen II through hsa-miR-181a-5p/hsa-miR-138-5p- INPP5E axis in chondrocytes. These results suggest that SNHG12 might maintain the cell cycle of chondrocytes through hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E axis.
In conclusion, SNHG12 upregulates INPP5E via inhibiting hsa-miR-181a-5p/hsa-miR-138-5p in chondrocytes. INPP5E increasing autophagy of chondrocytes via de-activating the mTOR-primary cilia-mTOR loop. Our results provide a theoretical basis for the treatment of patients with cartilage abnormalities related diseases.