Silencing of circ-PRKCH protects against lipopolysaccharide (LPS)-evoked chondrocyte damage and extracellular matrix loss by the miR-140-3p/ADAM10 axis

. Circular RNAs (circRNAs) have been implicated in the pathology of osteoarthritis (OA). Nev-ertheless, the precise actions of circRNA protein kinase C eta (circ-PRKCH, hsa_circ_0032131) on OA pathogenesis are still undiscovered. Cell viability and apoptosis were determined using Cell Counting-8 Kit (CCK-8) assay and flow cytometry, respectively. The levels of circ-PRKCH, microRNA (miR)-140-3p and a-disintegrin and metallopeptidase domain 10 (ADAM10) mRNA were tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Targeted interplays among circ-PRKCH, miR-140-3p and ADAM10 were verified by dual-luciferase reporter assay. c irc-PRKCH was up-regulated in OA tissues and lipopolysaccharide (LPS)-evoked C-28/I2 cells. circ-PRKCH knockdown alleviated LPS-evoked cell injury and extracellular matrix (ECM) loss in C-28/I2 cells. Mechanistically, circ-PRKCH acted as a miR-140-3p sponge. Moreover, the silencing of circ-PRKCH exerted a protective role in LPS-evoked C-28/I2 cells by up-regulating miR-140-3p. ADAM10 was a direct target of miR-140-3p, and miR-140-3p overexpression mitigated LPS-evoked C-28/I2 cell injury and ECM loss by down-regulating ADAM10. Furthermore, circ-PRKCH mediated ADAM10 expression via sponging miR-140-3p in C-28/I2 cells. Our present study suggested that circ-PRKCH silencing alleviated LPS-evoked chondrocyte injury and ECM loss partially through the miR-140-3p/ADAM10 axis.


Introduction
Osteoarthritis (OA), the most prevalent joint disorder, affects the health in the elderly population worldwide (Martel-Pelletier et al. 2016). OA is mainly caused by extravagant chondrocyte death and extracellular matrix (ECM) loss in articular cartilage (Charlier et al. 2019). Despite developed primary care and surgery, effective treatments remain insufficient (Oo et al. 2018). A better understanding for the mechanisms of the occurrence and progression of OA will help to make a breakthrough in OA innovative therapies.
Circular RNAs (circRNAs) are a novel class of noncoding RNAs with backsplice junction via covalently linked 3'-5' ends, which serve as key players in normal development and disease (Patop et al. 2019). Recent studies also highlight that some circRNAs directly interact with microRNAs (miRNAs) through sequestration (Verduci et al. 2019), and numerous circRNAs implicate in the occurrence and progression of OA (Zhou et al. 2018;Xiao et al. 2019). Zhou et al. (2019b) uncovered that circRNA.33186 was overexpressed in the OA animal and cell model, and the depletion of circRNA.33186 restrained OA progression via mediating matrix metalloproteinase-13 (MMP-13) expression by sponging miR-127-5p. Liu et al. (2016) underscored that circRNA related to the chondrocyte ECM (circRNA-CER) accelerated chondrocyte inflammation, and its down-regulation promoted ECM formation through targeting the miR-136/MMP13 axis. Shen and colleagues demonstrated that circRNA serpin family E member 2 (circ-SERPINE2) level was reduced in the OA cartilage tissues, and the elevated circSERPINE2 expression weakened chondrocyte apoptosis and enhanced ECM formation by the regulation of the miR-1271-5p/E26 transformation-specific-related gene (ERG) axis (Shen et al. 2019). As for circRNA protein kinase C eta (circ-PRKCH, hsa_circ_0032131), derived from PRKCH gene which is associated with OA pathogenesis (Wang et al. 2015), it was discovered to be up-regulated in the peripheral blood of patients with OA ). Nevertheless, the function and mechanism of circ-PRKCH on OA pathogenesis are still undiscovered.
miRNAs have been widely recognized to be relevant to OA pathology (Malemud 2018). miR-140-3p was reported to be the most significantly up-regulated miRNA in healthy cartilage (Karlsen et al. 2014). Earlier researches had uncovered that miR-140-3p expression was strongly decreased in OA and was negatively associated with OA severity (Ntoumou et al. 2017;Yin et al. 2017). Moreover, the up-regulation of miR-140-3p played a protective role in OA aggravation (Al-Modawi et al. 2019). When we used several computational methods to help identify the mechanism of circ-PRKCH in OA pathogenesis, we found two complementary sites among circ-PRKCH, miR-140-3p and a-disintegrin and metallopeptidase domain 10 (ADAM10), which prompted us to inspect the miR-140-3p/ADAM10 axis as a molecular mediator of circ-PRKCH in OA progression.
In the current project, we initially constructed an in vitro model of OA by lipopolysaccharide (LPS)-evoked chondrocytes. Subsequently, we investigated the function and mechanism of circ-PRKCH on LPS-evoked chondrocyte injury and ECM loss.

Tissue samples
All procedures were approved by the Ethics Committee of Weifang People's Hospital. 30 OA cartilage samples were collected from the tissues excised during knee replacement surgery from OA patients (range = 46-72 years, mean age = 62.5 years) at Weifang People's Hospital. Thirty healthy knee cartilage samples were taken from volunteers (range = 40-76 years, mean age = 59.7 years, no known history of OA or rheumatoid arthritis) who underwent an amputation due to an accidental injury. The fresh tissue samples were stored at −80°C in RNAlater stabilization solution (Invitrogen, Merelbeke, Belgium). Written informed consent was provided by all subjects.

Measurement of cell viability and apoptosis
C-28/I2 cells were subjected to LPS treatment as described above or various transfections followed by 5 μg/ml of LPS challenge for 24 h. Cell viability was tested using a colorimetric Cell Counting-8 Kit (CCK-8, Yesen, Shanghai, China) based on the recommendation of producers. The number of viable cells was proportional to the 450 nM absorbance detecting by a microplate reader (Viktor X3, Perkin Elmer, Turku, Finland). Cell apoptosis assessment was carried out by flow cytometry using the Invitrogen Annexin V-FITC/ propidium iodide (PI) Kit as recommended by the manufacturers. A total of ~10,000 events per sample was used for the analysis of the apoptotic rate by a flow cytometer (BD Biosciences, Erembodegem, Belgium).

qRT-PCR
Total RNA from cartilage samples and C-28/I2 cells was extracted by homogenization in TRIzol reagent (Invitrogen) and was quantified by a Bioanalyzer (Bio-Rad, Glattbrugg, Switzerland) at 260 nm. For circ-PRKCH and ADAM10 mRNA quantification, cDNA was generated using the Bio-Rad iScript Kit and qRT-PCR was performed using Bio-Rad SYBR Green Supermix as recommended by the manufacturers. The TaqMan MicroRNA Reverse Transcription Kit and TaqMan universal master mix (Applied Biosystems, Courtaboeuf, France) were used for miR-140-3p quantification. Analysis was conducted using the 2 -ΔΔCt approach (Rivetti di Val Cervo et al. 2012) with β-actin or U6 as an internal reference gene. The PCR primers for circ-PRKCH, miR-140-3p and ADAM10 mRNA were listed in Table 1.

Statistical analysis
Each treatment was carried out at least three biological and technical replications, and data were reported as mean ± standard error of the mean. The differences between the different groups were compared using a Student's t-test or oneway analysis of variance (ANOVA) followed by the Tukey's multiple comparison test. The Spearman test was used for the determination of correlation among circ-PRKCH, miR-140-3p and ADAM10 in OA samples. Statistical significance was defined at p < 0.05.

LPS induced cell injury and ECM loss of C-28/I2 cells
To construct an in vitro model of OA, C-28/I2 cells were treated with various concentrations of LPS. CCK-8 and flow cytometry assays revealed that in contrast to the negative control, cell viability was significantly reduced (Fig. 1A) and cell apoptosis was remarkably promoted (Fig. 1B)   stimulation with 5 and 10 μg/ml of LPS. Moreover, Western blot data showed that LPS led to a decrease in the levels of proliferating marker ki-67 and anti-apoptotic protein Bcl-2 in C-28/I2 cells (Fig. 1C), supporting LPS-mediated viability suppression and apoptosis promotion. ELISA analyses showed that LPS treatment led to a striking increase in the production of pro-inflammatory cytokines IL-8, IL-1β and TNF-α in a dose-dependent manner in C-28/I2 cells ( Fig. 1D-F), indicating the enhanced impact of LPS on cell inflammation. These data together indicated that LPS induced chondrocyte damage by inhibiting cell viability and promoting cell apoptosis and inflammation. Furthermore, LPS (5 and 10 μg/ml) stimulation resulted in decreased levels of Aggrecan and Col2A1, the two main components of cartilage ECM (Fig. 1G).

circ-PRKCH was up-regulated and miR-140-3p was downregulated in OA tissues and LPS-evoked C-28/I2 cells
To preliminary observation the involvement of circ-PRKCH and miR-140-3p in OA, we determined their expression levels in OA tissues and LPS-evoked C-28/I2 cells. As shown by qRT-PCR, in comparison to the corresponding control group, circ-PRKCH level was prominently elevated in OA tissues ( Fig. 2A), and LPS stimulation dose-dependently increased the expression of circ-PRKCH in C-28/I2 cells (Fig. 2B). Conversely, OA patients had a lower miR-140-3p expression than that of control (Fig. 2C), and miR-140-3p level was significantly reduced by LPS treatment in C-28/I2 cells (Fig. 2D). Interestingly, an inverse correlation between circ-PRKCH level and miR-140-3p expression was discovered in OA tissues (Fig. 2E).

Silencing of circ-PRKCH mitigated LPS-evoked cell injury and ECM loss in C-28/I2 cells
To explore whether circ-PRKCH could affect OA progression, we then performed loss-of-function analyses using si-circ-PRKCH. Transient introduction of si-circ-PRKCH, but not the si-NC control, dramatically decreased the level of circ-PRKCH, which was elevated by LPS in C-28/I2 cells (Fig. 3A). Function experiments results revealed that in contrast to the negative control, LPS-evoked anti-viability and pro-apoptosis effects were notably abolished by circ-PRKCH  (Fig. 3B-D). Moreover, the enhanced impact of LPS on IL-8, IL-1β and TNF-α production was significantly reversed by the silencing of circ-PRKCH in C-28/I2 cells (Fig. 3E). Additionally, LPS-mediated Aggrecan and Col2A1 diminishment in C-28/I2 cells was prominently abrogated by circ-PRKCH silencing (Fig. 3F).

circ-PRKCH acted as a miR-140-3p sponge and circ-PRKCH silencing exerted a protective role in LPS-evoked C-28/I2 cells by miR-140-3p
We next determined the mechanism by which circ-PRKCH regulated LPS-evoked cell injury and ECM loss. Bioinfor-matic analysis for the interacted miRNAs of circ-PRKCH showed that it harbored seven nucleotides of sequence complementarity to the miR-140-3p seed region (Fig.  4A). Transient transfection of miR-140-3p mimic, but not the miR-NC control, remarkably reduced the activity of luciferase reporter gene fused on the wild-type circ-PRKCH reporter (WT-circ-PRKCH, Fig. 4B). However, little change was observed in luciferase activity of mutanttype circ-PRKCH construct (MUT-circ-PRKCH) in the presence of miR-140-3p mimic (Fig. 4B). qRT-PCR data showed that circ-PRKCH expression was notably increased by the corresponding overexpression plasmid (Fig. 4C). Moreover, in comparison to their counterparts, miR-140- 3p expression was significantly increased by circ-PRKCH silencing and was highly decreased when circ-PRKCH upregulation in C-28/I2 cells (Fig. 4D). These data together strongly pointed the role of circ-PRKCH as a sponge of miR-140-3p.

ADAM10 was directly targeted and repressed by miR-140-3p
miRNAs exert biological function through silencing gene expression. To identify the targets of miR-140-3p, the star-Base v.3 online software was used, and the data revealed a putative target sequence for miR-140-3p in the 3'UTR of ADAM10 (Fig. 5A). Transfection of ADAM10 3'UTR reporter construct (WT-ADAM10 3'UTR) in the presence of miR-140-3p mimic led to a striking reduction in luciferase activity, and this effect was completely abolished by the mutation of the target region (MUT-ADAM10 3'UTR, Fig.  5B). The data of qRT-PCR and Western blot assays showed that in comparison to their counterparts, ADAM10 was highly expressed at both mRNA and protein levels in OA tissues and LPS-evoked C-28/I2 cells (Fig. 5C-F). Excitingly, ADAM10 mRNA expression was inversely correlated with miR-140-3p level in OA tissues (Fig. 5G). qRT-PCR data also revealed that miR-140-3p was up-regulated in miR-140-3p mimic-transfecting cells (Fig. 5H). Additionally, ADAM10 level was significantly reduced by miR-140-3p overexpression and elevated following miR-140-3p depletion in C-28/ I2 cells (Fig. 5I).

Overexpression of miR-140-3p alleviated LPS-evoked C-28/I2 cell injury and ECM loss by down-regulating ADAM10
Transfection of ADAM10 overexpression plasmid led to a prominent elevation in ADAM10 expression at both mRNA and protein levels in C-28/I2 cells (Fig. 6A,B). Subsequent experiments data revealed that in contrast to the miR-NC group, miR-140-3p overexpression triggered a significant enhancement in cell viability and a distinct repression in cell apoptosis (Fig. 6C-E), as well as a striking reduction in the production of IL-8, IL-1β and TNF-α (Fig. 6F) in LPS-treated C-28/I2 cells. Moreover, the elevated miR-140-3p expression resulted in increased Aggrecan and Col2A1 levels in LPS-induced C-28/I2 cells (Fig. 6G). Nevertheless, these effects of miR-140-3p overexpression were remarkably abrogated by ADAM10 up-regulation in LPS-induced C-28/ I2 cells (Fig. 6C-G).

Discussion
OA is a debilitating joint disease with a high morbidity in the elderly population; however, little effective strategies are available to prevent OA development because of the inadequate understanding of OA pathology (Ebell 2018). Recently, circRNAs have been underscored as essential players in the pathogenesis of a wide variety of human diseases (Han et al. 2018). Nevertheless, it is still largely undefined about their roles in the occurrence and progression of OA. In this investigation, we explored whether circ-PRKCH affected LPS-evoked chondrocyte injury and ECM loss and whether the miR-140-3p/ADAM10 axis was involved in the regulatory networks of circ-PRKCH in OA.
LPS could promote chondrocyte inflammation and degeneration and trigger ECM expression, thereby contributing to OA progression (Wang 2018;Qing et al. 2019). Here, firstly, we successfully constructed an in vitro model of OA by LPS. Interestingly, we discovered that circ-PRKCH was highly expressed in OA cartilage samples and LPS elevated the expression of circ-PRKCH in C-28/I2 chondrocyte cells, supporting the finding by Wang et al. (2019). For the first time, we disclosed that the silencing of circ-PRKCH retarded LPS-evoked chondrocyte injury and ECM loss, as evidenced by the promotion in cell viability and the suppression in cell apoptosis and inflammation, as well as the increase in Aggrecan and Col2A1 levels.
circRNAs exert critical function in human diseases via mediating gene expression through acting as effective miR-NA sponges (Panda 2018). We then predicted the interacted miRNAs of circ-PRKCH using the CircInteractome database (Dudekula et al. 2016), and we verified that circ-PRKCH functioned as a sponge of miR-140-3p. Increasing evidence has shown that miR-140-3p plays a key role in a series of Zhu et al. (2018) reported that miR-140-3p level was decreased in peripheral artery disease, and it regulated the In-Stent Restenosis by targeting B cell lymphoma-2. Liu et al. (2019) underscored that the elevated miR-140-3p expression accelerated fracture healing by activating the Wnt pathway. Moreover, miR-140-3p was identified to hinder the progression of multiple tumors, such as breast cancer and colorectal cancer (Jiang et al. 2019;Zhou et al. 2019a). In this work, we observed a prominent downregulation of miR-140-3p in OA samples and LPS-evoked chondrocytes, in agreement with former studies (Ntoumou et al. 2017;Yin et al. 2017). We also validated that miR-140-3p overexpression exerted a protective effect in LPS-evoked chondrocyte injury and ECM loss, consistent with recent work (Al-Modawi et al. 2019). More interestingly, we were first to highlight that circ-PRKCH silencing alleviated LPSevoked chondrocyte injury and ECM loss by up-regulating miR-140-3p. We subsequently testified that ADAM10 in C-28/I2 cells was directly targeted and repressed by miR-140-3p. Among these predicted targets of miR-140-3p by the starBase v.3 software, ADAM10 caught our attention owing to its essential effect in skeletal growth and bone remodeling in humans and mice (Dallas et al. 1999;Mizuno et al. 2020). ADAM10 expression was reported to be increased in OA cartilage, and overexpressed ADAM10 led to cartilage injury (Chubinskaya et al. 2001;Yang et al. 2017). Moreover, the elevated ADAM10 expression could promote CD44 cleavage, giving rise to ECM loss in human chondrocytes (Kobayakawa et al. 2016). Additionally, ADAM10 was involved in the regulation of miR-20a on human arthritis progression and inflammation (Xie et al. 2020). Herein, we first uncovered that miR-140-3p overexpression alleviated LPS-evoked chondrocyte injury and ECM loss by down-regulating ADAM10. Zafar and colleagues underscored that miR-140-3p was correlated with OA pathogenesis via targeting ADAMTS5 (Rasheed et al. 2018). Furthermore, for the first time, we highlighted that circ-PRKCH protected against ADAM10 repression by acting as a miR-140-3p sponge. Previous evidence had shown that ADAM10 regulated the activation of Notch and NF-κB signaling pathways (Toonen et al. 2016;Alabi et al. 2018), which are implicated in OA progression (Saito et al. 2017). Therefore, more investigations about the relationship between the circ-PRKCH/miR-140-3p/ADAM10 axis and the two pathways on OA pathogenesis will be explored in further work. Besides, the current work was limited to in vitro investigations, and more in vivo studies about the new mechanism using the OA animal model is expected to be carried out in further work.