SNHG12 Promotes Autophagy by Blocking the mTOR-Primary Cilia-mTOR Loop via Activating the miR-181a-5p/miR-138-5p-INPP5E Axis in Chondrocyte

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Introduction
It has been reported that there are numerous diseases associated with cartilage abnormalities, including chondrocalcinosis, arthropathies, and relapsing polychondritis, which can severely impact human health [1].Chondrocytes are the only cell type found in normal cartilage, and a thorough understanding of their pathology and function is crucial for both cartilage repair and engineering [2].Primary cilia are multifunctional sensory organelles that regulate various signal transduction pathways and cellular activities, playing a critical role in the regulation of chondrogenesis [3].
Inositol polyphosphate-5-phosphatase E (INPP5E) is a transporter protein located on the primary cilia membrane, and it plays an important role in regulating the development and homeostasis of primary cilia [4].INPP5E is also responsible for maintaining the stability of primary cilia [5].However, mutations or absences of INPP5E can lead to primary ciliary signal transduction defciencies [4].Primary cilia, acting as mechanical sensors, are instrumental in limiting rapamycin complex 1 (mTORC1) signal transduction by converting mechanical signals into chemical activity [6].As a result, INPP5E may be involved in mTORC1 signal transduction by regulating primary cilia.
Moreover, a potential link between primary cilia and autophagy has recently been proposed, which could increase our understanding of the pathogenesis of cartilage-related diseases [7].Autophagy is an intracellular degradation system that plays a role in maintaining intracellular energy metabolism homeostasis and has been shown to modulate the function of damaged chondrocytes [8].In addition, each step of autophagy is regulated by the target protein mTORC1 [9].Terefore, INPP5E may be involved in regulating autophagy via primary cilia-mTORC1 signal transduction.
Te competitive endogenous RNA (ceRNA) hypothesis proposes that transcripts which exhibit shared microRNA (miRNA) binding sites compete for posttranscriptional control [10].However, research on the mechanism of ceRNA involving INPP5E is limited.miR-181a-5p and miR-138-5p are microRNAs that have been implicated in autophagy [11][12][13][14].Tey are known to regulate gene expression by binding to the 3′ untranslated region (UTR) of their target mRNAs.One of the targets of miR-181a-5p and miR-138-5p is INPP5E.Studies have shown that SNHG12 acts as a competing endogenous RNA (ceRNA) for miR-181a-5p and miR-138-5p [15,16].As a result, we speculate that SNHG12 may compete with INPP5E mRNA for binding to these microRNAs, thereby sequestering them and preventing their binding to INPP5E 3′ UTR.Tis ceRNA activity of SNHG12 may lead to increased expression of INPP5E, resulting in altered mTOR signaling and potential downstream efects on autophagy, which may reveal the relationship between SNHG12 and the miR-181a-5p/miR-138-5p-INPP5E axis, suggesting a regulatory network in which SNHG12 acts as a ceRNA to modulate the expression of INPP5E by competing with miR-181a-5p and miR-138-5p.Te purpose of this study was to explore the crucial connections between ceRNA, INPP5E, primary cilia, autophagy, mTORC1, and chondrocytes and to provide new insights into the physiological mechanisms associated with chondrocytes.

Bioinformatics Information.
Te next generation sequencing of 23 cartilage knee samples was conducted using the Afymetrix Human Gene 1.1 ST Array (transcript version) platform (HuGene-1_1-st).Te R language software package "limma" was utilized to identify diferentially expressed genes (DEGs) between the INPP5E-low expression (N � 11) and INPP5E-high expression (N � 12) groups.A signifcance threshold of P < 0.05 and fold change ≥2 were applied.Te Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.kegg.jp/kegg/rest/keggapi.html) was employed to obtain the latest KEGG pathway gene annotation as the background for mapping the genes.Enrichment analysis was performed using the R software package "clusterProfler" (version 3.14.3),and P < 0.05 was considered as a signifcant diference.For gene set enrichment analysis (GSEA), we obtained the GSEA software (version 3.0) from the GSEA Database (https://software.broadinstitute.org/gsea/index.jsp).We downloaded c2.cp.kegg.v7.4.symbols.gmtfrom the Molecular Signatures Database (https://www.gseamsigdb.org/gsea/downloads.jsp) to evaluate the pathways and molecular mechanisms.A signifcance threshold of P < 0.05 was applied.

Cell
Culture.Te C28/I2 cell line, composed of normal human chondrocytes, was provided by Zhongqiao Xinzhou Biotechnology Co., LTD (Shanghai, China).Te cells were cultured in the RPMI-1640 medium, supplemented with 10% FBS and 100 U/mL penicillin and streptomycin, all provided by Zhongqiao Xinzhou Biotechnology Co., LTD, Shanghai, China.Te cells were maintained in a 5% CO 2 humidifed incubator at 37 °C.

Cell Transfection and
Treatment. 1 * 10 6 C28/I2 cells were inoculated into 6-well plates and transfected with the corresponding plasmid or small interfering RNA (siRNA) using lip3000.Te details of the plasmids and siRNAs used are presented in Supplement Table 1.Follow-up experiments were conducted 72 hours after transfection.All reagents were provided by Guangdong Ruibo Biotechnology Co., LTD., China.

Real-Time Fluorescence Quantitative PCR (RT-PCR).
Total RNA was extracted from the cells using Trizol.Te total RNA was reverse-transcribed into cDNA using RT Master Mix for qPCR (gDNA digester plus, MedChemExpress, China).PCR detection was performed using POWER SYBR GREEN PCR MASTER (4368708, Applied Biosystems, USA).Te primer sequences used in this study are provided in Supplementary Table 2. Te relative expression levels were calculated using the 2 −△△Ct method.

Western Blotting (WB).
Te proteins present in C28/I2 cells were extracted using the radioimmunoprecipitation assay bufer (RIPA) and quantifed through the bicinchoninic acid (BCA) kit (C503021, Sangong Bioengineering Co., LTD., Shanghai, China).Te proteins were then added to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transported to polyvinylidene difuoride membranes (Millipore, USA).Te transferred membranes were blocked with Tris-bufered saline with Tween 20 2 International Journal of Clinical Practice (TBS-T) (0.1% Tween 20 and TBS) containing 5% skim milk for 30 minutes and were subsequently incubated overnight at 4 °C with primary antibodies (refer to Supplement Table 3).Following this, the membranes were washed three times for 10 minutes with TBS-T and incubated for an hour at 25 °C with HRP-conjugated secondary antibodies (refer to Supplement Table 3).Visualization of the protein bands was achieved by the application of enhanced chemiluminescence using an enhanced chemiluminescence (ECL) kit (AB133406, Abcam, USA).Images were obtained with SigmaTel software V2.0, and protein density was computed through the use of Image Lab software V3.0.

SNHG12 Deactivates the PI3K-Akt-mTOR Signaling Pathway via the hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E
Axis In Vitro.Compared with the control or NC group, the mRNA expression of PI3K, Akt, and mTOR was enhanced in the mimic-1 group.Compared with the mimic-1 group, the mRNA expression of PI3K, Akt, and mTOR was reduced in the mimic-1 + OE-INPP5E group (Figure 5(a)).Compared with the control or NC group, the protein expression of p-PI3K, p-Akt, and p-mTOR was enhanced in the mimic-1 group.Compared with the mimic-1 group, the protein expression of p-PI3K, p-Akt, and p-mTOR was reduced in the mimic-1 + OE-INPP5E group (Figure 5(b)).However, there was no signifcance of the protein expression of PI3K, Akt, and mTOR among the control, NC, mimic-1, and mimic-1 + OE-INPP5E groups in chondrocytes (Figure 5(b)).Compared with the control or NC group, the mRNA expression of PI3K, Akt, and mTOR was enhanced in the mimic-2 group.Compared with the mimic-1 group, the mRNA expression of PI3K, Akt, and mTOR was reduced in the mimic-2 + OE-INPP5E group (Figure 5(c)).Compared with the control or NC group, the protein expression of p-PI3K, p-Akt, and p-mTOR was enhanced in the mimic-1 group.Compared with the mimic-2 group, the protein expression of p-PI3K, p-Akt, and p-mTOR was reduced in the mimic-2 + OE-INPP5E group (Figure 6(d)).However, there was no signifcance of the protein expression of PI3K, Akt, and mTOR among control, NC, mimic-2, and mimic-2 + OE-INPP5E group in chondrocytes (Figure 5(d)).Compared with the control or NC group, the mRNA expression of PI3K, Akt, and mTOR was enhanced in the si-SNHG12 group.Compared with the si-SNHG12 group, the mRNA expression of PI3K, Akt, and mTOR was reduced in the si-SNHG12 + OE-INPP5E group (Figure 5(e)).Compared with the control or NC group, the protein expression of p-PI3K, p-Akt, and p-mTOR was enhanced in the si-SNHG12 group.Compared with the si-SNHG12 group, the protein expression of p-PI3K, p-Akt, and p-mTOR was reduced in the si-SNHG12 + OE-INPP5E group (Figure 6(f)).However, there was no signifcance of the protein expression of PI3K, Akt, and mTOR among control, NC, si-SNHG12, and si-SNHG12 + OE-INPP5E groups in chondrocytes (Figure 5(f)).Together, silencing SNHG12 activated the PI3K-Akt-mTOR signaling pathway via the hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E axis in chondrocytes.

SNHG12 Promotes the Expression of Collagen II and Cyclin D1 via the hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E
Axis In Vitro.Compared with the control or NC group, the mRNA expression of COL2A1 and CCND1 was reduced and the protein expression of collagen II and cyclin D1 was reduced in the si-INPP5E group (Figures 6(a) and 6(b)).Compared with the control or NC group, the mRNA expression of COL2A1 and CCND1 was reduced and the protein expression of collagen II and cyclin D1 was reduced in the mimic-1 group (Figures 6(c   (i) StarBase database was used to predict the lncRNAs that might bind to hsa-miR-181a-5p and hsa-miR-138-5p.(j) RT-PCR assay was used to detect the expression of SNHG12 in control, NC, si-SNHG12-1, si-SNHG12-2, and si-SNHG12-3 groups.(k) RT-PCR assay was used to detect the expression of hsa-miR-181a-5p and hsa-miR-138-5p in control, NC, and si-SNHG12-3 groups.(l, m) Te dual luciferase reporting assay was used to verify the binding of SNHG12 to hsa-miR-181a-5p and hsa-miR-138-5p.Notes: * p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the control.

Discussion
Primary cilia are horn-like sensory organelles that play a crucial role in regulating the function of chondrocytes [21].INPP5E regulates mitosis and is responsible for the ciliary disassembly of primary cilia [5].However, the impact of INPP5E on primary cilia in chondrocytes has not yet been reported.IFT88 is a core component of the intrafagellar transport complex B and is essential for the construction of cilia [22].Downregulation or loss of IFT88 is known to impair cilia occurrence [23].Ace-tubulin, a notable protein, regulates ciliary peristalsis and controls its stability [24].Our study found that silencing INPP5E inhibited the expression of both IFT88 and ace-tubulin, thereby indicating that the inhibition of INPP5E reduces primary cilia formation in chondrocytes.
Te impact of hsa-miR-181a-5p or hsa-miR-138-5p on primary cilia has yet to be documented.In this current study, the overexpression of hsa-miR-181a-5p or hsa-miR-138-5p was found to inhibit IFT88 and ace-tubulin in chondrocytes, implying that autophagy of chondrocytes is inhibited by these miRNAs through the inhibition of primary cilia.Furthermore, overexpression of hsa-miR-181a-5p or hsa-miR-138-5p was found to counteract the promoting efect of INPP5E on IFT88 and ace-tubulin in chondrocytes.Terefore, the downregulation of chondrocyte autophagy can be attributed to the inhibition of inPP5E-induced primary cilia by hsa-miR-181a-5p or hsa-miR-138-5p.International Journal of Clinical Practice  CeRNAs regulate the transcripts of shared miRNAs in a mutually inhibiting manner at the posttranscriptional level.Tis regulation creates a network between mRNAs and noncoding RNAs such as miRNAs, lncRNAs, and circRNAs.In this study, it was discovered that SNHG12 binds with hsa-miR-181a-5p or hsa-miR-138-5p, inhibiting their expression.SNHG12 has been reported to promote autophagy in previous studies, and this study confrmed its role in chondrocytes.Furthermore, silencing SNHG12 inhibits the promoting efect of INPP5E on Beclin-1, LC3 I, or LC3 II in chondrocytes.Tus, SNHG12 promotes chondrocyte autophagy through the hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E axis.In addition, this study found that SNHG12 promotes the expression of IFT88 and ace-tubulin in chondrocytes, a fnding not previously reported.Silencing SNHG12 also inhibits the promoting efect of INPP5E on IFT88 and ace-tubulin in chondrocytes.Terefore, SNHG12 promotes chondrocyte autophagy by promoting INPP5Einduced primary cilia formation.
Te inhibition of mTORC1 signaling by primary cilia has been demonstrated in previous studies [6].In our study, we observed an increase in protein expression of p-mTOR after treating chondrocytes with CH.Interestingly, treating chondrocytes with Rapa also led to elevated expression of acetubulin.Tis suggests the presence of an mTOR-primary cilia-mTOR loop in chondrocytes.mTOR is a highly conserved kinase that plays a crucial role in regulating autophagy [31].In this study, we discovered that inhibiting mTOR promotes autophagy in chondrocytes.Furthermore, inhibiting INPP5E reduced the enhancing efect of Rapa on autophagy.Terefore, INPP5E promotes chondrocyte autophagy by inhibiting the mTOR-primary cilia-mTOR loop.
Collagen II is a cross-linked copolymer that forms the core fber network during chondrogenesis [32].Te function of articular cartilage relies on the longevity of collagen II [33].Silencing INPP5E inhibits the expression of collagen II in chondrocytes, while silencing SNHG12 or overexpression of hsa-miR-181a-5p or hsa-miR-138-5p inhibits the promoting efect of INPP5E on collagen II in chondrocytes.Tese fndings suggest that SNHG12 promotes collagen II through the hsa-miR-181a-5p/hsa-miR-138-5p/INPP5E axis in chondrocytes.Chondrocytes require attachment to a matrix rich in collagen II for their proliferation and maturation [34], and therefore, SNHG12 might play a role in these processes.Dicam stimulates chondrocyte proliferation and maturation via the hedgehog signaling pathway in primary cilia [35].Inhibition of the PI3K/AKT/mTOR signaling pathway impedes chondrocyte proliferation [36].Terefore, SNHG12 may promote chondrocyte proliferation and maturation by activating the mTOR signal via INPP5Emediated primary cilia formation.
In conclusion, our study suggests that SNHG12 upregulates INPP5E by inhibiting hsa-miR-181a-5p/hsa-miR-138-5p in chondrocytes.Tis, in turn, increases autophagy in chondrocytes by deactivating the mTOR-primary cilia-mTOR loop.Tese fndings provide a promising foundation for the development of targeted treatments for patients with cartilage-related diseases.

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International Journal of Clinical Practice LC3 I/II were investigated.INPP5E knockdown decreased the levels of ace-tubulin and LC3 I/II, while mTOR activation induced their expression, which was reversed by INPP5E silencing (Figure2(e)).Furthermore, in the CHtreated group, there was also a reduction in ace-tubulin and LC3 I/II expression, which was rescued by rapamycin treatment (Figure2(e)).Tese results suggest that the regulation of autophagy by INPP5E occurs through the modulation of primary cilia-induced mTOR inactivation.In summary, the present study provides evidence that INPP5E inhibition reduces autophagy by promoting PI3K-Akt-m-TOR signaling activation, whereas activated PI3K-Akt-m-TOR signaling further downregulates autophagy by inhibiting primary cilia.Tese fndings shed new light on the mechanisms underlying the regulation of autophagy and primary cilia in chondrocytes.
(a)).Overexpression of hsa-miR-181a-5p inhibited the mRNA (Figure 3(b)) and protein (Figure 3(c)) expression of INPP5E in chondrocytes.Compared with the mimic-NC group, the relative intensity of fuorescence in the mimic-1 group was reduced signifcantly in INPP5E-WT chondrocytes.However, there was no signifcant diference of the relative intensity of fuorescence between the mimic-NC and mimic-1 groups in INPP5E-Mut chondrocytes via the dual luciferase reported assay (Figure 3(d)).Compared with the control or NC group, the expression of hsa-miR-138-5p was increased signifcantly in the mimic-2 group (Figure 3(e)).Overexpression of hsa-miR-138-5p inhibited the mRNA (Figure 3(f )) and protein (Figure 3(g)) expression of INPP5E in chondrocytes.

Figure 1 :
Figure 1: Silencing INPP5E activates the PI3K-Akt-mTOR signaling pathway in vitro.(a) We divided the samples into the INPP5E-low expression (N � 11) group and the INPP5E-high expression (N � 12) group in the GSE43191 dataset.Using P < 0.05 and fold change ≥2 as screening criteria, a hub of 998 DEGs were identifed.(b) KEGG analysis and (c) GSEA analysis were used to analysis the pathway mechanism involved in these 998 DEGs.(d) RT-PCR assay was used to detect the expression of INPP5E.(e) WB assay was used to detect the expression of INPP5E.(f ) RT-PCR assay was used to detect the expression of PI3K, Akt, and mTOR.(g) WB assay was used to detect the expression of PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR.Notes: * p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the control.
(a)) and protein (Figure 4(b)) expression of INPP5E was enhanced signifcantly in the overexpression (OE)-INPP5E group.Compared with the control or NC group, the mRNA (Figure 4(c)) and protein (Figure 4(d)) expression of INPP5E was reduced signifcantly in the si-INPP5E group.Compared with the control or NC group, the mRNA (Figure 4(e)) and protein (Figure 4(f )) expression of IFT88, Beclin1, LC3 I, and LC3 II was reduced signifcantly in the mimic-1 group.Compared with the mimic-1 group, the mRNA (Figure 4(e)) and protein (Figure 4(f )) expression of IFT88, Beclin1, and LC3 I was enhanced signifcantly in the mimic-1 + OE-INPP5E group.Compared with the control or NC group, the mRNA (Figure 4(g)) and protein (Figure 4(h)) expression of IFT88, Beclin1, LC3 I, and LC3 II was reduced signifcantly in the mimic-2 group.Compared with the mimic-2 group, the mRNA (Figure 4(g)) and protein (Figure 4(h)) expression of IFT88, Beclin1, and LC3 I was enhanced signifcantly in the mimic-2 + OE-INPP5E group.Compared with the control or NC group, the mRNA (Figure 4(i)) and protein (Figure 4(j)) expression of IFT88, Beclin1, LC3 I, and LC3 II was reduced signifcantly in the si-SNHG12 group.Compared with the si-SNHG12 group, the mRNA (Figure 4(i)) and protein (Figure 4(j)) expression of IFT88, Beclin1, and LC3 I was enhanced signifcantly in the si-SNHG12 + OE-INPP5E group.Compared with the control or NC group, the protein expression of ace-tubulin and LC3 I/II was increased signifcantly in the OE-INPP5E group.Compared with the OE-INPP5E group, the protein expression of ace-tubulin and LC3 I/II was decreased signifcantly in the si-SNHG12 + OE-INPP5E group, mimic-1 + OE-INPP5E group, or mimic-2 + OE-INPP5E group (Figure 4(k) ) and 6(d)) and mimic-2 group (Figures 6(e) and 6(f )).Compared with the mimic-1 group or the mimic-2 group, the mRNA expression of COL2A1 and CCND1 was increased and the protein expression of collagen II and cyclin D1 was increased in the mimic-1 + OE-INPP5E group (Figures6(c) and 6(d)) and the mimic-2 + OE-INPP5E group (Figures 6(e) and 6(f )), respectively.Compared with the control or NC group, the mRNA expression of COL2A1 and CCND1 was reduced and

Figure 2 :
Figure 2: Silencing INPP5E inhibits autophagy via regulating the mTOR-primary cilia-mTOR loop in vitro.(a) RT-PCR assay was used to detect the mRNA expression of IFT88, Beclin1, MAP1LC3A, and MAP1LC3B in control, NC, and si-INPP5E-2 groups.(b) WB assay was used to detect the protein expression of IFT88, Beclin1, LC3 I, and LC3 II in control, NC, and si-INPP5E-2 groups.(c) RT-PCR assay was used to detect the mRNA expression of IFT88, Beclin1, MAP1LC3A, MAP1LC3B, PI3K, Akt, and mTOR in control and CH-treated groups.(d) WB assay was used to detect the protein expression of IFT88, Beclin1, LC3 I, LC3 II, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR in control and CH-treated groups.(e) Ace-tubulin was used to label primary cilia, and LC3 II was used to label autophagy via immunofuorescence assay.Notes: * p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the control or between the indicated groups.

Figure 3 :
Figure 3: SNHG12 promotes the expression of INPP5E via hsa-miR-181a-5p/hsa-miR-138-5p in vitro.(a) RT-PCR assay was used to detect the expression of hsa-miR-181a-5p in control, mimic-NC, and mimic-1 groups.(b) RT-PCR assay was used to detect the expression of INPP5E in control, mimic-NC, and mimic-1 groups.(c) WB assay was used to detect the expression of INPP5E in control, mimic-NC, and mimic-1 groups.(d) Te dual luciferase reporting assay was used to verify the binding of INPP5E to hsa-miR-181a-5p.(e) RT-PCR assay was used to detect the expression of hsa-miR-138-5p in control, mimic-NC, and mimic-2 groups.(f ) RT-PCR assay was used to detect the expression of INPP5E in control, mimic-NC, and mimic-2 groups.(g) WB assay was used to detect the expression of INPP5E in control, mimic-NC, and mimic-2 groups.(h) Te dual luciferase reporting assay was used to verify the binding of INPP5E to hsa-miR-138-5p.(i)StarBase database was used to predict the lncRNAs that might bind to hsa-miR-181a-5p and hsa-miR-138-5p.(j) RT-PCR assay was used to detect the expression of SNHG12 in control, NC, si-SNHG12-1, si-SNHG12-2, and si-SNHG12-3 groups.(k) RT-PCR assay was used to detect the expression of hsa-miR-181a-5p and hsa-miR-138-5p in control, NC, and si-SNHG12-3 groups.(l, m) Te dual luciferase reporting assay was used to verify the binding of SNHG12 to hsa-miR-181a-5p and hsa-miR-138-5p.Notes: * p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the control.

Figure 4 :
Figure 4: Silencing SNHG12 promotes primary cilia and autophagy via the hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E axis in vitro.(a, b) RT-PCR and WB assay were used to detect the expression of INPP5E in control, NC, and OE-INPP5E groups.(c, d) RT-PCR and WB assay were used to detect the expression of INPP5E in control, NC, and si-SNHG12 groups.(e, f) RT-PCR assay was used to detect the mRNA expression of IFT88, Beclin1, MAP1LC3A, and MAP1LC3B, and WB assay was used to detect the protein expression of IFT88, Beclin1, LC3 I, and LC3 II in control, NC, mimic-1, and mimic-1 + OE-INPP5E groups.(g, h) RT-PCR assay was used to detect the mRNA expression of IFT88, Beclin1, MAP1LC3A, and MAP1LC3B, and WB assay was used to detect the protein expression of IFT88, Beclin1, LC3 I, and LC3 II in control, NC, mimic-2, and mimic-2 + OE-INPP5E groups.(i, j) RT-PCR assay was used to detect the mRNA expression of IFT88, Beclin1, MAP1LC3A, and MAP1LC3B, and WB assay was used to detect the protein expression of IFT88, Beclin1, LC3 I, and LC3 II in control, NC, si-SNHG12-1, and si-SNHG12-1 + OE-INPP5E groups.(k) Ace-tubulin was used to label primary cilia, and LC3 II was used to label autophagy via immunofuorescence assay.Notes: * p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the control or between the indicated groups.

Figure 6 :
Figure 6: Silencing SNHG12 inhibits the expression of collagen II and cyclin D1 via the hsa-miR-181a-5p/hsa-miR-138-5p-INPP5E axis in vitro.(a) RT-PCR assay was used to detect the expression of COL2A1 and CCND1 in control, NC, and si-INPP5E groups.(b) WB assay was used to detect the expression of collagen II and cyclin D1 in control, NC, and si-INPP5E groups.(c) RT-PCR assay was used to detect the expression of COL2A1 and CCND1 in control, NC, mimic-1, and mimic-1 + OE-INPP5E groups.(d) WB assay was used to detect the expression of collagen II and cyclin D1 in control, NC, mimic-1, and mimic-1 + OE-INPP5E groups.(e) RT-PCR assay was used to detect the expression of COL2A1 and CCND1 in control, NC, mimic-2, and mimic-2 + OE-INPP5E groups.(f ) WB assay was used to detect the expression of collagen II and cyclin D1 in control, NC, mimic-2, and mimic-2 + OE-INPP5E groups.(g) RT-PCR assay was used to detect the expression of COL2A1 and CCND1 in control, NC, si-SNHG12, and si-SNHG12 + OE-INPP5E groups.(h) WB assay was used to detect the expression of collagen II and cyclin D1 in control, NC, si-SNHG12, and si-SNHG12 + OE-INPP5E groups.* p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the control or between the indicated groups.