Long non-coding RNA MCM3AP-AS1 protects chondrocytes ATDC5 and CHON-001 from IL-1β-induced inflammation via regulating miR-138-5p/SIRT1

Figures & data

Table 1. The primer sequences used in the present work

Name	Primer sequences
MCM3AP-AS1	Forward: 5ʹ-GCTGCTAATGGCAACACTGA-3’
	Reverse: 5ʹ-AGGTGCTGTCTGGTGGAGAT-3’
MiR-138-5p	5ʹ-AGCTGGTGTTGTGAATCAGG-3ʹ
SIRT1	Forward: 5ʹ-GTATTTATGCTCGCCTTGCTG-3’
	Reverse: 5ʹ-TGACAGAGAGATGGCTGGAA-3’
U6	Forward: 5ʹ -CTCGCTTCGGCAGCACA-3’
	Reverse: 5ʹ -AACGCTTCACGAATTTGCGT-3’
GAPDH	Forward: 5′-ACGGATTTGGTCGTATTGGG-3 ′
	Reverse: 5′-CGCTCCTGGAAGATGGTGAT-3 ′

Figure 1. IL-1β induces inflammatory injury to chondrocytes in vitro. (a, b) CHON-001 and ATDC5 cells were treated with IL-1β (0 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL), and the viability of the cells was measured by CCK-8 assay. (c, d) Flow cytometry was employed to measure the apoptosis rate of CHON-001 and ATDC5 cells. ns.: not statistically significant

*P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control.

Table 2. Relationship between MCM3AP-AS1 expression and clinicopathological characteristics of knee OA

Characteristic	Cases	MCM3AP-AS1		P-value
		Low (n = 15)	High (n = 15)
Age (yr)
≥ 60	17	7	10	0.269
< 60	13	8	5
Gender
Male	11	6	5	0.704
Female	19	9	10
K-L category
III-Ⅳ	14	10	4	0.028*
I–II	16	5	11

Figure 2. MCM3AP-AS1 expression in OA. (a, b) qRT-PCR showed that MCM3AP-AS1 was significantly down-regulated in the cartilage tissues of OA patients and OA models in vitro.

***P < 0.001 compared with the control.

Figure 3. Overexpression of MCM3AP-AS1 promotes the viability of chondrocytes, inhibits apoptosis and inflammatory response in OA models in vitro. (a) qRT-PCR showed that the MCM3AP-AS1 overexpression model was successfully constructed. (b, c) CCK-8 assay showed that overexpression of MCM3AP-AS1 significantly promoted the viability of chondrocytes. (d, e) Transwell assay showed that overexpression of MCM3AP-AS1 significantly promoted the migration of chondrocytes. (f, g) Flow cytometry assay showed that overexpression of MCM3AP-AS1 significantly inhibited apoptosis of chondrocytes. (H, I, J) ELISA assay showed that overexpression of MCM3AP-AS1 significantly inhibited the levels of IL-6, IL-8 and TNF-α in OA models in vitro.

*P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control.

Figure 4. MiR-138-5p is the target of MCM3AP-AS1. (a) StarBase database showed that miR-138-5p may be a potential target for MCM3AP-AS1. (b) Dual-luciferase reporter gene assay depicted that miR-138-5p mimics can significantly reduce the luciferase activity of wild-type MCM3AP-AS1 reporter, and has no effect on that of mutant MCM3AP-AS1 reporter. (c) qRT-PCR suggested that miR-138-5p was up-regulated in the cartilage tissues of OA patients. (d) qRT-PCR indicated that IL-1β treatment induced miR-138-5p expression in chondrocytes. (e) qRT-PCR data indicated that MCM3AP-AS1 and miR-138-5p expression levels were negatively correlated in the cartilage tissues of OA patients. (f) MiR-138-5p expression in the chondrocytes of MCM3AP-AS1 overexpression group were significantly reduced. ns.: not statistically significant

**P < 0.01, and ***P < 0.001 compared with the control.

Figure 5. MiR-138-5P reverses the role of MCM3AP-AS1 in OA chondrocytes. (a, b) CCK-8 assay results showed that the effect of MCM3AP-AS1 overexpression on cell proliferation could be offset by miR-138-5 mimics. (c) Transwell assay indicated that the effect of MCM3AP-AS1 overexpression on the migration of chondrocytes could be counteracted by miR-138-5p (d) Flow cytometry showed that the decrease in apoptosis rate induced by overexpression of MCM3AP-AS1 can be offset by miR-138-5 mimics. (E, F, G, H) ELISA results showed that the contents of inflammatory cytokines after cotransfection of miR-145-5p was significantly higher than those in MCM3AP-AS1 overexpression group

*P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6. MCM3AP-AS1 regulates SIRT1 expression through miR-138-5p. (a) SIRT1 contained a binding sequence for miR-138-5p. (b) Dual-luciferase reporter gene assay confirmed that miR-138-5p mimics reduced the luciferase activity of the wild-type SIRT1 reporter, but had no effect on the luciferase activity of the mutant SIRT1 reporter. (c, d) RIP assay showed that miR-138-5p could directly bind with SIRT1 mRNA. (e) The RNA pull-down assay was performed to validate the interaction between miR-138-5p and SIRT1 mRNA. (f) qRT-PCR showed that SIRT1 mRNA was significantly down-regulated in OA cartilage tissues. (g) There was a negative correlation between miR-138-5p and SIRT1 mRNA expression levels in OA cartilage tissues. (h) There was a positive correlation between MCM3AP-AS1 and SIRT1 mRNA expression levels in OA cartilage tissues. (i) Overexpression of MCM3AP-AS1 could significantly increase SIRT1 expression. After co-transfection of MCM3AP-AS1/miR-138-5p, SIRT1 expression was significantly reduced. ns.: not statistically significant

*P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 7. Graphic abstract: MCM3AP-AS1 functioned through the miR-138-5p/SIRT1 axis in chondrocytes to regulate the progression of OA, showing protective effects