LncRNA MEG3 inhibits rheumatoid arthritis through miR‐141 and inactivation of AKT/mTOR signalling pathway

Abstract Rheumatoid arthritis (RA) is a chronic inflammation mediated by autoimmune responses. MEG3, a kind of long noncoding RNA (lncRNA), participates in cell proliferation in cancer tissues. However, the correlation between MEG3 and RA is yet unclear. Therefore, to clarify how MEG3 works in RA, we performed a series of experiments using RA samples. We found that MEG3 was downregulated in the fibroblast‐like synoviocytes of RA patients (RA‐FLS), in comparison with healthy subjects. MEG3 was also down‐regulated evidently in lipopolysaccharide (LPS)‐treated chondrocyte. As part of our experiments, MEG3 was overexpressed in chondrocyte by transfection with lentivirus containing sequences encoding MEG3. In addition, in presence of LPS, reductions were identified not only in the cell proliferation, but also in the generation of interleukin‐23 (IL‐23), which, however were reversed in the lentivirus (containing MEG3‐encoding sequences)‐transfected chondrocytes. Up‐regulated MEG3 resulted in an increase the level of Ki67. Moreover, MEG3 was negatively correlated with miR‐141, and miR‐141 was up‐regulated in LPS‐treated chondrocyte. Inhibitory effects of MEG3 overexpression, mentioned above, were partially abolished by overexpressed miR‐141. Further, animal experiment also showed the inhibitory effect of MEG3 in overexpression on the AKT/mTOR signaling pathway. In‐vivoexperiments also showed that cell proliferation was facilitated by MEG3 overexpression with inhibited inflammation. In summary, the protective role of MEG3 in RA was proved to be exerted by the increase in the rate of proliferation, which might correlate to the regulatory role of miR‐141 and AKT/mTOR signal pathway, suggesting that MEG3 holds great promise as a therapeutic strategy for RA.


Abstract
Rheumatoid arthritis (RA) is a chronic inflammation mediated by autoimmune responses. MEG3, a kind of long noncoding RNA (lncRNA), participates in cell proliferation in cancer tissues. However, the correlation between MEG3 and RA is yet unclear.
Therefore, to clarify how MEG3 works in RA, we performed a series of experiments using RA samples. We found that MEG3 was downregulated in the fibroblast-like synoviocytes of RA patients (RA-FLS), in comparison with healthy subjects. MEG3 was also down-regulated evidently in lipopolysaccharide (LPS)-treated chondrocyte.
As part of our experiments, MEG3 was overexpressed in chondrocyte by transfection with lentivirus containing sequences encoding MEG3. In addition, in presence of LPS, reductions were identified not only in the cell proliferation, but also in the generation of interleukin-23 (IL-23), which, however were reversed in the lentivirus (containing MEG3-encoding sequences)-transfected chondrocytes. Up-regulated MEG3 resulted in an increase the level of Ki67. Moreover, MEG3 was negatively correlated with miR-141, and miR-141 was up-regulated in LPS-treated chondrocyte. Inhibitory effects of MEG3 overexpression, mentioned above, were partially abolished by overexpressed miR-141. Further, animal experiment also showed the inhibitory effect of MEG3 in overexpression on the AKT/mTOR signaling pathway. In-vivo experiments also showed that cell proliferation was facilitated by MEG3 overexpression with inhibited inflammation. In summary, the protective role of MEG3 in RA was proved to be exerted by the increase in the rate of proliferation, which might correlate to the regulatory role of miR-141 and AKT/mTOR signal pathway, suggesting that MEG3 holds great promise as a therapeutic strategy for RA.

K E Y W O R D S
AKT/mTOR, long noncoding RNA, MEG3, miR-141, rheumatoid arthritis

| INTRODUC TI ON
Rheumatoid arthritis (RA) is a chronic disease mediated by autoimmune responses with manifestations like systemic inflammation. 1,2 Various studies have shown that genetic and environmental risk factors together contribute to the development of RA. [3][4][5][6] An increasing number of studies are identifying a correlation between the cytokine networks and the RA pathogenesis. 7,8 At present, tremendous achievement has been seen in developing effective treatment of RA, in which suppressing the inflammatory responses is the most effect method. 5,9,10 However, further studies are necessary to discover the mechanism in inflammatory responses of RA. 11 Long non-coding RNAs (lncRNAs), transcripts in length longer than 200 nc, show the crucial role in inflammation. 12,13 For example, in the collagen, antibody induced arthritis mouse model of RA, shikonin can suppress the inflammation by targeting specific lncRNAs, 14 and in presence of quercetin, the apoptosis of fibroblast-like synoviocytes (FLS) in RA was also blocked, in which the involvement of lncRNA MALAT1 plays a critical role. [15][16][17][18][19][20][21][22] In this study, we attempted to discover how MEG3 affects the proliferation and inflammation of LPS-treated chondrocytes in RA.

| Cell culture
Chondrocytes were separated from cartilaginous tissues of male SD rats (200-280 g). (detailed in the Appendix S1).

| CCK-8 assay and crystal violet staining
CCK-8 assay and crystal violet staining were performed according to manufactures' instruction (detailed in the Appendix S1).

| Western blot analysis
See Appendix S1.

| Statistics
GraphPad Prism V was utilized for statistical analysis. The data were expressed as the mean ± standard error of the mean (SEM). All experiments were conducted a minimum of three times. A P value of <.05 suggested statistical significance.

| Upregulation of MEG3 in RA
To identify the divergence of MEG3 expression in the synovial tissues of healthy and RA subjects, we examined MEG3 expression in these subjects and found significant upregulation of MEG3 expression in the synovial tissue of RA patients ( Figure 1A). Moreover, MEG3 expression was up-regulated in the FLS obtained from the RA synovial tissue ( Figure 1B). Thus, upregulation of MEG3 expression is conducive to RA.
To investigate how MEG3 affects LPS-treated changes in chondrocytes, we determined the changes in MEG3 level upon LPS treatment. Results showed MEG3 expression was markedly decreased in LPS-treated chondrocytes (Figure 2A). To find out how MEG3 affects the LPS-treated changes in proliferation and inflammation of chondrocytes, we overexpressed MEG3 using lentivirus vectors ( Figure 1D).As shown in Figure 1E  found that overexpressed MEG3 up-regulated Ki67 and PCNA in LPS-treated chondrocytes ( Figure 1G). Moreover, IL-23 and IL-17, the pro-inflammatory cytokines, were down-regulated upon MEG3 overexpression ( Figure 1H). These results indicate that MEG3 protects chondrocytes in the presence of LPS through relieving the inhibitory effect on proliferation as well as down-regulating the release of pro-inflammatory cytokines.

| Effect of MEG3 on LPS-treated chondrocytes was reversed by miR-141 overexpression
It was predicted that there was a possible site on MEG3 sequences that could bind to miR-141 (Figure 2A), suggesting that they could be correlated. As shown in Figure 2B

| MEG3 enhanced cell proliferation and suppressed inflammation in vivo
To further clarify the function of MEG3, we carried out in vivo experiments in RA model of rats that were established by infusing LV-MEG3 subcutaneously. In Figure 2H Figure 2L). Thus, we inferred that MEG3 down-regulates miR-141, promotes cell proliferation, reduces Th17 cells and inactivates AKT/ mTOR signal pathway in vivo.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.