Treatment effect of CDKN1A on rheumatoid arthritis by mediating proliferation and invasion of fibroblast-like synoviocytes cells

Abstract

The objective of the present study was to evaluate the role of CDKN1A in rheumatoid arthritis (RA). Related gene expression data screened from Gene Expression Omnibus (GEO) were processed with network analysis. Protein-protein interaction was analysed through string database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure mRNA and microRNA expression. Cell proliferation and cell cycle were tested by MTT assay and flow cytometry, respectively. Transwell migration and invasion assay was used to test cell migration and invasion. CDKN1A screened by bioinformatics methods showed differential expression in RA cells compared with healthy controls (HC), and was at an important position in the protein-protein interaction network of RA. Compared with the HC group, CDKN1A was down-regulated in human RA synovium tissues and human fibroblast-like synoviocytes (HFLS). Contrary to CDKN1A silencing, CDKN1A over-expression significantly inhibited the proliferation and invasion of HFLS-RA, arrested HFLS-RA in G0/G1 phase and down-regulated the expressions of tumour necrosis factor (TNF)-α and interleukin (IL)-6, while it up-regulated the expression of IL-10. CDKN1A over-expression could also suppress phosphorylated signal transducers and activators of transcription 1 (pSTAT-1) expression. MiR-146a, highly expressed in RA tissues, could regulate CDKN1A negatively. Anti-146a suppressed cell proliferation and invasion, and at the same time enhanced IL-10 expression but inhibited IL-6, TNF-α and pSTAT-1 expression. The results indicated that CDKN1A over-expression, which could be enhanced by miR-146a suppression, inhibited the proliferation of invasion in HFLS-RA. This was probably a result of suppressed pSTAT-1, IL-6 and TNF-α expression and enhanced IL-10 expression.

Keywords: CDKN1A; MiR-146a; human fibroblast-like synoviocytes; rheumatoid arthritis.

Figure 1

Screening of differentially expressed genes (DEGs) and core genes in rheumatoid arthritis (RA). (a) Analysis of differentially expressed genes in four data sets. Four sets of gene expression data containing the samples in the RA group and the healthy control (HC) group were downloaded from the Gene Expression Omnibus (GEO) database for analysis. Twelve common DEGs, including CDKN1A, were found among the four data sets, which might be associated with RA disease. (b) Analysis of gene–gene interaction was performed between the DEGs and hot genes of RA through the string website. Cluster analysis was carried out using k‐means clustering, and it was found that CDKN1A, signal transducers and activators of transcription 1 (STAT‐1) and C‐C chemokine receptor type 5 (CCR5) were at the core position in the cluster network of RA hot genes.

Figure 2

Cyclin‐dependent kinase inhibitor 1A (CDKN1A) expression in rheumatoid arthritis (RA) tissues and human fibroblast‐like synoviocytes (HFLS). (a) CDKN1A mRNA was decreased significantly in the RA group compared with the healthy control (HC) group. (b) The level of CDKN1A was inhibited in the HFLS‐RA group. (c) Compared with the HFLS group, the CDKN1A protein level was also inhibited in the HFLS‐RA group. *P < 0·05; **P < 0·01.

Figure 3

The role of cyclin‐dependent kinase inhibitor 1A (CDKN1A) in proliferation, migration and invasion of human fibroblast‐like synoviocytes rheumatoid arthritis (HFLS‐RA). (a) Compared with the scramble and NC groups, the growth rate in the CDKN1A^– group was increased significantly (*P < 0·05), and the difference in cell proliferation was greater with the prolongation of culture time. (b) The majority of cells in the CDKN1A⁺ group were arrested in the G1/S phase compared with the scramble and NC groups. (c) Compared with the scramble and NC groups, migration and invasion of HFLS‐RA were increased significantly by CDKN1A silencing and inhibited clearly by CDKN1A over‐expression. (d) The expression of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was lower and the expression of IL‐10 was higher to different degrees after over‐expressing CDKN1A in HFLS‐RA (*P < 0·05; **P < 0·01). There was no obvious change in the level of signal transducers and activators of transcription 1 (STAT‐1). (e) Compared with the negative siRNA scramble cell line, the expression of TNF‐α and IL‐6 was higher and the level of IL‐10 was lower after CDKN1A silencing. (f) Over‐expression of CDKN1A reduced the protein expression of phosphorylated STAT‐1 (pSTAT‐1) significantly, while CDKN1A silencing increased it. *P < 0·05.

Figure 4

MiR‐146a directly targeted cyclin‐dependent kinase inhibitor 1A (CDKN1A). (a) Relative miR‐146a expression in synovium samples was higher in the rheumatoid arthritis (RA) group compared with the healthy control (HC) group. (b) Relative miR‐146a expression and relative CDKN1A expression showed a negative correlation in synovium samples. (c) The level of miR‐146a was also found higher in human fibroblast‐like synoviocytes (HFLS)‐RA than that in HFLS. (d) Luciferase activity decreased only in the CDKN1‐wild‐type (WT)⁺ miR‐146a group. (e) After transfected with anti‐146a, the level of miR‐146a was suppressed in HFLS‐RA. (f) The expression of CDKN1A mRNA was promoted. (g) The expression of CDKN1A protein was up‐regulated in the anti‐146a group while anti‐146a⁺ CDKN1A^– reversed it. *P < 0·05; **P < 0·01.

Figure 5

Inhibition of miR‐146a reversed the effects of cyclin‐dependent kinase inhibitor 1A (CDKN1A) silencing on human fibroblast‐like synoviocytes rheumatoid arthritis (HFLS‐RA). (a) The growth rate in the anti‐146a group showed an obvious down‐regulation compared with the scramble and NC groups, while it was reversed in the anti‐146a⁺ CDKN1A^– group. (b) The cells in the anti‐146a group were arrested mainly in the G1/S phase, while anti‐146a⁺ CDKN1A^– reversed it. (c) Migration and invasion of HFLS‐RA was inhibited significantly in the anti‐146a group and clearly reversed in anti‐146a⁺ CDKN1A^–. (d,e) The expression of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was suppressed in the anti‐146a group, while the expression of IL‐10 was promoted. These influences were reversed significantly in the anti‐146a⁺ CDKN1A^– group. (f) The expression level of phosphorylated signal transducer and activator of transcription 1 (pSTAT‐1) protein was lower in the anti‐146a group and there was no significant difference in STAT‐1 protein. Anti‐146a⁺ CDKN1A^– reversed the inhibition of pSTAT‐1 protein in HFLS‐RA. *P < 0·05.