Introduction:
MiR-101 rs7536540 may influence the expression of miR-101 and another polymorphism, rs2227513, which in turn affects the expression of interleukin (IL)-22, a gene with an essential role in the pathogenesis of rheumatoid arthritis (RA). However, studies on the combined effect of these polymorphisms are still scarce.

Material and methods:
DAS28 (ESR) and Clinical Disease Activity Index (CDAI) scores were measured to evaluate the severity of RA. Quantitative real-time PCR was performed to analyze the expression of miR-101 and IL-22 mRNA. ELISA and Western blot were carried out to examine the expression of IL-22 protein. MTT assay and flow cytometry were used to assess the cellular proliferation and apoptosis of fibroblast-like synoviocytes (FLS) incubated in the synovial fluid (SF) collected from RA patients carrying differential genotypes. Immunofluorescence was performed to measure the expression of p-STAT3. Luciferase assay was carried out to explore the inhibitory role of miR-101 in the expression of IL-22.

Results:
The severity of RA was progressively increased in patients with rs7536540 GG + rs2227513 AA, rs7536540 GG + rs2227513 AG, rs7536540 CC/CG + rs2227513 AA and rs7536540 CC/CG + rs2227513 AG genotypes. The CC/CG alleles at rs7536540 were correlated with up-regulated miR-101 expression in the serum and SF of RA patients, whereas both CC/CG alleles at rs7536540 and AG alleles at rs2227513 were correlated with elevated expression of IL-22. Incubation of FLS with SF isolated from RA patients carrying the CC/CG alleles at rs7536540 and AG alleles at rs2227513 remarkably increased the cell proliferation and inhibited the apoptosis of FLS. Luciferase assay demonstrated that the expression of IL-22 was notably suppressed by miR-101 in THP-1 cells.

Conclusions:
In this study, we investigated the combined effect of polymorphisms rs7536540 and rs2227513 on the expression of IL-22 and the proliferation of FLS as well as their association with the severity of RA, so as to gain a deep insight into the molecular mechanisms underlying the severity of RA.