Evaluation Of Migration And Invasion Of The RA-FLS And The Relative Expression Of The RA-associated MiRNAs

Objectives:In order to evaluate the migration and invasion of RA-FLS and relative expression of the RA-associated miRNAs, providing the evidence for further studying the role of miRNAs in pathogenesis of RA and the new targets for the treatment of RA in clinic.Subject:Human fibroblast-like synoviocytes, rheumatoid arthritis fibroblast-like synoviocytesMethods:Both MH7A and HFLS were cultured in the DMEM supplemented with 15% heat-inactivated fetal bovine serum (FBS), and passaged when cells were grown to the confluence of 80%, and the passage numbers of the MH7A and HFLS for the research were kept between from passage 3 to 6. Cell viability was detected by trypan blue exclusion assay. Transwell cell migration assay and Transwell cell invasion assay were used to evaluate the migration and invasion of the synoviocytes. Total RNA was extracted from the MH7A and HFLS using the Trizol Reagent, and RNA concentrations were determined. The homopolymeric tailing reaction of Poly (A) and reverse transcription to cDNA were accomplished using the SYBR(?) PrimeScriptTM miRNA RT-PCR Kit (Takara, RR716) according to the supplier’s instruction. Eleven kinds of RA-associated miRNAs and the internal miRNA control U6 snRNA control were selected was determined, The primers of the miRNAs were designed according to the instructions of SYBR(?) PrimeScriptTM miRNA RT-PCR Kit after searching the miRNAs database on the internet (http://www.mirbase.org), and the primers of internal miRNA control U6 snRNA were also designed. After the oligonucleotides synthesized, the levels of miRNAs were determined by Real-time PCR following the manufacturer’s protocol and performed in an ABI 7500 real-time PCR system (Applied Biosystem, USA). The relative expression of the indicated miRNAs was calculated using the 2-ΔΔCT method, and normalized to the expression of internal miRNA control U6 snRNA.Results:The HFLS with multi-coners were in long-fusiform, and the type of the synoviocytes was bigger with several projections, vague border and different sizes. While the MH7A were arraying in regular pattern. Cell viability was tested by trypan blue exclusion assay, and the viability of the trypsinized both MH7A and HFLS was over 90%, thus, these cells could be used in the following experiments. Resuilts from the Transwell migration assay and the Transwell invasion assay showed that the MH7A had higher ability of migration and invasion than HFLS. Eleven kinds of miRNAs were selected for our research, including miR-132,-155,-203,-223,-124,-15a,-16,18a,-19a,-26a and miR-146a. The levels of the indicated miRNAs from total RNA extracts were quantified using RT-qPCR, normalized to U6. The markedly upregulation of miR-132,-155,-203,-223 and miR-124 was shown in MH7A, and the relative expression of them was 2.328,3.882,6.020,1.343 and 1.831 folds, respectively, as compared to those in HFLS (P<0.05). Meanwhile, we also demonstrated that the significant downregulation of miR-15a,-16,18a,-19a,-26a and miR-146a in MH7A, and the relative expression of them were 0.625,0.367,0.742,0.602,0.533 and 0.424 folds, respectively, as compared to those in HFLS (P<0.05).Conclusions:1. The rheumatoid arthritis fibroblast-like synoviocytes had higher ability of migration and invasion than human fibroblast-like synoviocytes.2. The findings presented here revealed that 11 kinds of the RA-associated miRNAs were differently expressed between MH7A and HFLS, suggesting that differentiated expression of the miRNAs in RA-FLS might regulating the secretion of MMPs and inflammatory factors directly or indirectly. This might be novel molecular targets for the diagnosis, monitors and treatment of RA in clinic.