LncRNA LOC100912373 modulates PDK1 expression by sponging miR-17-5p to promote the proliferation of fibroblast-like synoviocytes in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a common autoimmune disease and characterized by chronic inflammation, abnormal synovial cell proliferation, and joint swelling and tenderness, and it causes patients substantial pain. To date, the pathogenesis of RA remains unclear, and specific treatment is still lacking in the clinic. Evidence from previous research indicated that the long noncoding RNA (lncRNA) LOC100912373 is a key lncRNA and involved in RA. However, our understanding of the specific mechanism of lncRNA LOC100912373 in RA development and progression is still in its infancy. In this study, fibroblast-like synoviocytes (FLSs) were cultured by enzyme-dispersed and substrate-attached explant methods. The MTT method, flow cytometry and transmission electron microscopy were used to determine the effect of lncRNA LOC100912373 on FLSs. The expression of key genes such as lncRNA LOC100912373, miR-17-5p, PDK1 and AKT in FLSs was detected by RT-qPCR, immunofluorescence and Western blot. The localization of lncRNA LOC100912373 was determined by fluorescence in situ hybridization. The specific targeting relationship between lncRNA LOC100912373 and miR-17-5p/PDK1 was verified by RNA immunoprecipitation and luciferase reporter gene analysis. The results showed that lncRNA LOC100912373 localized in the cytoplasm and was highly expressed in the synovial tissues and FLSs of AA rats. LncRNA LOC100912373 overexpression promoted the proliferation of FLSs. In addition, lncRNA LOC100912373 could bind to miR-17-5p, and the expression of lncRNA LOC100912373 was negatively correlated with miR-17-5p and positively correlated with PDK1/AKT. In conclusion, lncRNA LOC100912373 may upregulate the expression of PDK1 by sponging miR-17-5p, accelerating the phosphorylation of AKT and inducing the proliferation of FLSs, thus promoting the occurrence and development of RA.

Keywords: Rheumatoid arthritis; ceRNA theory; fibroblast-like synoviocytes; lncRNA LOC100912373/miR-17-5p/PDK1 axis.

Figure 1

Observation of synovial cell morphology by optical microscopy and fluorescence microscopy (A-C ×40, D-F ×200). (A) Primary culture for 3 days. (B) Primary culture for 7 days. (C) FLSs sub-cultured 3 times. (D-F) Immunocytochemical staining of FLSs.

Figure 2

Expression and localization of lncRNA LOC100912373 in synovial tissues and FLSs. A: Expression of lncRNA LOC100912373 in the synovium. B: Expression of lncRNA LOC100912373 in FLSs. ##P<0.01 compared with the control group. C: Localization of lncRNA LOC100912373 in FLSs by FISH (×200). a: CY3 fluorescence staining, b: DAPI fluorescence staining, c: Merge.

Figure 3

Detection of cell transfection and lncRNA LOC100912373 expression under conditions of overexpression or interference. (A) Observation of cell transfection results under a fluorescence microscope (×100), (a) pcDNA3.1-LOC100912373 group, (b) siRNA group. (B) The relative expression of lncRNA LOC100912373 after overexpression was detected by qRT-PCR. (C) The relative expression of lncRNA LOC100912373 after interference was detected by qRT-PCR. ##P<0.01 compared with the model group.

Figure 4

The effect on the proliferation and cell cycle of FLSs after lncRNA LOC100912373 overexpression and interference. (A) The OD (570 nm) of each group at different reaction times. (B) Quantification of the cell cycle results. ##P<0.01 compared with the control group, **P<0.01 compared with the model group. (C) The phase of the cell cycle was detected by flow cytometry. (a) Control group, (b) model group, (c) pcDNA3.1-LOC100912373 group, (d) pcDNA3.1-NC group, (e) siRNA group, (f) siRNA-NC group.

Figure 5

Observation of the ultrastructure of FLSs by transmission electron microscopy after lncRNA LOC100912373 overexpression and siRNA interference (×12000). A: control group. B: model group. C: pcDNA3.1-LOC100912373 group. D: pcDNA3.1-NC group. E: siRNA group. F: siRNA-NC group. In the model group, MARK1 indicates nuclear membrane, MARK2 indicates autophagy AP, MARK3 indicates mitochondrial, MARK4 indicates rough endoplasmic reticulum, MARK5 indicates heterochromatin, MARK6 indicates nucleus.

Figure 6

Changes in miR-17-5p expression in FLSs after lncRNA LOC100912373 overexpression and siRNA interference. ##P<0.01 compared with the control group, **P<0.01 compared with the model group.

Figure 7

Changes in PDK1 protein and mRNA expression in FLSs after lncRNA LOC100912373 overexpression and interference. A: Changes in PDK1 protein expression were observed by immunofluorescence technique (×200). B: Changes in PDK1 mRNA expression were observed by qRT-PCR. C: Changes in PDK1 protein expression were observed by Western blot. D: Semiquantitative analysis of PDK1 protein. ##P<0.01 compared with the control group, **P<0.01 compared with the model group.

Figure 8

Changes in AKT protein and mRNA expression in FLSs after lncRNA LOC100912373 overexpression and interference. A: Changes in p-AKT protein expression were observed by immunofluorescence technique (×200). B: Changes in AKT mRNA expression were observed by qRT-PCR. C: Changes in AKT and p-AKT protein expression were observed by Western blot. D: Semiquantitative analysis of p-AKT/AKT protein. ##P<0.01 compared with the control group, **P<0.01 compared with the model group.

Figure 9

LncRNA LOC100912373 can combine with miR-17-5p and target PDK1. A: Schematic comparison between lncRNA LOC100912373 and the “seed sequence” in miR-17-5p. B: The binding relationship between lncRNA LOC100912373 and miR-17-5p verified by dual luciferase reporter gene assay, ##P<0.01 compared with the NC group. C: Schematic comparison between miR-17-5p and the “seed sequence” in PDK1. D: The binding relationship between PDK1 and miR-17-5p verified by dual luciferase reporter gene assay, #P<0.05 compared with the NC group. E: The enrichment of lncRNA LOC100912373 and miR-17-5p verified by RIP assay. ##P<0.01 compared with the pcDNA3.1 group, **P<0.01 compared with the pcDNA3.1-LOC100912373 group.

Figure 10

LncRNA LOC100912373 targets miR-17-5p and upregulates PDK1/AKT to promote the proliferation of FLSs, leading to the occurrence and development of RA.