Activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting siRNA.

BACKGROUND: Fibroblast-like synoviocytes (FLSs), resident mesenchymal cells of synovial joints, play an important role in the pathogenesis of rheumatoid arthritis (RA). Dickkopf-1 (DKK-1) has been proposed to be a master regulator of bone remodeling in inflammatory arthritis. Here, potential impairation on the activity of FLSs derived from RA to small interfering RNAs (siRNAs) targeting DKK-1 was investigated. METHODS: siRNAs targeting DKK-1 were transfected into FLSs of patients with RA. Interleukin (IL)-1ß, IL-6, IL-8, matrix metalloproteinase (MMP) 2, MMP3, MMP9, transforming growth factor (TGF)-ß1, TGF-ß2 and monocyte chemoattractant protein (MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Invasion assay and H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation, respectively. Western blotting was performed to analyze the expression of nuclear factor (NF)-κB, interleukin-1 receptor-associated kinase (IRAK)1, extracellular regulated protein kinases (ERK)1, Jun N-terminal kinase (JNK) and ß-catenin in FLSs. RESULTS: DKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs (P < 0.01). siRNAs induced a significant reduction of the levels of IL-6, IL-8, MMP2, MMP3 and MMP9 in FLSs compared to the control group (P < 0.05). DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs (P < 0.05). Important molecules of pro-inflammatory signaling in FLSs, including IRAK1 and ERK1, were decreased by the inhibition of DKK-1 in FLSs. In contrast, ß-catenin, a pivotal downstream molecule of the Wnt signaling pathway was increased. CONCLUSIONS: By inhibiting DKK-1, we were able to inhibit the proliferation, invasion and pro-inflammatory cytokine secretion of FLSs derived from RA, which was mediated by the ERK or the IRAK-1 signaling pathway. These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

Upregulated microRNA-15b alleviates ovarian cancer through inhitbition of the PI3K/Akt pathway by targeting LPAR3.

Ovarian cancer characterizes as the fourth leading consequence of death associated with cancer for women. Accumulating evidence underscores the vital roles of microRNAs (miRNAs) in preventing ovarian cancer development. Besides, induction of the phosphatidylinositol-3 kinase/serine/threonine kinase (PI3K/Akt) pathway associated with the ovarian cancer cell migration and invasion. The study aims to examine the effects of miR-15b on the proliferation, apoptosis, and senescence of human ovarian cancer cells by binding to lysophosphatidic acid receptor 3 (LPAR3) with the involvement of the PI3K/Akt pathway. The positive expression of LPAR3 protein was detected by immunohistochemistry. Then the interaction between miR-15b and LPAR3 was examined. The possible role of miR-15b in ovarian cancer was explored using gain- and loss-of-function experiments. Subsequently, the functions of miR-15b on PI3K/Akt pathway, proliferation, migration, invasion, senescence and apoptosis of ovarian cancer cells were assessed. Furthermore, in vivo tumorigenicity assay in nude mice was performed. LPAR3 was overexpressed, whereas miR-15b was poorly expressed in ovarian cancer tissues. LPAR3 is a direct target of miR-15b. Restored miR-15b promoted Bax expression, apoptosis, and senescence, inhibited expression of LPAR3 and Bcl-2, the extent of PI3K and Akt phosphorylation, as well as ovarian cancer cell proliferation, migration, and invasion. Further, tumor growth was observed to be prevented by miR-15b overexpression. Collectively, our study demonstrates that miR-15b represses the proliferation and drives the senescence and apoptosis of ovarian cancer cells through the suppression of LPAR3 and the PI3K/Akt pathway, highlighting an antitumorigenic role of miR-15b.

Reversal of cisplatin resistance by microRNA-139-5p-independent RNF2 downregulation and MAPK inhibition in ovarian cancer.

Some microRNAs (miRs) are dysregulated in cancers, and aberrant miR expression has been reported to correlate with chemoresistance of cancer cells. Therefore, the present study aims at investigating the effects of microRNA-139-5p (miR-139-5p) on cisplatin resistance of ovarian cancer (OC) with involvement of ring finger protein 2 (RNF2) and the mitogen-activated protein kinase (MAPK) signaling pathway. OC tissues were obtained from 66 primary OC patients. The cisplatin-sensitive A2780 and cisplatin-resistant A2780/DDP cell lines were collected for construction of RNF2 silencing and overexpressed plasmids. Cell vitality and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin V-FITC/propidium iodide double-staining, respectively. Next, expression of RNF2, extracellular signal-related kinase, and p38 was determined by quantitative reverse transcription-quantitative polymerase chain reaction and Western blot analysis. Finally, the volume of xenograft tumors in BALB/c nude mice was detected. RNF2 and miR-139-5p were identified to be involved in OC. In addition, MAPK activation and RNF2 were related to cisplatin resistance of OC. miR-139-5p was downregulated in cisplatin-resistant OC tissues, and miR-139-5p overexpression could inhibit cell vitality, reduce cisplatin resistance, and promote apoptosis of OC cells. Furthermore, miR-139-5p combined with MAPK inhibitors more obviously reduced cisplatin resistance of OC. Taken together, this study demonstrated that miR-139-5p overexpression combined with inactivation of the MAPK signaling pathway can reverse the cisplatin resistance of OC by suppressing RNF2. Thus, miR-139-5p overexpression might be a future therapeutic strategy for OC.

MicroRNA-374b inhibits cervical cancer cell proliferation and induces apoptosis through the p38/ERK signaling pathway by binding to JAM-2.

Cervical cancer (CC) remains a highly prevalent cancer and mortality globally among women globally. The aim of the present study was to assess the ability of miR-374b to regulate CC cells through JAM-2, whilst exploring whether the underlying mechanism and its relation to the p38/ERK signaling pathway. During the study, microRNA-374b (miR-374b) was observed to have been expressed at a low level among CC tissues. Hence, a series of miR-374b mimics, miR-374b inhibitors, siRNA against JAM-2, SB202190 (an inhibitor for p38), and PD98059 (an inhibitor for ERK) were introduced to treat CC Siha cells and normal cervical Ect1/E6E7 cells. MTT, flow cytometry, scratch test, and transwell assays were applied to determine cell viability, apoptosis, migration, and invasion. The inhibitory role of the p38/ERK signaling pathway was observed in the CC cells treated with miR-374b mimics or siRNA against JAM-2. miR-374b mimic exposure was found to reduce cell viability, migration, and invasion, but induce apoptosis. MiR-374b inhibitor exposure was observed to have induced effects on the CC cells in a contrary manner to those induced by that of the miR-374b mimics. The key findings of the study demonstrated that miR-374b significantly inhibits cell proliferation, migration, and invasion through the blockade of the p38/ERK signaling pathway activation, as well as negatively binding to JAM-2, highlighting its potential as a therapeutic target for CC.

Elevated Levels of Soluble ST2 were Associated with Rheumatoid Arthritis Disease Activity and Ameliorated Inflammation in Synovial Fibroblasts.

BACKGROUND: Much evidence has demonstrated that interleukin (IL)-33 plays an important role in rheumatoid arthritis (RA). However, there have been limited studies about soluble ST2, a receptor for IL-33, in RA. The aims of this study were to detect the levels of ST2 in the serum and synovial fluid of RA patients and to reveal the association of these levels with disease activity and the function of ST2 in RA. METHODS: A total of 56 RA patients and 38 age-matched healthy controls were enrolled in this study. Synovial fluid samples were collected from another 30 RA patients and 20 osteoarthritis patients. Serum and synovial fluid levels of ST2 were measured by ELISA. In addition, the levels of ST2 in the serum of RA patients before and after therapy were detected. The function of ST2 in RA was revealed by the results of an in vitro cell assay, where recombinant ST2 proteins were used to treat peripheral blood mononuclear cells (PBMCs) and RA synovial fibroblasts (RASFs). RESULTS: Serum-soluble ST2 levels were significantly higher in RA patients (127.14 ± 61.43 pg/ml) than those in healthy controls (78.37 ± 41.93 pg/ml, P < 0.01). Synovial fluid-soluble ST2 levels (41.90 ± 33.58 pg/ml) were much higher in RA patients than those in osteoarthritis patients (19.71 ± 16.72 pg/ml, P < 0.05). RA patients who received effective therapy for 6 months showed decreased serum-soluble ST2 levels (113.01 ± 53.90 pg/ml) compared to baseline (139.59 ± 68.36 pg/ml) (P = 0.01). RA patients with high disease activity had higher serum-soluble ST2 levels (162.02 ± 56.78 pg/ml) than those with low disease activity (94.67 ± 40.27 pg/ml, P = 0.001). Soluble ST2 did not affect IL-1ß, IL-6, IL-8, or tumor necrosis factor-α (TNF-α) expression in PBMCs from RA patients. However, soluble ST2 ameliorated the expressions of IL-33 and IL-1ß but not that of IL-6, IL-8, or TNF-α in resting RASFs. Interestingly, in the RASFs stimulated by TNF-α plus IL-1ß, soluble ST2 showed extensive suppressive effects on the expression of IL-6, IL-8, and TNF-α. CONCLUSION: Elevated levels of ST2 in the serum and synovial fluid were associated with disease activity and ameliorated IL-33 expression and IL-33-induced inflammation in RASFs, suggesting that soluble ST2 might be a potential therapeutic candidate for RA.

Serum IgA against type 3 muscarinic acetylcholine receptor is a novel marker in diagnosis of Sjögren's syndrome.

BACKGROUND: Antibodies against type 3 muscarinic acetylcholine receptor (M3R) are involved in the pathogenesis of Sjögren's syndrome (SS), but the clinical value of them in SS patients has been controversial. The aims of this study were to: (1) establish an improved enzyme-linked immunosorbent assay (ELISA) to detect IgA antibodies against M3R; (2) evaluate the value of IgA antibodies against the second extracellular loop of M3R205-220 (c2M3RP) in diagnosis of SS. METHODS: To increase the ELISA sensitivity, c2M3RP was coupled to bovine serum albumin (BSA) by the glutaraldehyde method and a 96-well microplate was treated by ultraviolet rays before coated. Concentrations of anti-c2M3RP, anti-SSA, and anti-SSB were measured in the sera of 240 individuals: 91 patients with primary SS and 149 controls (16 secondary SS, 27 systemic lupus erythematosus, 40 rheumatoid arthritis and 66 healthy controls). Diagnostic properties of anti-c2M3RP were determined by receiver-operating characteristic curve analysis. RESULTS: The prevalence of serum IgA anti-c2M3RP antibodies in patients with pSS (46%, 42/91) was significantly higher than that in patients with systemic lupus erythematosus (19%, 5/27), in rheumatoid arthritis (15%, 6/40) and in healthy controls (5%, 3/66). However, there was no significant difference between the two SS groups (P = 0.727). The diagnostic performance of IgA anti-M3RP antibodies was similar to anti-SSA assay, but had 22% higher sensitivity than anti-SSB. By analyzing of IgA anti-c2M3RP antibodies, combination of anti-SSA and anti-SSB resulted in increased sensitivity, whereas their specificity was not significantly changed. CONCLUSIONS: The improved anti-c2M3RP ELISA is a novel, sensitive, and specific serological test for the diagnosis of SS. The combined application of anti-c2M3RP, anti-SSA and anti-SSB tests can improve the laboratory diagnosis of SS. The IgA anti-c2M3RP antibodies may serve as a novel diagnostic marker for SS.

Diagnostic significance of measuring antibodies to cyclic type 3 muscarinic acetylcholine receptor peptides in primary Sjogren's syndrome.

OBJECTIVE: SS is an autoimmune disease characterized by salivary and lacrimal gland dysfunction leading to dry mouth (xerostomia) and dry eyes (xerophthalmia). Anti-muscarinic acetylcholine type-3 receptor (anti-M3R) autoantibodies have been shown to be a good serum marker in primary SS (pSS). The aim of this study was to assess the clinical correlations of anti-M3R-derived peptide antibodies in patients with pSS. METHODS: Sequences of the first to fourth cycle-M3R (c1M3R-c4M3R)-derived peptide was synthesized by a solid-phase technique on an Applied Biosytems Peptide Synthesizer. Synthesized cM3R peptide (cM3RP) was used as substrate in an ELISA to detect IgG anti-cM3RP antibodies in serum samples of patients and controls. The clinical and biological parameters of the diseases were also evaluated. The EULAR SS disease activity index (ESSDAI) score was used to measure disease activity in patients with primary SS. RESULTS: (i) Anti-c2M3RP antibodies were highly prevalent in pSS patients, and the titre is much higher than anti-c1,3,4M3RP antibodies. (ii) The prevalence of anti-c2M3RP antibodies in pSS, SLE, RA and healthy controls was 62.2, 7.1, 5.3 and 1.6%, respectively. The prevalence of anti-linear-2-M3RP antibodies in pSS, SLE and RA patients and healthy controls were 56.1, 20.0, 14.7 and 9.4%. (iii) The specificity of anti-c2M3RP antibodies was 95.1%, much higher than that of linear polypeptide (84.7%) for pSS diagnosis. (iv) In pSS patients, anti-c2M3RP positivity had significantly increased frequency in patients who were RF or ANA positive, and had several haematological abnormalities, such as leucopenia, anaemia and thrombocytopenia. Furthermore, the ESSDAI score was significantly higher in anti-c2M3RP-positive pSS patients (P < 0.05). CONCLUSION: Anti-c2M3RP antibody was highly specific for patients with pSS. The presence of anti-c2M3RP antibody in pSS indicates that c2M3RP may act as an autoantigen that may play a role in the pathogenesis of pSS.