Mechanism of mirna-21-5p on apoptosis of IL-1β- induced rat. synovial cells

Transcription

1 Mechanism of mirna-21-5p on apoptosis of IL-1β- induced rat synovial cells Zhen Li 1,2,3,4, Xiaofu Li 4, Yi Wang 4, Qingjun Wei 1,2,3,* 1 Orthopaedic Department, the First Affiliated Hospital of Guangxi Medical University, Guangxi Medical University, Guangxi, , China. 2 Research Centre for Regenerative Medicine and Guangxi Key Laboratory of Regenerative Medicine, Guangxi Medical University, Guangxi, , China. 3 Collaborative Innovation Center of Guangxi Biological Medicine, Guangxi Medical University, Nanning, , China 4 Orthopaedic Department, People s Hospital of Mianzhu, Mianzhu, , China * Corresponding author qingjunwei7703@sina.com Abstract: The aim of this study is to investigate the role of mirna-21-5p in the apoptosis of rat synovial cells induced by IL-1β. The synovial cells of rats were cultured in vitro and identification of type II collagen and proteoglycan were stained by toluidine blue method; The synovial cells were divided into normal control group (NC group), control group (Control group), blank plasmid transfection group (BL group) and transfection group (mirna group) four groups. Control, BL and mirna three groups of cells were treated by IL-1 beta, the cell proliferation rate was detected by MTT, while RT-PCR and Westem-blot were used to detect the expression of Bax and Bcl-2. All cells were able to secrete protein polysaccharide and collagen type II. MTT detection found that after IL-1 beta treatment, the cell proliferation rate of mirna group was significantly lower than that of Control group(54.7±3.5 vs 31.6±2.9), the difference was statistically significant (P < 0.05); Compared with the control group, BaxmRNA and protein expression levels of mirna group were significantly lower(1.00±0.31 vs 0.75±0.15, P < 0.05),Bcl-2 mrna and protein expression levels of mirna group were significantly higher (2.24±1.02 vs 1.00±0.64, 3

2 P < 0.05). In conclusion, mirna-21-5p can inhibit the expression of Bcl-2 and promote the expression of Bax, The protective effect of sliding mode cells induced by IL-1β. A new way is to treat rheumatoid arthritis. Key words: Rheumatoid arthritis; mirna-21-5p; Bax; Bcl-2 4

3 Introduction Rheumatoid arthritis (RA) is an autoimmune disease caused by dysfunction of the immune system. The cause of the disease is mainly due to the proliferation of inflammatory factors and sliding mode cells which cause damage to articular cartilage [1]. Interleukin may process plays an important role and can promote the proliferation of synovial cells in RA pathology. Therefore, how to effectively inhibit synovial cell abnormal proliferation of become the key points of the control and treatment of RA disease [2, 3]. In vitro cell research, often through the IL-1β induced synovial cells to simulate the RA pathological process. MiRNA is a kind of small RNA, a large number of basic research confirmed that mirna had a certain role in the regulation of tumor cells [4-7]. Related studies had indicated that the over-expression of mirna-21-5p had inhibitory effect on tumor proliferation, however, the mechanism of mirna-21-5p in RA was relatively limited. This study aimed to study the effect of mirna-21-5p on the proliferation of synovial cells induced by IL-1β. Materials and Methods Isolation and Culture of rat synovial cells Taking 5 SPF Wistar male rats, after ether anesthesia, Cartilage slice, cutting into pieces and cleaning by PBS. Centrifugal 5min at the speed of 8000r/min, After removing the supernatant, the type II collagenase with 5% FBS concentration of 0.04% was added, Stayed overnight at 37 DEG C in water bath, Using 200 mesh filter, Centrifugal 5min at the speed of 8000r/min, Discard supernatant, Culturing in DMEM medium containing 10%FBS, Temperature was controlled at 37, Culture in the incubator with 5% CO 2, Culture medium replacement time is 72h. Cells were detected in all third generations. Phenotype identification Climb the tablet with PBS cleaning 2 times, adding a concentration of 4% of the formaldehyde, fixed at room temperature 20min. Decting the expression of type II collagen and protein polysaccharide in cells. 5

4 Cell treatment The synovial cells were divided into four groups: NC group, Control group, BL group and mirna group. BL group: the use of liposome 2000 (Invitrogen, USA) as the carrier will slow virus transfection into the synovial cells.mirna group: the use of liposome 2000 (Invitrogen, USA) as a vector to mirna-21-5p transfected into the synovial cells, according to the database defined by mirna-21-5p transfection plasmid ( group and Control group were not transfected. Synovial cells were inoculated in 6 well plates until the completion of the 80%~90% based fusion, replaced by 0.5%FBS medium, Control group, group BL and group mirna were incubated by 10ng/ml IL-1 β for 1 h. MTT testing To determine the cell proliferation rate by using the four - methyl - (MTT) - (Sigma) - Kit instructions in the kit. RT-PCR Measuring mrna expression of Bax and Bcl-2 by RT-PCR method, using β-actin as internal control. Primer sequence were shown in Table 1. Western Blot testing According to Bcl-2, Bax and GAPDH antibody specification steps from the United States Cruze Snta, all groups of cells were detected. Statistical analysis and methods All data were expressed by mean±standard( x±s)deviation method, The data were used by SPSS19.0 soft, single factor analysis (ANOVA) and LSD method were used to analyze all the data. P < 0.05 means significantly difference. Results Synovial cell identification It was found that the cultured cells were able to secrete type II collagen by immunocytochemistry (Shown in Fig 1), and toluidine blue stained cells were purple (Shown in Fig 2). 6

5 Fig1. Type II collagen Fig2. Protein polysaccharide The mir-21-5p expressions in each groups The mir-21-5p gene expression of NC and mirna groups were significantly increased compared with control group (P < 0.05); There were no significant difference between BL and Control groups (P>0.05), Shown in Fig 3. 7

6 Fig3. mir-21-10p gene expression of four groups (flod) **: P<0.05, compared with Control group MTT testing Cell increment rates of NC group and mirna group (20.0±8.4 and 31.6±5.1)% were significantly lower than Control group (54.7±3.5)% (P<0.05); There were no significant differences between BL and Control groups (P>0.05),shown in Fig 4. Fig 4. Comparison of the proliferation rate of synovial cells ( x±s,%) **: P<0.05, compared with Control group Comparing gene and protein expression of Bax and Bcl-2 in difference groups Comparing with Control group, Bax mrna and protein expression of NC and mirna groups were significantly increased (1.00±0.31 vs 0.75±0.15, P<0.05); Bcl-2 mrna and protein expression of NC and mirna groups were significantly decreased (2.24±1.02 vs 1.00±0.64,P<0.05). The data was shown in Fig 5 and Fig 6. 8

7 Fig5. Gene and protein expressions of Bcl-2 in four groups (Fold). **: P<0.05, compared with Control group Fig6. Gene and protein expressions of Bax in four groups (fold) **: P<0.05, compared with Control group Discussion Inflammatory factor IL-1β induced by rat synovial cells, so that it can secrete a variety of inflammatory mediators, so as to establish the model of RA inflammatory cells in vitro. In this study, we isolated and cultured primary synovial cells from rats and used IL-1β- induced method to study the mechanism of mirna-21-5p in the pathogenesis of RA. So far, a large number of studies have confirmed that mirna-21-5p has a positive significance in regulating the proliferation of malignant tumor cells[8-10]. 9

8 RA is a kind of disease which is caused by the abnormal proliferation of synovial cells, which causes damage to the articular cartilage as the main clinical feature[11]. Therefore, we hypothesized that mirna-21-5p overexpression might have a protective effect on RA. IL-1β is a cytokine, which plays an important role in the pathogenesis of RA[12]. IL-1β can be used as a stable inducer in in vitro RA study[13]. Present study found that after mirna-21-5p transfection of mirna group synovial cell proliferation rate was significantly lower than that in the control group and BL group. The result indicates that the expression of mirna-21-5p can be effectively inhibited IL-1 induced proliferation of synovial cells. It was also found that there was no significant difference between the Control group and the BL group, the results showed that the cell damage caused by the carrier was not less. In order to further explain the mechanism of mirna-21-5p in the whole process, we further detect the expression of related proteins and mrna. Bcl-2 and Bax are two kind of cell proliferation and apoptosis is closely related to. if the expression of Bax increased, the same source and Bax dimers are formed, the cells have a tendency to apoptosis and inhibits the proliferation; if the expression of Bcl-2 in excess, and Bax together to form a heterodimer, which inhibits apoptosis and promote the proliferation, so when the expression of Bax was increased by decreasing the expression of Bcl-2 and can effectively restrain the proliferation of cells[14,15]. In this study, we found that compared to the transfected synovial cells after induced by IL-1β, and not transfected mirna-21-5p synovial cells and the Bax protein expression was significantly increased, and Bcl-2 are significantly reduced. Therefore, we speculate that probably is due to the mirna-21-5p the over expression of leads to increased expression of Bax, bcl-2 expression decreased, thus making the proliferation of synovial cells under effective control. This study confirmed that mirna-21-5p expression in the process of controlling RA disease played a key role in the process of controlling the disease of RA. The development of disease course may be related to the low expression of mirna-21-5p. Therefore, effectively improve the mirna-21-5p expression to 10

9 promote Bax expression increased and bcl-2 expression decreased, which can effectively control with RA in synovial cell hyperplasia, for the treatment of RA and RA treatment drug research provides new ideas. Reference: 1. Wellby M L, Kennedy J A, Pile K, et al: Serum interleukin-6 and thyroid hormones in rheumatoid arthritis.metabolism 50: , Nishimoto N,Ito A,Ono M,et al: IL-6 inhibits the proliferation of fibroblastic synovial cells from rheumatoid arthritis patients in the presence of soluble IL-6 receptor. Int Immunol. 12: , Nakahara H,Song J,Sugimoto M,et al: Anti-interleukin 6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis.arthritis Rheumatism 48: , Chen W, Zeng W, Li X, et al: MicroRNA-509-3p increases the sensitivity of epithelial ovarian cancer cells to cisplatin-induced apoptosis[j]. Pharmacogenomics 17: , Wu ZW, Liu YF, Wang S, et al: mirna-146a induces vascular smooth muscle cell apoptosis in a rat model of coronary heart disease via NF-κB pathway. Genetic Mol Res 14: , Saha MN, Abdi J, Yang Y, et al: mirna-29a as a tumor suppressor mediates PRIMA-1Met-induced anti-myeloma activity by targeting c-myc. Oncotarget 11: , Zhang JF, Shi LL, Zhang L, et al: MicroRNA-25 Negatively Regulates Cerebral Ischemia/Reperfusion Injury-Induced Cell Apoptosis Through Fas/FasL Pathway. J Mol Neurosci, 58: , Yamamichi N, Shimomura R, Inada K, et al: Locked nucleic acid in situ hybridization analysis of mir-21 expression during colorectal cancer development. Clin Cancer Res 15: , Papagiannakopoulos T, Shapiro A, Kosik KS: MicroRNA-21 targets a network of 11

10 key tumor-suppressive pathways in glioblastoma cells. Cancer Res 68: , Ren J, Zhu D, Liu M, et al: Downregulation of mir-21 modulates Ras expression to promote apoptosis and suppress invasion of Laryngeal squamous cell carcinoma. Eur J Cancer 46: , Charles-Schoeman C, Bonzalez-Gay MA, Kaplan I, et al: Effects of tofacitinib and other DMARDs on lipid profiles in rheumatoid arthritis: implications for the rheumatologist. Semin Arthritis Rheum 46: 71-80, KAPOOR M,MARTEL-PELLETIER J,LAJEUNESSE D,et al: Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol, 7: 33-42, SHAKIBAEI M, JOHN T, SEIFA R TH C, et al: Resveratrol inhibits IL-1β-induced stimulation of caspase-3 and cleavage of PARP in human articular chondrocytes in vitro.ann N Y Acad Sci 1095: , Wang J, Beauchemin M, Bertrand R: Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints. Cell Signal 23: , Gu X, Yao Y, Cheng R, et al: Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-x(L) subcellular distribution. Apoptosis 16: ,