ABSTRACT
Non-coding RNAs (ncRNAs), which are thought to regulate articular cartilage through endochondral osteogenesis, consist of mRNA-interfering complementary RNA (miRNA) and long non-coding RNAs (lncRNA). More and more experimental evidence reveals the role of ncRNAs in chondrocyte differentiation and the pathogenesis of several skeletal diseases, including osteoarthritis. In the past few years, increasingly sophisticated DNA sequencing methods and a large number of sepigenetic modifications have greatly contributed to our understanding of the pathophysiological mechanisms of osteoarthritis. Recent studies have revealed that RNA interacts with RNA-binding proteins, regulates gene transcription and protein translation, and is involved in various pathological processes in OA, promising to be a therapeutic target for osteoarthritis.
ABSTRACT
Objective:To investigate the effect and molecular mechanism of procyanidin on the proliferation, apoptosis and reactive oxygen species (ROS) level of gastric cancer cell line SNU-1 in vitro. Methods:SNU-1 cells were divided into control group and 12.5, 50.0, 200.0 μg/ml procyanidin groups. The effect of procyanidin on the proliferation of SNU-1 cells was detected by CCK-8 assay. The apoptosis level and ROS positive rate of cells were detected by flow cytometry, and 2 mmol/L glutathione was added to SNU-1 cells added with 200.0 μg/ml procyanidin to detect the apoptosis level and ROS positive rate of cells. The expression of apoptosis-related protein in cells was detected by Western blotting.Results:The results of CCK-8 experiment showed that the proliferation activities of SNU-1 cells in the control group and the 12.5, 50.0, 200.0 μg/ml procyanidin groups were 3.69±0.30, 3.29±0.41, 0.91±0.39, 0.45±0.22 respectively, with a statistically significant difference ( F=279.84, P<0.001) . Compared with the control group, the proliferation activities of SNU-1 cells in the three procyanidin groups were significantly inhibited ( P=0.006, P<0.001, P<0.001) . The results of flow cytometry showed that the early apoptosis rates of SNU-1 cells in the control group and the 12.5, 50.0, 200.0 μg/ml procyanidin groups were (0.00±0.00) %, (0.00±0.00) %, (0.09±0.07) % and (0.45±0.22) % respectively, with a statistically significant difference ( F=7.14, P=0.003) . The 50.0 and 200.0 μg/ml procyanidin groups increased significantly compared with the control group ( P=0.003, P=0.007) . The late apoptosis rates of SNU-1 cells in the four groups were (0.00±0.00) %, (0.01±0.00) %, (6.98±0.77) % and (33.32±2.78) % respectively, with a statistically significant difference ( F=654.28, P=0.003) . The 50.0 and 200.0 μg/ml procyanidin groups increased significantly compared with the control group ( P<0.001, P<0.001) . The positive rates of ROS in SNU-1 cells in the four groups were (0.02±0.01) %, (0.10±0.05) %, (1.15±0.26) % and (1.58±0.22) % respectively, with a statistically significant difference ( F=162.24, P<0.001) . The 50.0 and 200.0 μg/ml procyanidin groups increased significantly compared with the control group ( P<0.001, P<0.001) . The positive rates of ROS in SNU-1 cells in the 200.0 μg/ml procyanidin group and the glutathione intervention group were (1.25±0.63) % and (0.13±0.02) % respectively, with a statistically significant difference ( t=5.39, P=0.001) . The early apoptosis rates of the two groups were (10.56±3.24) % and (2.09±0.24) % respectively, and the late apoptosis rates were (29.65±6.01) % and (23.63±1.52) % respectively, with statistically significant differences ( t=2.61, P=0.048; t=3.97, P=0.012) . The expressions of Bcl-2 protein in SNU-1 cells in the control group and the 12.5, 50.0, 200.0 μg/ml procyanidin groups were 1.00±0.00, 0.83±0.05, 0.60±0.14 and 0.41±0.23 respectively, with a statistically significant difference ( F=10.63, P=0.004) . The 50.0 and 200.0 μg/ml procyanidin groups decreased significantly compared with the control group ( P<0.001, P<0.001) . Conclusion:Procyanidin can inhibit proliferation and promote apoptosis of gastric cancer SNU-1 cells in vitro, which may be achieved by increasing intracellular ROS levels and reducing Bcl-2 protein expression.
ABSTRACT
Objective:To investigate the expressions of miR-20a-5p and lysine (K) demethylase 6B (KDM6B) in osteosarcoma tissues and the effects of miR-20a-5p targeting KDM6B on the proliferation, migration and invasion of osteosarcoma cells and tumor growth.Methods:The clinicopathological and paracancerous tissues of 20 patients with osteosarcoma admitted to the First Affiliated Hospital of Chinese Medical University from January 2017 to March 2019 were collected. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of miR-20a-5p and KDM6B mRNA in tissues. The osteosarcoma MG63 cells were divided into control group, mimic NC group, miR-20a-5p mimic group, and NC+ empty vector group, miR-20a-5p+ empty vector group, miR-20a-5p+ KDM6B group. The expression levels of miR-20a-5p and KDM6B mRNA of all groups were detected by qRT-PCR. Western blotting was used to detect the expression level of KDM6B. CCK-8 assay, cell scratch test and Transwell test were used to detect cell proliferation, migration and invasion ability. According to the random number table method, nude mice were divided into NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group, with 5 mice in each group. Tumor growth ability was detected by tumor xenograft nude mouse models.Results:The relative expression level of miR-20a-5p mRNA in osteosarcoma tissues was 0.55±0.27, and that in paracancerous tissues was 1.22±0.28, with a statistically significant difference ( t=7.701, P<0.001). The relative expression level of KDM6B mRNA in osteosarcoma tissues was 1.66±0.19, and that in paracancerous tissues was 1.00±0.15, with a statistically significant difference ( t=12.219, P<0.001). After transfection of miR-20a-5p, KDM6B mRNA and protein expression levels decreased with the increase of miR-20a-5p expression level. After miR-20a-5p transfection for 48 h, the cell proliferation abilities of the blank control group, mimic NC group and miR-20a-5p mimic group were 0.83±0.04, 0.81±0.03 and 0.52±0.01 ( F=89.655, P<0.001), compared with the blank control group and mimic NC group, the cell proliferation ability was significantly inhibited in the miR-20a-5p mimic group (both P<0.001). The cell proliferation abilities of NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group were 0.83±0.05, 0.52±0.01 and 0.67±0.05 ( F=43.919, P<0.001), compared with the NC+ empty vector group, the cell proliferation ability was significantly inhibited in the miR-20a-5p+ empty vector group ( P<0.001); compared with the miR-20a-5p+ empty vector group, the cell proliferation ability of miR-20a-5p+ KDM6B group increased significantly ( P<0.001). The scratch healing rates of the blank control group, mimic NC group and miR-20a-5p mimic group were (32.51±2.73)%, (30.26±3.22)% and (13.52±1.77)% ( F=46.314, P<0.001), compared with the control group and the mimic NC group, the scratch healing rate of the miR-20a-5p mimic group was significantly decreased (both P<0.001). The scratch healing rates of NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group were (31.34±3.11)%, (12.15±1.64)% and (28.93±2.89)% ( F=47.511, P<0.001), compared with the NC+ empty vector group, the scratch healing rate of the miR-20a-5p+ empty vector group was significantly decreased ( P<0.001); compared with the miR-20a-5p+ empty vector group, the scratch healing rate of miR-20a-5p+ KDM6B group was significantly increased ( P=0.001). The numbers of transmembrane cells in the blank control group, mimic NC group and miR-20a-5p mimic group were 114±16, 108±11 and 42±6 ( F=36.282, P<0.001), compared with the control group and mimic NC group, the number of transmembrane cells of the miR-20a-5p mimic group was significantly decreased (both P<0.001). The numbers of transmembrane cells in the NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group was 143±11, 39±4 and 139±12 ( F=112.120, P<0.001), compared with the NC+ empty vector group, the number of transmembrane cells of the miR-20a-5p+ empty vector group was significantly decreased ( P<0.001); compared with the miR-20a-5p+ empty vector group, the number of transmembrane cells of the miR-20a-5p+ KDM6B group was increased significantly ( P<0.001). The tumor volumes of mice for 21 d in the NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group were (1 667.50±250.40) mm 3, (129.20±21.00) mm 3 and (775.41±77.51) mm 3 respectively, with a statistically significant difference ( F=77.651, P<0.001). The tumor weights of the 3 groups were (1.35±0.18) g, (0.12±0.01) g and (0.61±0.03) g respectively, with a statistically significant difference ( F=104.191, P<0.001). Conclusion:The expression of miR-20a-5p is significantly decreased in osteosarcoma tissues, and the expression of KDM6B is significantly increased in osteosarcoma tissues. Overexpression of miR-20a-5p may inhibit the proliferation, migration and invasion of osteosarcoma cells and tumor growth by targeting to reduce the expression of KDM6B.