ABSTRACT
Objective@#To explore the expression of microRNA-17-5p (miR-17-5p) in esophageal squamous cell carcinoma (ESCC) and its effects on cell proliferation and invasion ability.@*Methods@#Real-time quantitative PCR (RT-qPCR) was used to detect the miR-17-5p level in ESCC tissues and cells. MiR-17-5p inhibitor and negative control (NC) were transfected into EC9706 and TE1 cells, and miR-17-5p expression was examined by using RT-qPCR. Cell counting kit-8 (CCK-8) and EdU were conducted to detect cell proliferation and Transwell chamber was used to investigate cell invasion ability. Dual-luciferase reporter assay was used to detect the direct interaction of miR-17-5p and retinoblastoma-like protein-2 (RBL2). Western blot and RT-qPCR were used to detect the expression of RBL2 in ESCC tissues, respectively. Finally, the correlation between RBL2 and miR-17-5p was analyzed.@*Results@#The miR-17-5p level in ESCC tissues was 4.222±0.392, significantly higher than 1.081±0.046 in normal esophageal epithelial tissues (P<0.001). The expressions of miR-17-5p in ESCC cells, including EC9706, Eca109, TE1, KYSE450, KYSE70 and KYSE520, were 13.84±1.266, 6.453±0.293, 11.41±0.520, 2.613±0.548, 5.251±0.239 and 4.251±0.195, respectively, all obviously higher than (1.007±0.079) in normal esophageal epithelial cell Het-1A (P<0.05). The miR-17-5p level in patients with ESCC Ⅲ~Ⅳ was 5.094±0.562, markedly higher than 2.934±0.364 in patients with ESCCⅠ~Ⅱ(P<0.01). Moreover, the miR-17-5p level in ESCC patients with lymph node metastasis was 5.523±0.634, markedly higher than 3.533±0.461 in ESCC patients without lymph node metastasis (P<0.05). The survival ratio of ESCC patients with higher miR-17-5p level was evidently lower than that of ESCC patients with lower miR-17-5p level (P<0.05). MiR-17-5p inhibitor significantly downregulated the miR-17-5p expression in EC9706 and TE1, which suppressed cell proliferation and invasion ability. Dual-luciferase reporter assay revealed that co-transfection of 3′ untranslated region-wild type (3′UTR-WT) of RBL2 and miR-17-5p mimic significantly reduced luciferase activity in EC9706 and TE1 cells (P<0.01), which implicated that RBL2 was the direct target of miR-17-5p. The result of western blot revealed that RBL2 protein expressions in miR-17-5p group of EC9706 and TE1 cells were 0.936±0.055 and 0.923±0.048, obviously higher than 0.087±0.019 and 0.102±0.010 in control group (P<0.001). The expression of RBL2 in ESCC tissues was 0.219±0.510, markedly lower than 0.983±0.324 in normal esophageal epithelial tissues (P<0.001). The miR-17-5p level exhibited a negative correlation with RBL2 level in ESCC tissues (r=-0.462, P<0.001). Downregulation of RBL2 reversed the miR-17-5p inhibitor induced suppression of cell proliferation and invasion ability.@*Conclusion@#MiR-17-5p participates in the carcinogenesis and development of ESCC, thus may be a potential therapeutic target for ESCC patients.
ABSTRACT
Objective To investigate the preventive effect of Xuebijing injection on acute lung injury induced by cardiopulmonary bypass (CPB) and the underlying mechanism. Methods ① In vivo experiment: 30 Sprague-Dawley (SD) rats were randomly divided into sham group, CPB group, Xuebijing pretreatment group (XBJ+CPB group) with 10 rats in each group. CPB model was reproduced in rats; and CPB was not performed in sham group, but only through arteriovenous puncture. In the XBJ+CPB group, 4 mL/kg Xuebijing injection was injected intraperitoneally 2 hours before CPB, sham group and CPB group were injected with equal volume of normal saline at the same time. The blood from femoral artery was analyzed 4 hours after operation, and the oxygenation index (PaO2/FiO2) was calculated. Then the rats were sacrificed to collect bronchoalveolar lavage fluid (BALF), and the lung permeability index (PPI) was calculated. The lung tissues were harvested, and the wet/dry weight ratio (W/D) of lung tissue was measured. The index of quantitative evaluation of alveolar injury (IQA) was measured. The levels of interleukins (IL-1, IL-6) and tumor necrosis factor-α(TNF-α) in lung tissue and BALF were measured by enzyme-linked immunosorbent assay (ELISA). The content of malondialdehyde (MDA) and the activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) in lung tissue were detected by biochemical method. The microRNA-17-5p (miR-17-5p) expression in lung tissue was determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR).② In vitro experiments: type Ⅱ alveolar epithelial cells (AECⅡ) were cultured in vitro, and they were randomly divided into control group (the cells were treated by preoperative serum of CPB in patients with ventricular septal defect), CPB group (the cells were treated by serum after CPB in patients), and XBJ+CPB group (Xuebijing injection 10 g/L+serum after CPB in patients). After 12 hours of culture in each group, the expression of miR-17-5p was detected by RT-qPCR. AECⅡ cells were transfected with miR-17-5p mimic, inhibitor or corresponding control oligonucleotide (negative control), respectively, to observe the effect of miR-17-5p on Xuebijing regulating CPB-induced apoptosis rate and caspase-3 activity. Results ① In vivo experiment: compared with the sham group, the PPI, lung W/D ratio, IQA, and IL-1, IL-6, TNF-α in lung tissue and BALF, as well as MDA content and MPO activity in lung tissue were significantly increased, PaO2/FiO2 and SOD activity in lung tissue were significantly decreased. The parameters of the XBJ+CPB group were significantly improved, suggesting that Xuebijing pretreatment could improve CPB-induced ALI in rats. The expression of miR-17-5p in lung tissue of the CPB group was significantly down-regulated as compared with sham group (2-ΔΔCt: 0.48±0.13 vs. 1.00±0.11, P < 0.05);while the expression of miR-17-5p in the XBJ group was significantly up-regulated as compared with the CPB group (2-ΔΔCt: 1.37±0.09 vs. 0.48±0.13, P < 0.05), indicating that the improvement of Xuebijing injection on lung injury after CPB might be related to miR-17-5p. ② In vitro experiment: the changes in miR-17-5p expression in each group of AECⅡ cells confirmed in vivo results. After transfection of miR-17-5p mimic, the apoptotic rate and caspase-3 activity of each group were significantly lower than those transfected with negative control, and the decrease was more significant in the XBJ+CPB group [apoptotic rate: (7.37±0.95)% vs. (12.60±1.90)%, caspase-3 (A value): 0.82±0.09 vs. 1.37±0.08, both P < 0.05]. After transfection of miR-17-5p inhibitor, the apoptotic rate and caspase-3 activity of each group were significantly more than those transfected with negative control [in the XBJ+CPB group: apoptotic rate was (16.30±1.86)% vs. (12.60±1.90)%, caspase-3 (A value) was 1.78±0.13 vs. 1.37±0.08, both P < 0.05]. This indicated that the apoptosis of AECⅡ cells cultured in serum after CPB was significantly reduced by miR-17-5p, and further reduced by the pretreatment with Xuebijing. Conclusions Xuebiing injection can reduce the inflammatory reaction and oxidative stress of lung tissue in rats with ALI induced by CPB, and improve oxygenation. The mechanism may be related to up-regulation of miR-17-5p expression in AECⅡ cells and inhibition of apoptosis of AECⅡ cells.