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1.
AIM To observe the changes of lipophagy during foam cells formation, and to determine the effect of lipophagy on the lipid content and cholesterol outflow of foam cells. METHODS Human THP-1 monocytes were induced by phorbol-12-myristate-13-acetate for 48 h to differentiate into macrophages, and then were incubated with 50 mg/L oxidized low-density lipoprotein （oxLDL） to form foam cells. Lipids in foam cell were stained by oil red O, and the lipid content was determined. The total cholesterol （TC） and free cholesterol （FC） levels in foam cells were measured by cholesterol testing kit. Cholesteryl ester （CE） and CE/TC ratio were calculated. The cholesterol efflux rate was detected by cholesterol efflux assay kit. The expression of autophagy-related proteins, including autophagy-related protein 5 （Atg5）, microtubule-associated protein 1 light chain 3 （LC3） and P62, were detected by Western blot. The colocalization of lipid droplets （LD） and LC3 was detected by immunofluorescence staining. The autophagy inducer rapamycin （Rap） or blocker 3-methyladenine （3MA） was used to intervene foam cells, and the expression of Atg5, LC3 and P62, the co-expression of LD and LC3, the cholesterol content and the cholesterol efflux rate were determined. RESULTS Formation of foam cells was observed at 24 h after stimulation with oxLDL at 50 mg/L, as indicated by intracellular CE/TC ratio exceeding 50%.Cholesterol efflux assay revealed that the cholesterol efflux rate increased within 24 h during foam cell formation but decreased after 48 h （P<0.05）. Western blot results displayed that the expression of Atg5 and LC3-II/LC3-I ratio were increased within 24 h of foam cell formation, but was deceased after 48 h （P<0.05）. The expression of P62 was decreased within 24 h but was increased at 48 h （P<0.05）. The colocalization of LD and LC3 was increased at 24 h but was decreased at 48 h after oxLDL stimulation. Treatment with Rap up-regulated the expression of Atg5 and LC3-II/LC3-I ratio, reduced the level of P62, increased the colocalization of LD and LC3, promoted the cholesterol efflux, anf reduced cholesterol content in foam cells （P<0.05）. On the contrary, 3MA inhibited the expression of Atg5, reduced LC3-II/LC3-I ratio, elevated the level of P62, decreased the colocalization of LD and LC3, reduced the outflow of cholesterol, increased the content of TC and CE, and elevated CE/TC ratio in foam cells （P<0.05）. CONCLUSION Lipophagy is enhanced at 24 h but decreased at 48 h during foam cell formation. Lipophagy inhibited foam cell formation by reducing cholesterol content and increasing cholesterol efflux.  相似文献   

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3.
AIM: To investigate the effect of microRNA-323 (miR-323) on the apoptosis of hypoxia-induced rat H9C2 cardiomyocytes and its mechanism. METHODS: The hypoxic injury model was established in the H9C2 cells. Anti-miR-323, pcDNA-FGF9 and si-FGF9 were transfected into the H9C2 cells and cultured under hypoxic condition for 48 h. The expression of miR-323 was detected by qPCR. The protein levels of fibroblast growth factor 9 (FGF9), cleaved caspase-3, c-Jun N-terminal kinase (JNK) and p-JNK were determined by Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The method of bioinformatics was applied to predict the target gene of miR-323, and dual-luciferase reporter assay was used for further validation. RESULTS: Hypoxia greatly reduced the viability of H9C2 cells at 12 h, 24 h and 48 h (P<0.05), and remarkably increased apoptotic rate and the protein level of cleaved caspase-3 in a time-dependent manner (P<0.05). The expression of miR-323 and the protein level of p-JNK were up-regulated and the expression of FGF9 was down-regulated in the H9C2 cells exposed to hypoxia (P<0.05). Down-regulation of miR-323 and over-expression of FGF9 obviously increased the viability of the H9C2 cells exposed to hypoxia, and decreased the apoptotic rate and the protein level of cleaved caspase-3 (P<0.05). FGF9 was the target gene of miR-323. Down-regulation of FGF9 reversed the attenuating effect of down-regulation of miR-323 on hypoxia-induced H9C2 cell injury. miR-323 regulated FGF9 and affected p-JNK level. CONCLUSION: miR-323 affects the viability and apoptosis of H9C2 cardiomyocytes under hypoxia by targeting FGF9 and regulating JNK signaling pathway.  相似文献   

4.
AIM To investigate the effect of β1-adrenergic receptor autoantibodies （β1-AA） on the rhythm of autophagy marker microtubule-associated protein 1 light chain 3 （LC3）, and the underlying mechanism of cardiomyocyte death. METHODS The test materials were Sprague-Dawley （SD） rats and H9c2 rat cardiomyocytes. The SD rats were randomly divided into immunization group and control group with 6 rats in each group. The H9c2 cells were randomly divided into control group, β1-AA group, lentivirus （LV）-NC group, and LV-shPer2 group （n=6）. Affinity chromatography was used for purification of β1-AA from rat serum. CCK-8 assay was used to observe the viability of cardiomyocytes treated with β1-AA for 24 h. The cells were synchronized by dexamethasone and then treated with β1-AA. The mRNA and protein levels of LC3 at different time points were determined by real-time PCR and Western blot, respectively. The Per2 protein level at different time points was also determined by by Western blot. JTK_CYCLE algorithm was used to estimate the circadian rhythm parameters. After destruction of LC3 circadian rhythm via LV-shPer2, CCK-8 assay was used to measure the viability of H9c2 cells. RESULTS High level of β1-AA in rat serum was found after active immunization compared with control group （P<0.05）. The viability of H9c2 cells in β1-AA group was significantly lower than that in control group （P<0.05）. The LC3 and Per2 rhythms were both disrupted in H9c2 cells induced by β1-AA （JTK_CYCLE P<0.05）. After LV-shPer2 infection, the LC3 rhythm was disrupted （JTK_CYCLE P<0.05） and the cell viability was reduced （P<0.05）. CONCLUSION β1-AA may induce the destruction of autophagy marker LC3 rhythm in rat cardiomyocytes and then promote cell death.  相似文献   

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6.
AIM To observe the changes of liver structure, the levels of transforming growth factor-β1 （TGF-β1）, microRNA-181a, LC3-II/-I, beclin-1 and collagen deposition in hepatic fibrosis （HF） rats induced by carbon tetrachloride （CCl4）, and the effect of microRNA-181a on autophagy of rat hepatic stellate cells （HSCs） induced by TGF-β1, and to explore the possible mechanism of microRNA-181a in regulating HSC activation and HF. METHODS Wistar rats （n=40） were randomly divided into 5 groups （with 8 in each）： control group （subcutaneous injection of olive oil, 3 mL/kg, twice a week）, and CCl4-induced HF groups of 2, 4, 6 and 8 weeks （subcutaneous injection of 40% CCl4, 3 mL/kg, twice a week for 2, 4, 6 and 8 weeks, respectively）. Masson staining was used to evaluate the changes of HF in rats. The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA. The level of microRNA-181a in rat liver tissues was detected by RT-qPCR. The protein levels of LC3-II/-I, beclin-1, α-smooth muscle actin （α-SMA）, collagen type I （Col I） and collagen type Ⅲ （Col Ⅲ） in rat liver tissues were measured by Western blot. HSC-T6 cells were transfected with microRNA-181a inhibitor, or pretreated with the autophagy inhibitor 3-methyladenine （3-MA）, before treatment with TGF-β1 to stimulate autophagy. The expression of microRNA-181a, LC3-II/-I, beclin-1, α-SMA, Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot. RESULTS The levels of TGF-β1, microRNA-181a, LC3-II/-I ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF, and microRNA-181a expression showed a positive correlation with autophagy-associated proteins （P<0.01）. MicroRNA-181a level was significantly increased, which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181a inhibitor, along with suppression of autophagy and cell activation （P<0.01）, which were similar to the effects of 3-MA treatment. CONCLUSION CCl4 promotes rat HF, the microRNA-181a expression of liver tissue, and autophagy in a time-dependent manner. Reducing the expression of microRNA-181a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1. The regulation of HSC autophagy by microRNA-181a may be involved in rat HF.  相似文献   

7.
2011 年春季定植的矮化中间砧苹果成品苗（3 年根 1 年干的‘宫藤富士’/SH6/平邑甜茶）为试材，设置 7 种不同的栽植密度（株行距分别为 1 m × 3 m、1.5 m × 3 m、2 m × 3 m、0.75 m × 4 m、1 m × 4 m、1.25 m × 4 m 和 1.5 m × 4 m），细纺锤形整枝修剪，自栽植第 2 年，连续 7 年调查 7 种栽植密度对树体生长、冠层光照分布、果实产量和品质的影响。随着树龄的增长，不同栽植密度下树干粗度和总枝量逐年增加，不同处理间树干粗度无显著差异，第 7 年 1 m × 3 m 和 0.75 m × 4 m 两个栽植密度下树体总枝量超过 140 万条 · hm-2，第 8 年均超过 140 万条 · hm-2。栽植前期（第 2 ~ 4 年）各栽植密度树体短枝比例不断增加，长枝比例不断减少，第 5 年各栽植密度枝类组成趋于稳定；综合稳产 3 年（第 6 ~ 8 年）树体的枝类组成数据，4 m 行距的短枝比例明显高于 3 m 行距，长枝比例略低。树体冠层平均相对光照强度由高到低的株行距处理依次为 1.5 m × 4 m（63.87%）、1.25 m × 4 m（61.44%）、2 m × 3 m（61.27%）、1 m × 4 m（59.19%）、0.75 m × 4 m（55.79%）、1.5 m × 3 m（53.67%）和 1 m × 3 m（49.37%）；相同栽植株数下，4 m 行距处理低光效（相对光照强度小于 40%）的区域比例显著小于 3 m 行距。比较前 5 年的累计产量，以行距 4 m 和 1 m × 3 m 的最高。综合稳产 3 年的结果情况，大果率（单果质量 > 200 g 的果实产量占总产量的比例）以 4 m 行距和 2 m × 3 m 的最高。各栽植密度下的果实的可溶性固形物含量、固酸比、果形指数和果实硬度均无显著差异。综上，采用 4 m 行距，1 ~ 1.25 m 株距，树体成形快，稳产后树体结构合理，冠层光照充足，低效光区比例少，前期产量高。  相似文献   

8.
AIMTo investigate whether adiponectin inhibits the decrease in autophagy of rat H9c2 cardiomy?ocytes induced by β1-adrenergic receptor (β1-AR) autoantibodies (β1-AA), and to explore its mechanism. METH?ODS: SD rats were actively immunized with β1-AR extracellular second loop (β1-AR-ECII) antigen peptide. Affinity chromatography was used to purify β1-AA in serum of the SD rats. The viability of H9c2 cells was measured by CCK-8 assay. The mRNA levels of LC3B and beclin-1 in the H9c2 cells were detected by real-time PCR. The protein levels of LC3-II, P62, AMP-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) were determined by Western blot. RESULTSPretreatment with adiponectin at 10 μg/L for 1 h reversed the decreased viability of H9c2 cells induced by β1-AA. Compared with control group, β1-AA decreased the mRNA expression of LC3B and beclin-1, decreased the protein level of LC3-II, and increased the expression of P62 protein in the H9c2 cells, suggesting that β1-AA decreased the autophagic flux in cardiomyocytes. Adiponectin obviously reversed β1-AA-induced decline in autophagic flux, and up-regulated the phosphorylation level of AMPK decreased by β1-AA. Treatment with AMPK inhibitor Compound C for 30 min, we observed that the mRNA expression of LC3B and beclin-1 and the protein level of LC3-II in the H9c2 cells decreased by β1-AA were not effectively reversed by adiponectin, but the increase in P62 protein expression was still effectively reversed, indicating that adiponectin increased autophagosome production dependent on the AMPK pathway, but increased autophagosome clearance independent on the AMPK pathway. CONCLUSION Adiponectin inhibits the decreased autophagy of H9c2 cardiomyocytes induced by β1-AA.  相似文献   

9.
AIM: To investigate the influence of programmed cell death 5 (PDCD5) on apoptosis and autophagy in the cardiomyocytes exposed to hypoxia/reoxygenation (H/R) and its potential mechanism.METHODS: H9c2 cells were exposed to H/R. PDCD5 was downregulated by RNA interference. The cell viability was measured by MTT assay. TUNEL assay was used to detect cell apoptosis. The mRNA and protein levels were determined by RT-qPCR and Western blot.RESULTS: The expression of PDCD5 was upregulated in the cardiomyocytes after H/R injury. Furthermore, H/R injury obviously reduced the cell viability and enhanced the apoptosis and autophagy of the cardiomyocytes. However, knockdown of PDCD5 increased the cell viability, and attenuated H/R-induced apoptosis, accompany with reduction of Bax and augment of Bcl-2 expression. Additionally, silencing PDCD5 markedly inhibited H/R-induced autophagy by regulating the expression of LC3-II/LC3-I and beclin-1. Moreover, downregulation of PDCD5 suppressed NF-κB signaling by redu-cing the protein level of p-P65.CONCLUSION: Silencing PDCD5 suppresses H/R-induced H9c2 cells apoptosis and autophagy by blocking NF-κB signaling pathway. The result indicates a new strategy for the prevention and treatment of myocardial I/R injury.  相似文献   

10.
AIM To investigate the effect of tetrandrine on the autophagy of human ovarian serous cystadenocarcinoma SKOV3 cells, and to explore its molecular mechanism. METHODS The SKOV3 cells were treated with various concentrations of tetrandrine, and the cell viability was measured by MTT assay. The formation of autophagolysosomes was observed by acridine orange staining under fluorescence microscope. The protein levels of LC3, mTOR, p-mTOR, Akt and p-Akt in the SKOV3 cells were determined by Western blot. The viability of the SKOV3 cells treated with tetrandrine alone or combined with autophagy inhibitor 3-methyladenine (3-MA) were measured by MTT assay. RESULTS Tetrandrine significantly inhibited the viability of SKOV3 cells in a dose-dependent manner (P<0.01). The results of acridine orange fluorescence staining showed that the number of intracellular autophagolysosomes with bright red fluorescence in the SKOV3 cells was significantly increased after tetrandrine treatment, while the autophagolysosomes were rarely observed in control group. The protein levels of LC3-II and P62 in the SKOV3 cells were significantly increased after tetrandrine treatment (P<0.01). Furthermore, treatment with tetrandrine resulted in significant down-regulation of phosphorylated form of mTOR and AKT in the SKOV3 cells (P<0.01), while total mTOR and AKT protein levels were not changed. Finally, combination of tetrandrine and 3-MA significantly decreased the cell viability compared with using tetrandrine alone (P<0.01). CONCLUSION The autophagy of human ovarian cancer SKOV3 cells were induced by tetrandrine and the molecular mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway．  相似文献   

11.
AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.  相似文献   

12.
AIM To investigate the effect of mangiferin on hypoxia/reoxygenation (H/R)-induced injury of human myocardial cells and its mechanism. METHODS Human myocardial AC16 cells were divided into normal group, H/R group and H/R + mangiferin (50, 100 and 200 μmol/L) treatment groups. The mRNA and protein expression levels of Kelch-like epichlorohydrin-associated protein-1 (Keap-1), Bax, Bcl-2, caspase-3, caspase-9 and superoxide dismutase 2 (SOD2) were detected by RT-qPCR and Western blot, respectively. The protein expression of nuclear factor E2-related factor 2 (Nrf-2) in nucleus was determined by Western blot. The expression of microRNA-432-3p (miR-432-3p) was detected by RT-qPCR. The generation of reactive oxygen speciess (ROS) in the cells was measured by DCFH-DA probing. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry. RESULTS No significant difference in the expression of miR-432-3p and Keap-1 between normal group and H/R group was observed. Compared with normal group, the nuclear translocation of Nrf-2, the ROS level, and the mRNA and protein expression of Bax, caspase-3 and caspase-9 were significantly increased in H/R group （P<0.05）. The mRNA and protein expression of SOD2 and Bcl-2, and the cell viability significantly decreased in H/R group compared with normal group, while the apoptosis was increased significantly （P<0.05）. Treatment with mangiferin resulted in an increase in the miR-432-3p expression and a decrease in the ROS level, and the expression of Keap-1, Bax, caspase-3 and caspase-9 was also inhibited as compared with H/R group （P<0.05）. The Nrf-2 nuclear translocation, and the protein levels of SOD2 and Bcl-2 in mangiferin treatment groups were significantly increased as compared with H/R group （P<0.05）. The cell viability was increased significantly, and the apoptosis was decreased significantly in mangiferin treatment groups as compared with H/R group （P<0.05）. The effects of mangiferin in middle- and high-dose groups were better than those in low-dose group, and no significant difference between middle- and high-dose groups was found. CONCLUSION Mangiferin inhibits the decrease in myocardial cell viability and the apoptosis induced by H/R injury. The mechanism may be related to the up-regulation of Nrf-2 antioxidant stress effect via enhancing the expression of miR-432-3p.  相似文献   

13.
AIM To investigate the effect of paeonol on the viability and migration ability of hepatocellular carcinoma cells and its molecular mechanism. METHODS Human hepatocellular carcinoma Hep3B cells was treated with paeonol at different concentrations (50, 100, 200 and 400 mg/L). The cell viability was measured by CCK-8 assay to determine the optimal drug concentration. The Hep3B cells were divided into normal control (NC) group, paeonol group, miR-NC group, miR-424-3p group, paeonol+anti-miR-NC and paeonol+anti-miR-424-3p group. The expression level of miR-424-3p was detected by RT-qPCR. The migration ability was detected by Transwell assay. The protein levels of cyclin D1, matrix metalloproteinase 2 (MMP2), MMP9 and PI3K/AKT signaling pathway-related molecules were determined by Western blot. RESULTS Paeonol intervention inhibited the viability of Hep3B cells in a concentration-dependent manner (P<0.05). The concentration of paeonol at 200 mg/L was selected for the following study. Paeonol intervention inhibited the protein expression of MMP2 and MMP9 in the Hep3B cells, and inhibited the migration ability of the Hep3B cells. Paeonol intervention promoted the expression of miR-424-3p in the Hep3B cells (P<0.05). Over-expression of miR-424-3p inhibited the expression of cyclin D1, MMP2 and MMP9 in the Hep3B cells and inhibited cell viability and migration ability (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on the viability and migration ability of the Hep3B cells (P<0.05). Paeonol inhibited phosphorylation levels of PI3K and AKT in the Hep3B cells and inhibited the activation of PI3K/AKT signaling pathway (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on PI3K/AKT signaling pathway in the Hep3B cells (P<0.05). CONCLUSION Paeonol inhibits the viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway.  相似文献   

14.
Lü Xin  TANG Wen-xue  GUO Liang 《园艺学报》2000,36(11):2081-2085
AIM To explore the effect of continuous renal replacement therapy （CRRT） on mRNA expression of autophagy-related molecules and the prognosis in the patients with acute kidney injury （AKI）. METHODS A total of 174 patients from our hospital who were diagnosed to have AKI and underwent CRRT between February 2015 and March 2018 were involved in this study. The plasma levels of interleukin-1β （IL-1β） and interleukin-6 （IL-6）, the serum creatinine （SCr） level, and the mRNA expression levels of autophagy-related molecules, including microtubule-associated protein 1 light chain 3-II （LC3-II）, autophagy-related protein 5 （Atg5） and beclin-1, in the monocytes from peripheral blood were compared before and after CRRT. According to the survival of AKI patients after 4 weeks of CRRT, the enrolled patients were divided into death group （n=43） and survival group （n=131）, and the mRNA expression levels of the above molecules were compared between the 2 groups. RESULTS After CRRT treatment, the plasma levels of IL-1β and IL-6, the level of SCr, and the mRNA expression levels of LC3-II, Atg5 and beclin-1 in the monocytes were significantly lower than those before CRRT treatment （P<0.05）. The mRNA expression levels of LC3-II, Atg5 and beclin-1 in death group were significantly higher than those in survival group （P<0.05）. The positive correlation between SCr and IL-1β, IL-6, LC3-II or beclin-1 was observed （P<0.05）, and no correlation between SCr and Atg5 was found （P>0.05）. CONCLUSION CRRT decreases the mRNA expression levels of autophagy-related molecules in the patients with AKI and reduces the autophagy activity, which is protective for the patients. These autophagy-related molecules may be applied as a potential markers to predict the prognosis of CRRT.  相似文献   

15.
AIM To investigate the effects of long non-coding RNA (lncRNA) LINC01503 on the viability, migration and invasion of lung cancer cells and its mechanism. METHODS Human lung carcinoma H1299 cells were divided into si-NC group (transfected with si-NC), si-LINC01503 group (transfected with si-LINC01503), pcDNA group (transfected with pcDNA), pcDNA-LINC01503 group (transfected with pcDNA-LINC01503), miR-NC group (transfected with miR-NC), miR-335-5p group (transfected with miR-335-5p mimics), si-LINC01503+anti-miR-NC group (co-transfected with si-LINC01503 and anti-miR-NC), si-LINC01503+anti-miR-335-5p group (co-transfected with si-LINC01503 and anti-miR-335-5p), miR-NC+WT-LINC01503 group (co-transfected with miR-NC and WT-LINC01503), miR-NC+MUT-LINC01503 group (co-transfected with miR-NC and MUT-LINC01503), miR-335-5p+WT-LINC01503 group (co-transfected with miR-335-5p and WT-LINC01503) and miR-335-5p+MUT-LINC01503 group (co-transfected with miR-335-5p and MUT-LINC01503). The expression of miR-335-5p and LINC01503 was detected by RT-qPCR. Western blot was used to detect protein expression. MTT assay was used to detect cell viability. Transwell assay was used to detect the migration and invasion abilities. Dual-luciferase reporter assay was used to confirm the targeted relationship between LINC01503 and miR-335-5p. RESULTS Compared with normal tissues, the expression of LINC01503 was significantly increased in the lung cancer tissues, and the expression of miR-335-5p was significantly decreased (P<0.05). Compared with stage I/II , the expression level of LINC01503 in the lung cancer tissues of stage III/IV was significantly increased, and the expression of miR-335-5p was significantly decreased (P<0.05). The patients with high expression of LINC01503 had lower short-term survival rates than those with low expression of LINC01503 (P<0.05). Compared with normal human bronchial epithelial cell line BEAS-2B, the expression of miR-335-5p in lung cancer cell lines H1299, A549 and SPC-A-1 were significantly decreased, and the expression of LINC01503 was significantly increased (P<0.05). Over-expression of miR-335-5p and inhibition of LINC01503 expression inhibited the viability, migration and invasion of H1299 cells, and inhibited the protein expression of cyclin D1, matrix metalloproteinase-2 （MMP-2） and MMP-9 (P<0.05). LINC01503 targeted and regulated miR-335-5p expression, and interfering with miR-335-5p expression reversed the inhibitory effect of inhibiting LINC01503 expression on the viability, migration and invasion of H1299 cells. CONCLUSION Inhibition of lncRNA LINC01503 inhibits the viability, migration and invasion of lung cancer cells. The mechanism may be related to the targeted regulation of miR-335-5p.  相似文献   

16.
AIM To investigate the effect of sinomenine (SN) on the damage of human neuroblastoma SK-N-SH cells induced by 1-methyl-4-4 phenylpyridine (MPP+) and its mechanism for exploring the pathogenesis of Parkinson disease. METHODS SN was used to treat MPP+-induced SK-N-SH cells. The levels of malondialdehyde (MDA) and glutathione (GSH) in cell culture supernatants were measured by ELISA. The apoptosis was analyzed by flow cytometry. The protein expression levels of Bcl-2 and Bax were determined by Western blot. The expression levels of long noncoding RNA ANRIL and microRNA-626 (miR-626) were detected by RT-qPCR. Dual-luciferase reporter assay was used to evaluate the relationship between ANRIL and miR-626. After ANRIL small interfering RNA was transfected into SK-N-SH cells, the effects of ANRIL expression knock-down on MPP+-induced SK-N-SH cell apoptosis, the protein expression levels of Bcl-2 and Bax, and the levels of MDA and GSH in cell culture supernatants were examined. RESULTS After treatment with MPP+, the apoptotic rate, Bax protein level and ANRIL expression in SK-N-SH cells were increased (P<0.05), and the Bcl-2 protein level and miR-626 expression were decreased (P<0.05). The level of MDA in cell culture supernatants was increased (P<0.05), and the level of GSH was decreased (P<0.05). After SN treatment or ANRIL expression knock-down, decreased apoptotic rate, Bax protein level and ANRIL expression (P<0.05), and increased Bcl-2 protein level and miR-626 expression in MPP+-induced SK-N-SH cells were observed (P<0.05). The level of MDA in the cell culture supernatants was decreased (P<0.05), and the level of GSH was increased (P<0.05). CONCLUSION SN attenuates MPP+-induced damage in SK-N-SH cells by regulating ANRIL/miR-626 signaling pathway.  相似文献   

17.
AIM: To investigate the effects of angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) on oxidative stress, autophagy and apoptosis in H9c2 cells, and to analyze the possible mechanism. METHODS: The rat H9c2 cells were cultured in vitro. The effect of AT1-AA at different concentrations for different time on the cell viability was measured by CCK-8 assay. Upon the optimum concentration (10-5 mol/L) and time point (24 h) determined in this stu-dy, the experssion levels of autophagy- and apoptosis-related proteins were detected by Western blot, and the levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) were examined by oxidative stress kits. RESULTS: AT1-AA decreased cell viability in a concentration- and time-dependent manner, promoted apoptosis, and up-regulated the levels of autophagy and oxidative stress (P<0.05). The apoptosis of H9c2 cells induced by AT1-AA was decreased after pretreatment with autophagy inhibitor 3-methyladenine (P<0.05). The levels of autophagy and apoptosis in the H9c2 cells pretreated with α-lipoic acid were decreased (P<0.05). Pretreatment with angiotensin Ⅱtype 1 receptor inhibitor telmisartan inhibited oxidative stress, and significantly decreased the levels of autophagy and apoptosis induced by AT1-AA in the H9c2 cells (P<0.05). CONCLUSION: AT1-AA induces autophagy and apoptosis of H9c2 cells through oxidative stress.  相似文献   

18.
AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O2) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.  相似文献   

19.
AIMTo investigate the role of SUMO-specific protease 3 (SENP3) in macrophage polarization and calcium phosphate (CaPO4)-induced abdominal aortic aneurysm (AAA) formation in mice. METHODS（1） Bone marrow-derived monocytes (BMDMs) in Senp3flox/flox (wild-type, WT) mice and Senp3flox/flox; Lyz2-Cre (monocyte-specific SENP3 knockout, i.e. conditioned knockout, cKO) mice were isolated and induced for M1 and M2 polarization. The mRNA and protein expression level of SENP3 were detected by RT-qPCR, Western blot and immunocytofluorescence, and the differential distribution of M1/M2 BMDMs from WT and cKO mice was analyzed. （2） CaPO4 was administrated to induce AAA model in 8~12-week-old male WT and cKO mice. The AAA incidence, survival rate and maximal aortic diameter were analyzed between the 2 groups. Aortic aneurysm tissues were collected for pathological analysis, and the expression levels of SENP3, interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), IL-6 and matrix metalloproteinases-9 (MMP-9) were measured by RT-qPCR and Western blot. Dihydroethidium staining in situ in frozen sections was used to analyze the production of reactive oxygen species (ROS). （3） To explore the potential mechanisms, Western blot and co-immunoprecipitation were used to verify the de-SUMO modification of mitogen-activated protein kinase kinase 7 (MKK7) induced by SENP3. Besides, BMDMs were transfected with Flag-MKK7 wild type (Flag-MKK7 WT) and SUMO-modified site K18 mutant (Flag-MKK7 K18R mutant), and then M1 polarization of the cells was induced. The protein levels of p-JNK and MMP-9 in the 2 groups were determined by Western blot. RESULTS(1) SENP3 expression was up-regulated in M1 polarized macrophages (P<0.01), but was down-regulated in M2 polarized macrophages (P<0.01). The expression of SENP3 was decreased during the transformation of M1 to M2 in the macrophages (P<0.01), but was significantly up-regulated during the opposite process (P<0.01). Besides, more M1 macrophages and less M2 macrophages after induction were observed in the BMDMs from cKO mice than those from WT mice. (2) SENP3 expression was up-regulated in AAA tissues (P<0.05). The AAA incidence of cKO mice was significantly reduced after CaPO4 induction (P<0.01), the survival rate was significantly improved (P<0.05), and maximal aortic diameter was significantly reduced in cKO group (P<0.01). The levels of IL-1β, IL-6 and TNFα, and the production of ROS were significantly down-regulated (P<0.01), meanwhile MMP-9 expression was also down-regulated in cKO mice (P<0.05). (3) the SUMO2/3 modification of MKK7 was reduced during M1 polarization, and MKK7 interaction with SENP3 was enhanced. Significantly up-regulated protein level of p-JNK and MMP-9 were verified in the M1 macrophages transfected with Flag-MKK7 K18R mutant (P<0.05). CONCLUSION SENP3 activates the MAPK/JNK pathway via de-SUMOylation of MKK7, regulates the M1/M2 polarization of macrophages and promotes the protein level of MMP-9, thus aggravating AAA formation.  相似文献   

20.
AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation; （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h; （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling, and the rest were treated the same as the H/R group; （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia; （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h, then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure, and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）, beclin 1, LC3, mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK, beclin 1, LC3-Ⅱ, p-mTOR and P62. RESULTS Compared with Con group, the cell viability in H/R, DMSO, 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK, beclin 1 and LC3 was significantly increased, and the protein levels of p-AMPK, beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased, and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R, DMSO and HX531 groups （P<0.05）, and no significant difference among H/R, DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R, and its mechanism may be related to the inhibition of autophagy.  相似文献   

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