Long non-coding RNA TTTY15 expression in osteosarcoma and its effect on viability and invasion ability of osteosarcoma cells

AIM:To observe the expression of long noncoding RNA TTTY15 in osteosarcoma tissues and cell lines and to explore its effect on the viability and invasion ability of osteosarcoma cell lines. METHODS:qPCR was used to detect the expression of TTTY15 in 11 cases of osteosarcoma and its adjacent tissues. The mRNA levels of TTTY15 in osteosarcoma cell lines (143B, Saos2, MG-63, U2OS and HOS) and human osteoblast cell line hFOB1.19 were also tested. TTTY15 was down-regulated after transfected with small interfering RNA in MG-63 cells, the cell line with the highest level of TTTY15. The effect of TTTY15 knockdown on the viability of MG-63 cells was measured by CCK-8 assay. The cell cycle distribution was analyzed by flow cytometry. The effect of TTTY15 knockdown on the cell invasion ability was detected by Transwell assay. The levels of miR-216b-5p and FOXM1 mRNA were detected by qPCR, and the changes of the related proteins were determined by Western blot. RESULTS:Compared with the adjacent tissues, the expression of TTTY15 increased in the osteosarcoma tissues (P<0.01). Compared with the human osteoblast cell line, the expression of TTTY15 increased in the osteosarcoma cell lines (P<0.05), and the level of TTTY15 in the MG-63 cells was the highest (P<0.01). After knockdown of TTTY15 expression in the MG-63 cells, the cell viability was decreased (P<0.05), cell cycle progression was inhibited (P<0.01), and the cell invasion ability was decreased (P<0.01). The expression of miR-216b-5p was increased (P<0.01) and the expression of FOXM1 mRNA was decreased (P<0.01). The protein expression of FOXM1, CDK4, cyclin D1, MMP-2 and N-cadherin was decreased, while the protein expression of E-cadherin was increased (P<0.05). CONCLUSION:The expression of TTTY15 is increased in the osteosarcoma tissues and cell lines. The low expression of TTTY15 inhibits the cell viability and invasion ability of osteosarcoma cells. The possible mechanism is that the knockdown of TTTY15 expression results in the increase in miR-216b-5p expression and the down-regulation of FOXM1 expression.     

Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133+ cell subset by promoting formation of Apaf-1/caspase-9 complex

AIM:To investigate the synergistic effect of resveratrol and 5-fluorouracil on osteosarcoma CD133⁺ cell subset.METHODS:Human osteosarcoma cell line MG-63 CD133⁺ cell subset and the corresponding CD133^- cell subset were treated with resveratrol and 5-fluorouracil.After treatment,the viability of MG-63 cells was measured by MTT assay.The apoptosis of MG-63 cells was analyzed by flow cytometry.Activation of caspase-9 and caspase-3,the expression of Apaf-1,and the release of cytochrome C were evaluated by Western blot.The interaction between Apaf-1 and pro-caspase-9 was detected by co-immunoprecipitation.RESULTS:The cell death and apoptosis of MG-63 CD133⁺ cell subset induced by 5-fluorouracil were significantly weaker than those in the corresponding MG-63 CD133^- cell subset.However,co-treatment with resveratrol significantly enhanced the effect of 5-fluorouracil on inhibiting the viability of MG-63 CD133⁺ cell subset.Mechanically,treatment with resveratrol upregulated the expression of Apaf-1.Transfection with Apaf-1 siRNA abolished the synergistic effect of resveratrol and 5-fluorouracil in MG-63 CD133⁺ cell subset.In addition,the results of co-immunoprecipitation indicated that the combination of resveratrol and 5-fluorouracil significantly induced the formation of Apaf-1/pro-caspase-9 complex,leading to the activation of caspase-9 in MG-63 CD133⁺ cell subset.CONCLUSION:Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133⁺ cell subset by promoting the formation of Apaf-1/caspase-9 complex.     

Effect of cytarabine on curcumin-induced autophagy in human acute myeloid leukemia cells

AIM:To observe whether autophagy occurs in curcumin-induced human acute myeloid leukemia KG1a cells in the presence of chemotherapeutic drug cytarabine and the possible mechanism. METHODS:KG1a cells were cultured in vitro. The ultrastructural changes of the cells were observed under transmission electron microscope. Autophagy was detected by acridine orange staining. The cell viability was measured by MTT assay. The cell cycle distribution was analyzed by flow cytometry. The expression of autophagy-related molecules beclin-1 and LC3 at mRNA and protein le-vels was determined by RT-qPCR and Western blot. RESULTS:Curcumin dose-dependently inhibited the viability of KG1a cells (P<0.05). The growth inhibition rate in combination group was significantly higher than that in single reagent group and control group (P<0.01). Electron microscopical observation showed that curcumin induced the occurrence of autophagosomes, and cytarabine increased curcumin-induced autophagosomes. Acridine orange staining showed that the combined treatment with cytarabine increased the autophagy induced by curcumin, and the number of autophagic acid vesicles and cells containing autophagic acid vesicles were increased. Curcumin blocked the cell cycle in the G₀/G₁ phase. The mRNA expression levels of beclin-1 and LC3 in combination group were significantly higher than those in single reagent group and control group(P<0.01). The results of Western blot showed that the protein expression of beclin-1 was significantly up-regulated in combination group (P<0.05), and the ratio of LC3-Ⅱ/LC3-I was higher than that in control group (P<0.01). CONCLUSION:Curcumin inhibits the viability of KG1a cells and induces autophagy. Cytarabine promotes autophagy, which is superior to curcumin alone. It may be related to the up-regulation of beclin-1 and LC3-Ⅱ by the two reagents.     

Effects of miR-337 targeting p53 expression on autophagy and migration ability of colon cancer cells

AIM: To investigate the effect of microRNA-337 (miR-337) on the autophagy and migration ability of colon cancer cells, and to explore its possible mechanism involving targeting p53 expression. METHODS: The me-thod of immunohistochemistry was used to detect the protein expression of beclin-1, LC3B and p53 in colon cancer tissues. The correlations between the protein expression of beclin-1/LC3B and clinicopathological features, and the correlations between the protein expression of p53 and beclin-1/LC3B were analyzed. After knock-down of p53 expression by small interfering RNA, the formation of autophagiosomes was observed under electron microscope in colon cancer cell line HCT116, and the protein expression of beclin-1 and LC3B was determined by Western blot. The miRNAs targeting p53 were predicted and screened by bioinformatics, and their expression in HCT116 cells was verified by RT-qPCR. Luciferase reporter assay was used to detect the regulatory effect of miR-337 on p53 gene. The protein expression of p53, beclin-1 and LC3B was determined by Western blot, and the migration ability of HCT116 cells after miR-337 over-expression was detected by Transwell assay. RESULTS: The protein expression of beclin-1 and LC3B in the colon cancer tissues was decreased, which was significantly related to the occurrence, development, invasion and metastasis of colon cancer. The expression of p53 was increased in the colon cancer tissues, which was negatively correlated with the protein expression of beclin-1 and LC3B. Knock-down of p53 gene expression increased the protein expression of beclin-1 and LC3B (P<0.05). Over-expression of miR-337 down-regulated the expression of p53, up-regulated the protein expression of beclin-1 and LC3B, and decreased the migration ability of HCT116 cells (P<0.05). CONCLUSION: miR-337 promotes autophagy and inhibits migration ability of colon cancer cells, and the mechanism may be related to targeted inhibition of p53 expression.     

Effect of miR-22 secreted by macrophage exosomes on cardiomyocyte autophagy under uremic toxin stimulation

AIM To investigate the effect of microRNA-22 (miR-22) secreted by macrophage exosomes on the autophagy of H9c2 cardiomyocytes under uremic toxin stimulation. METHODS The macrophage-derived exosomes stimulated by indoxyl sulfate (IS) were collected and co-cultured with H9c2 cells. The levels of miR-22 in the macrophages, macrophage-derived exosomes and H9c2 cells were detected by RT-qPCR. The viability of H9c2 cells was measured by CCK-8 assay. The expression of exosome surface marker protein CD63 and autophagy-related proteins LC3 and P62 was determined by Western blot. RESULTS Under IS stimulation, the expression of exosome surface marker protein CD63 in the macrophages was significantly higher than that in control group (P<0.05), and the levels of miR-22 in the macrophages and macrophage-derived exosomes were significantly increased (P<0.01). With the increase in macrophage exosome concentration, the viability of H9c2 cells was decreased gradually (P<0.05), and the stimulation of macrophage exosomes reduced P62 expression and promoted the conversion of LC3-I to LC3-II in a dose-dependent manner (P<0.05). Macrophage-derived exosomes increased the ratio of LC3-II to LC3-I but decreased P62 protein expression in the H9c2 cells transfected with miR-22 mimic compared with the cells transfected with corresponding negative control miRNAs (P<0.05). However, miR-22 inhibitor yielded contrasting results. CONCLUSION IS-stimulated macrophages increase expression of miR-22 in cardiomyocytes through exosomes, and promote autophagy of the cardiomyocytes.     

Repressive effect of miRNA-363 via SOX4 on human osteosarcoma cell growth and apoptosis

AIM: To detect the expression of miRNA-363 and SOX4 in osteosarcoma tissues and to investigate the effect of miRNA-363 on the viability and apoptosis of human osteosarcoma cell line MG-63.METHODS: Real-time PCR was used to detect the expression level and the relationship of miRNA-363 and SOX4 mRNA in the osteosarcoma tissues and the corresponding paratumorous tissues collected from 63 patients. The expression levels of miRNA-363 and SOX4 in osteosarcoma cell line MG-63 after transfected with miRNA-363 mimics were measured. The cell viability was measured by CCK-8 assay. Flow cytometry was used to monitor the changes of cell cycle and apoptosis. The changes of SOX4 and miRNA-363 expression levels in the MG-63 cells after transfection with SOX4 siRNA or pcDNA/SOX4 was detect by real-time PCR.RESULTS: The expressed level of miRNA-363 was lower, and the expression level of SOX4 was higher in the osteosarcoma tissues than those in the adjacent normal tissues. A significantly negative correlation between the expression levels of miRNA-363 and SOX4 was observed. The expression of miRNA-363 in the MG-63 cells after transfection with miRNA-363 mimics was significantly up-regulated, while the expression of SOX4 in the MG-63 cells was significantly down-regulated, with significant difference as compared with the cells transfected with miRNA-NC and control cells. The viability of MG-63 cells was inhibited, the cell cycle was arrested in G₀/G₁ phase, and the cell apoptosis was increased by transfection with miRNA-363 mimics. The relative protein expression levels of SOX4 in SOX4 siRNA group and pcNDA/SOX4 group were significantly different from those in negative control group, but the relative expression levels of miRNA-363 had no significant difference. Over-expression of SOX4 restored the viability of the MG-63 cells reduced by miR-363.CONCLUSION: The expression level of miRNA-363 is low in human osteosarcoma tissue. miRNA-363 may inhibits the viability of osteosarcoma cell line MG-63 and promotes cell apoptosis in vitro via inhibiting the SOX4 expression.     

miR-335 regulates proliferation of human osteosarcoma MG-63 cells by targeting Sp1

AIM：To investigate the regulatory role of miR-335 on the proliferation of human osteosarcoma cell line MG-63. METHODS：Luciferase reporter gene assay was used to detect the specific binding ability of miR-335 to Sp1 3’-untranslated region (UTR). Real-time PCR and Western blotting were used to evaluate the expression of Sp1 mRNA and protein, respectively. MTT assay was used to analyze the proliferation of MG-63 cells. RESULTS：The luciferase reporter gene assay showed that miR-335 targeted Sp1 3’-UTR. Western blotting and real-time PCR showed that miR-335 inhibited the protein expression of Sp1, but had no effect on the mRNA expression of Sp1. Transfection of Sp1 expression plasmid increased the protein and mRNA expression of Sp1. MTT assay showed the viability of MG-63 cells transfected with miR-335 was significantly decreased compared with negative control. Transfection of Sp1 expression plasmid partly reversed the inhibitory effect of miR-335 on the proliferation of MG-63 cells. CONCLUSION: miR-335 inhibits the proliferation of human osteosarcoma MG-63 cells, which may be related to its targeting on Sp1 3’-UTR and the subsequent down-regulation of Sp1 expression.     

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy. METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3 groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group. The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 mL/kg, suspended in corn oil, ig). After 24 h of operation, the rats from each group were sacrificed and their brains were collected. TTC staining and HE staining were used to define the infarct areas and brain structure. The autophagy-related proteins beclin-1, P62, LC3-Ⅱ/-I and Atg5 in the cortex in each group were compared by Western blot. In vitro, PC12 cells were divided into 3 groups:control group, CoCl₂ group and CoCl₂+EA pretreatment group. CoCl₂ at 800 μmol/L was added to the PC12 cells to induce an anoxic environment. The PC12 cells were pretreated with EA at 8 μmol/L and the cell viability was measured by CCK-8 assay. The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining. MDC staining and TMRE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively. The autophagy-related proteins in PC12 cells were also investigated. RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining. HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage. EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group. Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-Ⅱ/-I in the cortex were increased (P<0.01), and P62 protein expression was decreased (P<0.01) in HIE group. Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-Ⅱ/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01). In vitro, compared with CoCl₂ group, the PC12 cells in CoCl₂+EA pretreatment group showed a lower ROS level. Moreover, the cells in CoCl₂+EA pretreatment group exhibited higher mitochondrial membrane potential than that in CoCl₂ group. MDC staining in CoCl₂ group showed high value of fluorescence and increased number of autophagosomes. EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12 cells. Furthermore, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I in CoCl₂ group were higher (P<0.01), and the protein expression of P62 was lower (P<0.01) than those in control group. In CoCl₂+EA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with CoCl₂ group (P<0.01). CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.     

miR-181a regulates CSE-induced autophagy dysfunction and releases of pro-inflammatory factors in NR8383 alveolar macrophages

AIM:To investigate the effect of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced autophagy disorder and releases of pro-inflammatory factors in NR8383 rat alveolar macrophages. METHODS:The NR8383 cells were treatment with 5%,10% and 20% CSE. The release levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-8 were measured by ELISA. The level of miR-181a was detected by RT-qPCR. The numbers of autophagosomes were observed by Cyto-ID staining. The expression levels of LC3-Ⅱ, beclin-1 and p62 were determined by Western blot. NR8383 cells were pretreated with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin (Rapa) before treatment with 20% CSE, and the release levels of TNF-α, IL-6 and IL-8 were measured by ELISA. Furthermore, NR8383 cells were transfected with miR-181a mimic or miR-181a inhibitor before treatment with 20% CSE, and the release levels of TNF-α, IL-6 and IL-8, and the expression of LC3-Ⅱ, beclin-1 and p62 were detected by ELISA and Western blot, respectively. RESULTS:CSE increased release levels of pro-inflammatory factors and autophagy disorder in a concentration-dependent manner in the NR8383 cells (P<0.05). 3-MA increased CSE-induced releases of pro-inflammatory factors. However, Rapa partially reversed CSE-induced releases of pro-inflammatory factors. Additionally, miR-181a mimic inhibited CSE-induced releases of pro-inflammatory factors and promoted autophagy. However, miR-181a inhibitor increased CSE-induced releases of pro-inflammatory factors and autophagy disorder. CONCLUSION:miR-181a regulates CSE-induced releases of pro-inflammatory factor in the NR8383 cells, which may be related to the regulatory role of miR-181a in autophagy disorder.     

Inhibition of autophgay enhances resveratrol-induced apoptosis of human chondrosarcoma cells

AIM: To investigate whether autophagy is up-regulated when resveratrol (Res) induces apoptosis in chondrosarcoma, and to study the effects of autophagy inhibitor combined with Res on chondrosarcoma. METHODS: SW1353 cells were divided into 4 groups: control group, Res group, 3-methyladenine (3MA) group, and Res+3MA group. Electron microscopy was used to observe the autophagyosomes in control group and Res group. At the same time, the viability of the cells in the 4 groups was detected by CCK-8 assay. TUNEL staining and Western blotting (for determining the levels of cleaved caspase-3, Bax and Bcl-2) were used to reflect levels of apoptosis in all groups. The expression of autophagy-related proteins Beclin 1, LC3-Ⅱ and p62 was detected by Western blotting. RESULTS: Exposure of the cells to Res resulted in a decrease in cell viability and an increase in the level of apoptosis (P<0.05). Compared with control group, the level of apoptosis was increased but the autophagy was decreased (P<0.05). Compared with Res group, the cell viability and the level of autophagy were decreased and the level of apoptosis was increased (P<0.05). CONCLUSION: Resveratrol induces apoptosis and autophagy, and inhibition of autophgay enhances resveratrol-induced apoptosis in chondrosarcoma.     

Effect of remote ischemic postconditioning on autophagy of hippocampal neural cells after cardiopulmonary resuscitation in rats

AIM: To observe the effect of remote ischemic post-conditioning (RIPostC) on autophagy of hippocampal neural cells after cardiopulmonary resuscitation (CPR) in rats. METHODS: Male SD rats (n=45) were randomly divided into sham operation group (sham group), cardiac arrest (CA)/CPR group and RIPostC group, with 15 rats in each group. A CPR model of asphyxiated CA was established by clamping the tracheal tube. Neurological deficit scoring (NDS) was performed at different time points after return of spontaneous circulation (ROSC). The rats were sacrificed 24 h after ROSC and hippocampal tissues were removed. Western blot was used to detect autophagy markers LC3-Ⅱ/LC3-I and beclin-1 in the hippocampal tissues. The apoptosis was detected by TUNEL method. The formation of LC3 particles was observed by immunofluorescence. The ultrastructural changes of autophagosomes and mitochondria were observed under transmission electron microscope. RESULTS: Compared with sham group, the NDS scores of CA/CPR group were decreased, the protein expression of LC3-Ⅱ/LC3-I and beclin-1 was increased (P<0.05), and the apoptosis of the neural cells was increased (P<0.05). Compared with CA/CPR group, the NDS scores in RIPostC group was increased, the protein expression of LC3-Ⅱ/LC3-I and beclin-1 was decreased (P<0.05), and the neural cell apoptosis was decreased (P<0.05). The number of LC3 particles was decreased, intracellular autophagosome number was reduced, and the mitochondrial structure damage was alleviated. CONCLUSION: Remote ischemic post-conditioning improves neurological function in rats after CPR, which may be related to inhibition of excessive autophagy in hippocampus.     

Effects of PDCD5 on hypoxia/reoxygenation-induced autophagy and apoptosis of cardiomyocytes

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.     

NOD8 inhibits autophagy in human pancreatic cancer cells and effect of apoptosis on autophagy regulatd by NOD8

AIM: To explore whether NOD8 inhibits autophagy in human pancreatic cancer cells and its underlying mechanisms, and to investigate the effect of apoptosis on the autophagy regulated by NOD8. METHODS: The empty plasmid pEGFP-C2 and recombinant plasmid pEGFP-NOD8 were transfected into the Panc-1 cells using JetPRIME reagent.The untransfected cells served as control group. The protein levels of NOD8, autophagy-related proteins beclin-1 and LC3-II, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins Akt, p-Akt, mTOR and p-mTOR were determined by Western blot 48 h after transfection. Meanwhile, the number of LC3 spots was quantified by immunofluorescence staining. Furthermore, after a broad caspase inhibitor Z-VAD-FMK was applied to NOD8-over-expressing cells, the protein expression levels of beclin-1 and LC3-II were detected by Western blot and the number of LC3 spots was observed by immunofluorescence staining. RESULTS: The protein level of NOD8 in pEGFP-NOD8 group was significantly higher than that in control group and pEGFP-C2 group (P<0.01). The protein expression of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8 group were significantly decreased as compared with control group and pEGFP-C2 group. Moreover, the protein levels of p-AKT and p-mTOR in pEGFP-NOD8 group were higher than those in control group and pEGFP-C2 group, while no significant difference of mTOR and AKT protein expression was found among these 3 groups. Furthermore, the protein levels of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8+Z-VAD-FMK group were significantly increased compared with pEGFP-NOD8 group. CONCLUSION: NOD8 inhibits autophagy in the Panc-1 cells and its mechanism may be related to the activation of PI3K/Akt/mTOR pathways. Apoptosis enhances the inhibitory effect of NOD8 on autophagy.     

MicroRNA-98 suppresses viability and invasion ability of colorectal can-cer cells by targeting EZH2

AIM: To study the target relationship between microRNA-98 (miR-98) and enhancer of Zeste homolog 2 (EZH2), and the effect of miR-98 on the viability and invasion ability of colorectal cancer cells.METHODS: The target relationship between EZH2 and miR-98 was predicted by TargetScan software and confirmed by dual-luciferase reporter assay. The miR-98 mimic and miR-98 inhibitor were transfected into human colorectal cancer SW480 cells and SW620 cells. The protein expression level of EZH2 was determined by Western blot. The cell viability was measured by MTT assay, and the invasion ability was detected by Transwell assay. EZH2 over-expression vector was transfected into the colorectal cancer cells, and the cell viability and invasion ability were measured.RESULTS: miR-98 targeted EZH2 and down-regulated EZH2 protein expression in the SW480 cells and SW620 cells. miR-98 over-expression significantly decreased, while miR-98 knockdown dramatically increased the viability and invasion ability of SW480 cells and SW620 cells. Additionally, EZH2 over-expression enhanced the viability and invasion ability of SW480 cells and SW620 cells.CONCLUSION: miR-98 inhibits the viability and invasion ability of SW480 cells and SW620 cells by targeting EZH2, which may provide new therapeutic target and method for colorectal cancer treatment.     

Relationship between rat hepatic fibrosis and regulation of hepatic stellate cell autophagy by microRNA-181a

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.     

Effect of autophagy in SH-SY5Y cells injured by Aβ25-35

AIM: To explore whether the damage of neurons induced by amyloid β-protein (Aβ) is related to the regulation of autophagy and its mechanism based on Akt/mTOR pathway. METHODS: SH-SY5Y cells were incubated with Aβ_25-35 (5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L and 25 μmol/L) for 24 h, and the cell viability was measured by MTT assay. The protein levels of LC3-I, LC3-II, Akt, p-Akt, mTOR and p-mTOR in the SH-SY5Y cells were determined by Western blot. After the SH-5Y5Y cells were incubated with autophagy inducer rapamycin (Rapa) or autophagy inhibitor 3-methyladenine (3-MA) combined with Aβ_25-35 for 24 h, the cell viability and related protein expression were detected by the same methods above mentioned. RESULTS: Each concentration of Aβ_25-35 damaged SH-SY5Y cells and decreased the viability of SH-SY5Y cells. Aβ_25-35 increased the expression of autophagy marker protein LC3-II, increased the level of LC3-II/LC3-I, and down-regulated the phosphorylation level of Akt and mTOR proteins (P<0.05). When combined with autophagy inducer Rapa, the cell viability was not significantly affected, the expression of LC3-II protein was increased, LC3-II/LC3-I was increased significantly, and p-mTOR/mTOR level was decreased (P<0.05). When combined with autophagy inhibitor 3-MA, the protein expression of LC3-II and the level of LC3-II/LC3-I showed a downward trend, while the level of p-Akt/Akt was decreased (P<0.05). CONCLUSION: Aβ_25-35 may induce SH-SY5Y cell autophagy and injury by down-regulating phosphorylation levels of Akt and mTOR proteins.     

Effect of beclin-1 gene silencing on TuAKe-injured gastric cancer SGC-7901 cells

AIM: To observe the effect of beclin-1 silencing by the technique of RNA interference on the injury of human gastric cancer SGC-7901 cell by Sheliugu extract (the extract from tuber of Amorphophallus konjac, TuAKe). METHODS: To knock down the expression of beclin-1 gene, SGC-7901 cells were transfected with lentiviral vector carrying beclin-1-shRNA. The beclin-1 gene knock-down and non-knock-down SGC-7901 cells were treated with TuAKe. The cell viability was analyzed by CKK-8 assay. The percentages of apoptotic cells were detected by flow cytometry. The expression of beclin-1 and LC3 was detected by Western blot. RESULTS: The beclin-1 gene silencing decreased the protein expression of beclin-1 and increased the protein expression of LC3 in the SGC-7901 cells, leading to the decrease in cell viability and the increase in apoptotic rate (P<0.05). TuAKe increased the protein expression of beclin-1 and LC3 in the SGC-7901 cells, and decreased the protein expression of LC3 in the SGC-7901 cells with beclin-1 gene silencing, thus inhibiting the cell viability and increasing the apoptotic rate (P<0.05). CONCLUSION: Beclin-1 gene silencing inhibits the activation of beclin-1-related signaling pathway in gastric cancer SGC-7901 cells, and aggravates the injury of cell viability induced by TuAKe.     

Effect of Ginsenoside Rh2 on apoptosis of human osteosarcoma cell line MG-63

AIM: To investigate the effect of Ginsenoside Rh2(Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS: The cell viability was determined by MTT assay. MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining. The expression of Bcl-2, Bax, cytochrome C(Cyt C) and cleaved caspase-3 were measured by Western blot.RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner. Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased, while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group(P<0.05). The protein level of cleaved caspase-3 was also increased(P<0.05).CONCLUSION: Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway, suggesting that Rh2 is a novel approach for the treatment of osteosarcoma.     

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.     

CD147 affects autophagy of prostate cancer PC-3 cells

AIM：To study the autophagy of prostate cancer PC-3 cells induced by CD147 in vitro. ME-THODS：The method of amino acid starvation to induce autophagy was used. The expression of CD147was detected by Western blotting. To study the functional effects of CD147 on autophagy in prostate cancer PC-3 cells, the down-regulation of CD147expression was induced by the technique of RNAi. The conversion of autophagic marker protein LC3-I to LC3-II was determined by Western blotting. The cell death after starvation-induced autophagy was analyzed by trypan blue exclusion assay. RESULTS：The CD147 expression gradually increased in starvation-induced autophagy. The down-regulation of CD147 significantly increased the expression of autophagy-related protein LC3-II compared with control group. Meanwhile, the cell death rates increased from (19.3±3.1)% and (22.3±3.5)% in control groups to (38.4±3.1)% in silencing the expression of CD147in the PC-3 cells (P<0.05). CONCLUSION：CD147 inhibits starvation-induced autophgy and autophagy death in the prostate cancer PC-3 cells.