共查询到20条相似文献，搜索用时 531 毫秒
1.
AIM To investigate the effect of sinomenine （SIN） on the apoptosis of human rheumatoid arthritis （RA） fibroblast-like synoviocytes （FLS） and its molecular mechanism. METHODS Human RA FLS were isolated and cultured. The cells treated with lipopolysaccharide （LPS） at 100 mg/L was recorded as LPS group. The cells treated with SIN at 3.2 mmol/L and LPS at 100 mg/L were recorded as LPS+SIN group. The cells without any treatment served as blank group. The cells transfected with miR-con, miR-23b-3p, si-con and si-fibroblast growth factor 9 （FGF9） and treated with 100 mg/L LPS were recorded as LPS+miR-con group, LPS+miR-23b-3p group, LPS+si-con group and LPS+si-FGF9 group, respectively. The anti-miR-con, anti-miR-23b-3p, pcDNA and pcDNA-FGF9 were also transfected into RA FLS, and then the cells were treated with SIN at 3.2 mmol/L and LPS at 100 μg/mL. These cells were recorded as LPS+SIN+anti-miR-con group, LPS+SIN+anti-miR-23b-3p group, LPS+SIN+pcDNA group, LPS+SIN+pcDNA-FGF9 group, respectively. The cell viability was measured by CCK-8 assay. The number of colonies was accessed by colony formation experiment. The protein levels of FGF9, cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. The apoptosis was analyzed by flow cytometry. The expression of miR-23b-3p and FGF9 mRNA was detected by RT-qPCR. Dual-luciferase reporter assay was used to verify the targeting relationship between miR-23b-3p and FGF9. RESULTS Treatment with SIN promoted LPS-induced apoptosis of RA FLS, inhibited cell proliferation, up-regulated miR-23b-3p expression, and down-regulated FGF9 expression. miR-23b-3p targeted FGF9. Over-expression of miR-23b-3p or silencing of FGF9 inhibited LPS-induced proliferation and enhanced apoptosis of the RA FLS. Interfering with miR-23b-3p or over-expression of FGF9 reversed the effects of SIN on the proliferation and apoptosis of LPS-induced RA FLS. CONCLUSION Sinomenine induces RA FLS apoptosis and inhibits cell proliferation through miR-23b-3p/FGF9 signaling. 相似文献
2.
AIM To study the effect of microRNA-153-3p (miR -153 -3p ) knock-down on oxidative injury of H9C2 cells induced by H2O2 and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H2O2, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR -153 -3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H2O2 concentration (P< 0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H2O2 concentration (P< 0.05). Knock-down of miR -153 -3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H2O2 concentration (P< 0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P< 0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P< 0.01). Dual interference with Nrf2 and miR -153 -3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H2O2 (P< 0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H2O2 through up-regulating Nrf2/ARE signaling pathway. 相似文献
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 scutellarin （SCU） on oxidative stress and apoptosis induced by lipopolysaccharide （LPS） in human glomerular epithelial cells and its mechanism. METHODS Human glomerular epithelial cells were cultured in vitro , and were treated with LPS （1.0 mg/L） to establish a cell injury model. The cells were divided into normal control （NC） group, LPS group, NC+SCU group, LPS+SCU group, LPS+miR-NC group, LPS+microRNA-7-5p （miR-7-5p） group, LPS+SCU+anti-miR-NC group and LPS+SCU+anti-miR-7-5p group. Cell viability was detected by CCK-8 assay. Apoptosis was detected by flow cytometry. The intracellular malondialdehyde （MDA） content and superoxide dismutase （SOD） activity, and lactate dehydrogenase （LDH） activity in the cell culture supernatant were determined by kit. RT-qPCR was used to detect the expression level of miR-7-5p. RESULTS Compared with NC group, the cell viability, miR-7-5p expression and SOD activity in LPS group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P <0.05）. Compared with LPS group, the cell viability, miR-7-5p expression and SOD activity in LPS+SCU group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P <0.05）. Compared with LPS+miR-NC group, the cell viability and SOD activity in LPS+miR-7-5p group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P <0.05）. Compared with LPS+SCU+anti-miR-NC group, the cell viability and SOD activity in LPS+SCU+anti-miR-7-5p group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P <0.05）. CONCLUSION Scutellarin inhibits LPS-induced oxidative stress damage and apoptosis in glomerular epithelial cells via up-regulating miR-7-5p expression. 相似文献
5.
AIM: To investigate the effect of microRNA-486 (miR-486) on lipopolysaccharide (LPS)-induced apoptosis of alveolar epithelial cell A549. METHODS: A549 cells were treated with LPS, and the expression of miR-486 was detected by RT-qPCR. miR-486 mimics were transfected into LPS-induced A549 cells, and RT-qPCR was used to detect the up-regulation effect. The apoptotic rate was analyzed by flow cytometry and the protein levels of cleaved caspase-3 (C-caspase-3) and C-caspase-9 were determined by Western blot. The target gene prediction software was used to predict the target gene PTEN of miR-486. Luciferase reporter vector was used to identify the target relationship. pcDNA 3.1-PTEN and miR-486 mimics were co-transfected into A549 cells to detect the effect of PTEN up-regulation on apoptosis of miR-486 mimics transfected A549 cells stimulated with LPS. RESULTS: After LPS treatment, the expression of miR-486 in A549 cells was significantly decreased (P<0.05). Transfection of miR-486 mimics significantly up-regulated the expression of miR-486 in A549 cells stimulated with LPS (P<0.05). The apoptotic rate of A549 cells and the protein levels of C-caspase-3 and C-caspase-9 were significantly increased after LPS treatment (P<0.05). Up-regulation of miR-486 significantly down-regulated LPS-induced apoptosis of A549 cells (P<0.05). The expression of PTEN was negatively regulated by miR-486. Transfection of pcDNA 3.1-PTEN significantly increased the expression of PTEN, promoted the apoptosis and increased the protein levels of C-caspase-3 and C-caspase-9 in A549 cells stimulated with LPS after co-transfection with miR-486 mimics(P<0.05). CONCLUSION: miR-486 inhibits PTEN expression and reduces LPS-induced apoptosis of A549 cells. 相似文献
6.
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
7.
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
8.
AIM: To investigate the effect of microRNA-132 (miR-132) transfection on the lipopolysaccharide (LPS)-induced inflammation in rat alveolar macrophages. METHODS: The rat alveolar macrophage NR8383 cultured without pyrogen in vitrowere divided into blank control group, negative control group and transfected group. The cells in the 3 groups were transfected with phosphate buffer solution (PBS), Lipofectamine 2000 and synthesized miR-132 mimic respectively. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Real-time PCR was used to detect the expression of miR-132 in the cells. After NR8383 cells were stimulated with LPS for 6 h, the NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in NR8383 cells was assayed by Western blotting.RESULTS: After transfection, the expression of miR-132 was significantly higher than that in blank control group and negative control group. The growth of NR8383 cells in transfected group was significantly inhibited compared with blank control group and negative control group (P<0.05). After the cells were stimulated with LPS, the productions of NF-κB, TNF-α and IL-6 in transfected NR8383 cells were decreased compared with blank control group and negative control group (P<0.05).CONCLUSION: Transfection of alveolar macrophages with miR-132 significantly suppresses the cell growth, and inhibits inflammatory responses induced by LPS. 相似文献
9.
TIAN Xiao-gang ZHAO Lin ZHANG Chun-lin REN Jin-xia ZHAO Feng-ju YANG Wen-cui GOU Cai-xia 《园艺学报》2018,34(8):1461-1467
AIM: To investigate the molecular biological mechanisms by which microRNA-126 (miR-126) enhances the radiosensitivity of gastric cancer cells. METHODS: SGC-7901 cells were cultured in vitro. In order to over-express miR-126 in SGC-7901 cells, miR-126 mimic was transfected. The mRNA and protein levels of enhancer of zeste ho-molog 2 (EZH2) were detected by RT-qPCR and Western blot, respectively. The targeting relationship between miR-126 and EZH2 was determined by dual-luciferase reporter assay. To estimate the effect of EZH2 on miR-126-enhanced radiosensitivity of the SGC-7901 cells, the pcDNA3.1-EZH2 vector was also co-transfected with miR-126 mimic, and then CCK-8 assay and flow cytometry were used to detect the viability and apoptotic rate of the cells after radiation. RESULTS: Over-expression of miR-126 significantly inhibited the expression of EZH2 in SGC-7901 cells both at protein and mRNA levels (P<0.05). A direct targeting relationship between miR-126 and EZH2 was confirmed by dual-luciferase reporter assay. Compared with the cells only transfected with miR-126 mimic, co-transfection of pcDNA3.1-EZH2 with miR-126 mimic increased the viability but reduced the apoptosis of the cells treated by radiation (P<0.05). CONCLUSION: Targeting inhibition of EZH2 may be one of the mechanisms by which miR-126 enhances the radiosensitivity of gastric cancer cells. 相似文献
10.
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. 相似文献
11.
AIM:To investigate the role and mechanism of microRNA-25 (miR-25) in apoptosis of H9c2 cells induced by hypoxia/reoxygenation. METHODS:The H9c2 cells with over-expression of miR-25 were treated with hypo-xia/reoxygenation. Real-time PCR was used to detect the expression of miR-25 and high mobility group box-1 (HMGB1) mRNA. Western blot was performed to examine the protein expression levels of HMGB1, Bcl-2 and cleaved caspase-3. Flow cytometry was used to analyze the proportion of apoptotic cells and the cell cycle. Dual-luciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9c2 cells. Moreover, the H9c2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation to verify whether HMGB1 participated in the regulation of apoptosis of H9c2 cells. RESULTS:Over-expression of miR-25 significantly reduced the protein expression levels of HMGB1 and cleaved caspase-3, and increased the expression of Bcl-2 and the entrance into S phase in H9c2 cells induced by hypoxia/reoxygenation (P<0.01). The result of dual-luciferase reporter assay showed that compared with the control group, transfection with HMGB1-3' UTR-psi-CHECK2+miR-25 mimic strongly inhibited the luciferase activity (P<0.05). After the H9c2 cells transfected with HMGB1-shRNA was treated with hypoxia/reoxygenation, the expression of Bcl-2 was up-regulated, the expression of cleaved caspase-3 was down-regulated, and the cells in S phase were increased (P<0.05). CONCLUSION:miR-25 reduces apoptosis of H9c2 cells induced by hypoxia/reoxygenation, and its mechanism may be related with the inhibition of HMGB1 expression via interacting with its 3'-UTR. 相似文献
12.
AIM: To investigate the effect of microRNA-24-3p (miR-24-3p) on the viability and apoptosis of esophageal cancer cells. METHODS: The expression of miR-24-3p and KLF6 mRNA in the esophageal cancer cells TE11, Eca109 and EC9706 were detected by RT-qPCR. The protein expression of KLF6 was determined by Western blot. EC9706 cells were transfected with anti-miR-24-3p and KLF6 siRNA. The cell viability was measured by MTT assay, the apoptotic rate was analyzed by flow cytometry, and the proliferation, apoptosis and IL-6/STAT3 signaling pathways related proteins were determined by Western blot. The level of IL-6 was measured by ELISA. The dual luciferase reporter gene assay was used to verify the relationship between miR-24-3p and KLF6. RESULTS: The levels of miR-24-3p were up-regulated in the esophageal cancer cells TE11, Eca109 and EC9706 (P < 0.05), and the expression of KLF6 at mRNA and protein levels was down-regulated (P < 0.05). Knock-down of miR-24-3p expression inhibited the cell viability, induced apoptosis, and inhibited the protein levels of CDK4, cyclin D1, CDC25A, p-STAT3, Bcl-2 and IL-6, and promoted the protein expression of caspase-3 and Bax in EC9706 cells. CONCLUSION: miR-24-3p targets KLF6 gene to affect the viability and apoptosis of esophageal cancer cells by regulating IL-6/STAT3 signaling pathway. 相似文献
13.
XING Meng-yao MA Xiao-juan GONG Xue-li Maimaitizunong MAISUER YU Wen-yan ZHANG Xue-mei SUN Zhan 《园艺学报》2018,34(11):2101
AIM: To study the effect of p65 gene silencing by adeno-associated virus type 9 (AAV9)-mediated RNA interference on angiotensin Ⅱ (Ang Ⅱ; 10-6 mol/L for 24 h)-induced apoptosis of rat ventricular H9c2 myocytes, and to elucidate the possible mechanism. METHODS: The H9c2 cells were transfected with rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA at multiplicity of infection (MOI)=4×106 vg/cell. eGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of eGFP positive cells was determined by flow cytometry. The expression of p65 was determined by Western blot. CCK-8 assay was used to measured the viability of transfected H9c2 cells. The apoptosis of the cells transfected with the virus and with Ang Ⅱ stimulation was analyzed by flow cytometry. RESULTS: The cells began to exhibit eGFP expression on the 2nd day after transfection. The fluorescence intensity was increased over the time of transfection. eGFP expression reached the maximum on the 5th day, and the transfection efficiency was (52.7±1.9)% at this time point. Compared with blank control group, no significant effect of AAV9 on the viability of H9c2 cells was observed. In resting state, p65 in the H9c2 cells had a certain activity. After Ang Ⅱ stimulation, the activity of p65 was obviously increased, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited the expression of p65. The apoptosis of H9c2 cells in Ang Ⅱ stimulation group was significantly higher than that in blank control group, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited apoptosis of H9c2 cells. CONCLUSION: Transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibits the expression of p65 gene of NF-κB pathway in the H9c2 cells without causing cell growth inhibition, and reduces the apoptosis induced by Ang Ⅱ. 相似文献
14.
Inhibitory effect of anti-miR-196a on invasion and migration of human pancreatic cancer PANC-1 cells
ZHANG Shi-neng ZHUANG Xiao-hong TANG Jian ZHUANG Yan-yan HUANG Feng-ting CHENG Wen-bo CHEN Wen-jie 《园艺学报》2012,28(8):1367-1372
AIM:To investigate the expression of miR-196a in different pancreatic cancer cell lines and to observe the effect of anti-miR-196a on the biological behaviors of human pancreatic cancer PANC-1 cells. METHODS:The expression of miR-196a in the pancreatic cancer cells was examined by real-time quantitative PCR. Anti-miR-196a was chemically synthesized and transfected into PANC-1 cells by Lipofectamine 2000. The cell proliferation was measured by CCK-8 assay. The apoptosis was determined by flow cytometry. Matrigel invasion assay was performed to examine the migration and invasion of the tumor cells. The wild-type and mutant-type NF-κB inhibitor α(NFKBIA) 3'UTR luciferase reporter vectors were constructed. The relative activity of Renilla luciferase was detected to confirm the binding site of miR-196a on NFKBIA mRNA. RESULTS:The expression of miR-196a in human pancreatic cancer cell lines was significantly higher than that in human pancreatic ductal epithelial H6c7 cells. The expression of anti-miR-196a in miR-196a group was down-regulated. After transfected with miR-196a, no change of cell proliferation and apoptosis was observed, but the abilities of invasion and migration were reduced(P<0.01). Compared with negative control, wild-type NFKBIA3'UTR or mutant-type NFKBIA 3'UTR, cotransfection of anti-miR-196a and wild-type NFKBIA 3'UTR significantly increased the relative activity of Renilla luciferase. CONCLUSION:miR-196a is one of the oncomiRs and may be a target microRNA of human pancreatic cancer for gene therapy. 相似文献
15.
AIM:To study the effect of APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) on H9c2 cardiomyocyte injury induced by lipopolysaccharides (LPS). METHODS:The H9c2 cells were treated with LPS. RT-qPCR and Western blot were used to detect the expression of APPL1 in the H9c2 cells. The recombinant APPL1 lentiviral vector was used to transfect into the H9c2 cells. After LPS treatment, the over-expression efficiency was detected by RT-qPCR and Western blot. The viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein level of activated caspase-3 in the H9c2 cells was determined by Western blot. The content of malonaldehyde (MDA) in the H9c2 cells and the level of lactate dehydrogenase (LDH) in the culture medium were detected. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the level of reative oxygen species (ROS) in the H9c2 cells were also examined. RESULTS:The expression of APPL1 at mRNA and protein levels in LPS-treated H9c2 cells was decreased significantly (P<0.05). Over-expression of APPL1 by transfection of recombinant lentiviral vector significantly increased the level of APPL1 at mRNA and protein levels in the H9c2 cells with LPS treatment (P<0.05). LPS treatment reduced the viability, but increased the apoptotic rate of the H9c2 cells, the protein level of activated caspase-3, the content of MDA and the level of LDH in the culture medium. The activity of SOD and GSH-Px was reduced, while the level of ROS was increased as compared with control group (P<0.05). Over-expression of APPL1 elevated the viability of H9c2 cells treated with LPS, and the apoptotic rate and the protein level of activated caspase-3 were decreased. The content of MDA and the level of LDH in the culture medium were reduced, the activity of SOD and GSH-Px was elevated, and the level of ROS was reduced as compared with the H9c2 cells treated with LPS alone (P<0.05). CONCLUSION:Over-expression of APPL1 reduces oxidative damage and apoptosis of the H9c2 cells induced by LPS. 相似文献
16.
AIM: To elucidate the role of microRNA-34a (miR-34a) in transplantation of human adipose-derived stem cells (hADSCs) for treatment of Achilles tendonitis. METHODS: The viability of hADSCs transfected with miR-34a mimic and miR-34a inhibitor for 24 h, 48 h, 72 h and 96 h was measured by CCK-8 assay. A rat model of collagenase-induced Achilles tendonitis was established. The rats with Achilles tendonitis were divided into 5 groups:PBS group, hADSCs group, hADSCs+NC group, hADSCs+miR-34a group and hADSCs+anti-miR-34a group. The tendon tissues were isolated to detect stiffness, stress and maximum loading tension by biomechanical evaluation and to assess miR-34a expression level as well as the expression of collagen I, scleraxis (Scx) and tenascin C (TNC) at mRNA and protein levels by RT-qPCR and Western blot. RESULTS: miR-34a over-expression and knockdown suppressed and enhanced the viability of hADSCs in a time-dependent manner, respectively. Moreover, stiffness, stress and maximum loading tension in hADSCs group were increased compared with PBS group, while these biomechanical indexes in hADSCs+miR-34a group and hADSCs+anti-miR-34a group were improved and reduced as compared with hADSCs+NC group, respectively. Furthermore, up-regulation of collagen I, Scx and TNC expression at mRNA and protein levels was observed in hADSCs group as compared with PBS group. Meanwhile, miR-34a expression was increased but the expression of collagen I, Scx and TNC at mRNA and protein levels declined in hADSCs+miR-34a group. In contrast, reversed effects on the trends mentioned above were observed in hADSCs+anti-miR-34a group.CONCLUSION: miR-34a affects therapeutic outcome for Achilles tendonitis by regulating the viability and differentiation of hADSCs. 相似文献
17.
AIM To study the effects of extracts of Herba Taxilli (Sangjisheng, SJS) on the viability and apoptosis of osteoarthritic chondrocytes and the underlying mechanism. METHODS Human primary osteoarticular chondrocytes (RPOC) were divided into control group, interleukin-1β (IL-1β) group, IL-1β+low-dose extracts of SJS (SJS-L) group, IL-1β+medium-dose extracts of SJS (SJS-M) group, IL-1β+high-dose extracts of SJS (SJS-H) group, IL-1β+anti-miR-NC group, IL-1β+anti-miR-375 group, IL-1β+SJS-H+miR-NC group, IL-1β+SJS-H+miR-375 group. The cell viability was measured by MTT assay, apoptosis was analyzed by flow cytometry, miR-375 expression was detected by qPCR, and the protein levels of cyclin D1, P21, Bcl-2, Bax and caspase-3 were determined by Western blot. RESULTS Compared with control group, the viability of RPOC at 24 h, 48 h and 72 h and the protein expression levels of cyclin D1 and Bcl-2 were significantly decreased (P <0.05), the protein levels of P21, Bax and caspase-3, the apoptotic rate and the expression level of miR-375 were remarkably increased in IL-1β group(P <0.05). Compared with IL-1β group, the cell viability at 24 h, 48 h and 72 h and the protein expression of cyclin D1 in the RPOC were greatly increased (P <0.05), while the expression of P21 was significantly decreased in IL-1β+SJS-M group and IL-1β+SJS-H group(P <0.05).The apoptotic rate, Bax, caspase-3 protein and miR-375 expression were obviously decreased (P <0.05), and Bcl-2 protein level was significantly increased in IL-1β+SJS-H group compared with IL-1β group(P <0.05). Compared with IL-1β+anti-miR-NC group, the expression of miR-375, the protein levels of P21, Bax, caspase-3 and the apoptotic rate in the RPOC of IL-1β+anti-miR-375 group were markedly decreased (P <0.05), while the cell viability at 24 h, 48 h and 72 h and the protein levels of cyclin D1 and Bcl-2 were significantly increased (P <0.05). Over-expression of miR-375 reversed the effects of extracts of SJS on the viability and apoptosis of RPOC with IL-1β stimulation. CONCLUSION The extracts of Herba Taxilli promotes the viability and inhibits apoptosis of RPOC treated with IL-1β, which is related to the regulation of miR-375 expression. 相似文献
18.
AIM: To investigate the effects of microRNA-193 (miR-193) on the proliferation and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS: Cultured rat MSCs were transfected with pre-miR-193 or anti-miR-193 to regulate the expression of miR-193. The proliferation of the MSCs after transfection was evaluated by MTS assay, colorimetric BrdU cell proliferation assay and Ki-67 immunostaining. Cell apoptosis was analyzed by flow cytometry with Annexin V/PI staining. The effect of miR-193 on the expression of cell cycle-related proteins was evaluated by qRT-PCR. RESULTS: Transfection of pre-miR-193 or anti-miR-193 regulated the expression of miR-193 in MSCs effectively. Over-expression of miR-193 significantly promoted the proliferation of MSCs (P<0.05), and inhibition of miR-193 reduced the proliferation of MSCs (P<0.05). miR-193 had no significant effect on the apoptosis of MSCs (P>0.05). The result of qRT-PCR indicated miR-193 promoted the expression of cyclin-dependent kinase 2 (CDK2) significantly (P<0.01). CONCLUSION: miR-193 promotes the proliferation of MSCs possibly through the CDK2 pathway. 相似文献
19.
AIM: To explore the effect of hexokinase 2 (HK2) on lipopolysaccharide (LPS)-induced apoptosis of human lung epithelial BEAS-2B cells and the underlying mechanisms.METHODS: BEAS-2B cells were treated with LPS to induce cell injury, and the cell viability was examined by CCK-8 assay. Hoechst 33342 staining and Annexin V/PI double staining were used to analyze the apoptosis. The apoptotic pathway was identified by the specific inhibitor for caspase-8 or caspase-9. The releases of key mediators in mitochondrial apoptosis pathway were examined by Western blot. The effects of HK2 in these process were confirmed by HK2 over-expression followed by LPS treatment.RESULTS: CCK-8 assay showed that LPS treatment decreased the viability of BEAS-2B cells in a dose/time-dependent manner (P<0.01). The apoptosis of BEAS-2B cells was manifested by Hoechst 33342 and Annexin V/PI double staining. Pretreatment with z-LEHD-fmk, but not z-IETD-fmk, reversed the decreased cell viability under LPS stimulation. HK2 down-regulation was involved in LPS-induced apoptosis of the BEAS-2B cells. After HK2 over-expression, the cell viability was increased after LPS treatment. Releases of cytochrome C and apoptosis-inducing factor from mitochondrion to cytoplasm during apoptosis were also inhibited by HK2 over-expression.CONCLUSION: Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial cells. 相似文献
20.
AIM: To study the effect of microRNA (miR)-24 on chemotherapy sensitivity and its possible mechanisms in human lung adenocarcinoma A549 cells. METHODS: The expression of miR-24 in the A549 cells and A549/DDP cells was determined by real-time PCR. Transfection of miR-24 inhibitor was used to down-regulate the miR-24 level in the A549/DDP cells. The viability and apoptosis rate were measured by CCK-8 assay and flow cytometry, respectively. The protein levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cytochrome C (Cyt C), phosphorylated extracellular signal regulated kinase (p-ERK) and P53 were detected by Western blot. Luciferase reporter assay was used to predict and identify the target genes of miR-24. RESULTS: The expression of miR-24 was significantly higher in the A549/DDP cells than that in the A549 cells (P<0.05). miR-24 inhibitor induced cell apoptosis and increased the sensitivity of the A549/DDP cells to cisplatin. Furthermore, miR-24 inhibitor down-regulated the ratio of Bcl-2/Bax, while up-regulated the protein levels of P53, p-ERK, cleaved caspase-9, cleaved caspase-3 and Cyt C. Incubation with U0126, a specific ERK inhibitor, partly reversed the viability of miR-24 inhibitor transfected A549/DDP cells. Bioinformatics analysis demonstrated that p53 was a potential target gene of miR-24. Co-teansfection of miR-24 inhibitor and P53 siRNA in A549/DDP cells partially reversed the effect of miR-24 inhibitor on cell viabiltiy. CONCLUSION: Down-regulation of miR-24 increases the sensitivity of A549/DDP cells to cisplatin. The mechanism may be related to directly targeting p53 gene and over-activation of ERK/P53 signaling pathway, thus promoting apoptosis via mitochondrial apoptosis pathway. 相似文献