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OBJECTIVES@#Hyperhomocysteinaemia (Hcy) is an independent risk factor for cardiovascular and cerebrovascular diseases. MicroRNA (miR)-18a-5p is closely related to cardiovascular diseases. This study aims to investigate the effects of miR-18a-5p on homocysteine (Hcy)-induced myocardial cells injury.@*METHODS@#H9c2 cells were transfected with miR-18a-5p mimic/miR-18a-5p mimic negative control (NC) or combined with Hcy for intervention, and untreated cells were set as a control group. The transfection efficiency was verified by real-time RT-PCR, and cell counting kit-8 (CCK-8) assay was used to determine cell viability. Flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) levels. Western blotting was performed to measure the protein levels of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin1, p62, Bax, Bcl-2, and Notch2. Dual luciferase reporter assay was used to detect the interaction of miR-18a-5p with Notch2.@*RESULTS@#Compared with the control, treatment with Hcy or transfection with miR-18a-5p mimic alone, or combined treatment with Hcy and miR-18a-5p mimic/miR-18a-5p mimic NC significantly reduced the H9c2 cell viability, promoted apoptosis and ROS production, up-regulated the expressions of Bax and Beclin, down-regulated the expressions of Bcl-2, p62, and Notch2, and increased the ratio of LC3-II/LC3-I (all P<0.05). Compared with the combined intervention of miR-18a-5p mimic NC and Hcy group, the above indexes were more significantly changed in the combined intervention of miR-18a-5p mimic and Hcy group, and the difference between the 2 groups was statistically significant (all P<0.05). There is a targeted binding between Notch2 and miR-18a-5p.@*CONCLUSIONS@#MiR-18a-5p could induce autophagy and apoptosis via increasing ROS production in cardiomyocytes, and aggravate Hcy-induced myocardial injury. Notch2 is a target of miR-18a-5p.
Subject(s)
Rats , Animals , Apoptosis/genetics , Autophagy/genetics , bcl-2-Associated X Protein , MicroRNAs/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Reactive Oxygen Species , Myocytes, Cardiac/drug effects , Homocysteine/adverse effects , HyperhomocysteinemiaABSTRACT
Objective:To investigate the effect of lncRNA AC132217.4 on the proliferation and invasion of liver cancer MHCC97-H cells and its molecular mechanism.Methods:The TCGA database was used to analyze the differential expression of AC132217.4 in liver cancer tissue and adjacent tissue, and to analyze the relationship between the expression level of AC132217.4 and the overall survival of liver cancer patients. Transfection of pcDNA-AC132217.4 plasmid into MHCC97-H cells was defined as AC132217.4 group, transfection of pcDNA plasmid into MHCC97-H cells was defined as negative control (NC) group, respectively. The proliferation and invasion ability of MHCC97-H cells were detected by MTT method and Matrigel invasion assay. The binding site between AC132217.4 and miR-18a-5p was analyzed by starBase v2.0 software and dual luciferase reporter gene assay. Real-time quantitative PCR (RT-qPCR) detected the differential expression of miR-18a-5p in the two groups of MHCC97-H cells. The expression of epithelial-mesenchymal transition protein was detected by Western-blotting. Measurement data with normal distribution were expressed as mean±standard deviation ( ± s), and t-test was used for comparison between the two groups. Results:Compared with adjacent tissues, the expression of AC132217.4 was down-regulated in liver cancer tissues ( P<0.01). Compared with liver cancer patients with low expression of AC132217.4, the overall survival of liver cancer patients with high AC132217.4 expression was longer ( P<0.05). The pcDNA-AC132217.4 plasmid significantly inhibited the proliferation of MHCC97-H cells ( P<0.05). The number of invasive cells in the NC group and AC132217.4 group were (131.30±12.55) and (37.45±7.77), respectively. The pcDNA-AC132217.4 plasmid significantly inhibited the invasive ability of MHCC97-H cells ( t=6.36, P<0.01). AC132217.4 directly complemented miR-18a-5p ( P<0.01). The expression of miR-18a-5p in MHCC97-H cells in AC132217.4 group (1.04±0.30) was significantly lower than that in NC group (6.13±0.75) ( t=6.27, P<0.01). Compared with the NC group, the expressions of epithelial phenotype proteins Cytokeratin and Claudin-1 in MHCC97-H cells in AC132217.4 group were up-regulated, while the expressions of mesenchymal phenotype proteins Vimentin, Slug and Snail were down-regulated. Conclusions:The expression of AC132217.4 is low in liver cancer tissue, and it is related to the overall survival of liver cancer patients. AC132217.4 might inhibit the proliferation and invasion of liver cancer MHCC97-H cells by sponge miR-18a-5p.
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Long non-coding RNAs (lncRNAs) cancer susceptibility candidate 2 (CASC2) has been characterized as atumor suppressor in glioma. Although CASC2 may predict the prognosis of glioma patients, the role andmechanism of CASC2 in human glioblastoma remain to be fully illuminated. Expression of CASC2 and miR18a was detected using RT-qPCR. Cell growth was evaluated by MTT assay, colony formation assay, and flowcytometry; metastasis and epithelial-mesenchymal transition (EMT) were determined with transwell assay andWestern blot, respectively. The target binding between CASC2 and miR-18a was predicted on Starbasesoftware, and confirmed by luciferase reporter assay and RNA immunoprecipitation. Xenograft experimentmeasured tumor growth. As a result, CASC2 was downregulated and miR-18a was upregulated in glioblastomatumor tissues and cells (T98 and A172). Overexpression of CASC2 promoted apoptosis rate and E-cadherinexpression, but suppressed cell viability, colony-forming ability, migration, invasion, and expression ofN-cadherin and Vimentin in T98 and A172 cells, accompanied with tumor growth inhibition in vivo; whereas,silencing of CASC2 exerted the opposite effect on cell growth, metastasis and EMT of T98 and A172 cellsin vitro. However, reintroduction of miR-18a could reverse CASC2 upregulation-mediated suppression onabove cell behaviors in vitro. More importantly, miR-18a was a downstream target for CASC2, and wasnegatively regulated by CASC2. Collectively, this study demonstrated that CASC2 served as tumor suppressorin glioblastoma by inhibiting cell growth, metastasis and EMT both in vitro and in vivo partially via CASC2-miR-18a axis.
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Objective@#To investigate the expressional levels and diagnostic values of miR-18a and miR-21 in esophageal carcinoma.@*Methods@#The expressions of miR-18a and miR-21 in esophageal cancer tissues and adjacent tissues from 45 esophageal cancer patients, peripheral blood from 45 esophageal cancer patients and 50 healthy donors respectively were detected by RT-PCR. The expressions of miR-18a and miR-21 in normal esophageal epithelial cell HET-1A, esophageal cancer cell lines including ECA109, KYSE150 and TE1 were also detected. Chemiluminescence immunoassay was used to quantitatively detect the concentrations of carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), CYRFA21-1 and TPA (tissue polypeptide antigen) in peripheral blood serum from esophageal cancer patients and healthy controls. Meanwhile, the diagnostic effects of miR-18a and miR-21 on esophageal cancer were compared with those of tumor markers in serum.@*Results@#The expression levels of miR-18a and miR-21 in esophageal cancer cells ECA109, KYSE150 and TE1 were 1.64±0.17, 1.62±0.19, 1.46±0.12 and 20.52±1.48, 6.73±0.73, 1.43±0.19, respectively, higher than those in normal esophageal epithelial cells (both P<0.01). The expressions of miR-18a and miR-21 in esophageal cancer tissues were 32.48±28.62 and 8.67±11.98, respectively, significantly higher than those in adjacent tissues (all P<0.001). The expression levels of miR-18a and miR-21 in peripheral blood of patients with esophageal cancer were 12.66±11.92 and 9.15±8.14, respectively, significantly higher than those in the normal control group (both P<0.001). The receiver operating characteristic (ROC) curve analysis showed that the area under the curve of miR-18a and miR-21 for diagnosis of esophageal cancer were 0.948 and 0.913 5, respectively. Compared with traditional esophageal tumor markers, the expressions of miR-18a and miR-21 were more sensitive in the diagnosis of esophageal cancer. The sensitivity and accuracy of the expressions of miR-18a and miR-21 combined with traditional esophageal tumor markers in diagnosis of esophageal cancer can be further improved to 97.8% and 68.4%, respectively.@*Conclusion@#Our study reveals that the expressions of miR-18a and miR-21 play important roles in the diagnosis of esophageal cancer and may be potentially novel biomarkers.
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[Objective]To construct miR-18a overexpression and inhibition lentivirus vectors and to determine their effects on human nasopharyngeal cancer(NPC)cell line CNE1 and CNE2.[Methods]Designed the primers for Real-time polymerase chain(PCR)reaction to obtain the miR-18a premature gene.The premature gene and the siRNA oligo-nucleutides of miR-18a were connected to the lentivirus vector GV369 and GV280,respectively.The construction vectors were confirmed by DNA sequencing.Then,293T cell was infected with the vectors plus Helper 1.0 and pHelper 2.0 vec-tors to obtain recombinant lentivirus vector for miR-18a overexpression and inhibition. The NPC cell line CNE1 and CNE2 were infected with the successful recombinant lentivirus vectors.Puromycin was added to select the positive infect-ed cells. PCR method was used to detect the miR-18a expression level after infecting the recombinant lentivirus vector into the NPC cell line.[Results]A recombinant lentivirus vector expressing miR-18a interference oligonucleutides was obtained and confirmed by DNA sequencing.The virus titer was 3×108TU/mL,and the expression of its target gene ATM was downregulated in CNE1 and CNE2.A recombinant lentivirus vector expressing miR-18a premature gene was obtained and confirmed by DNA sequencing. The virus titer was 3×108TU/mL,and the miR-18a was overexpressed in CNE1 (20.3 fold upregulation,P<0.01)and CNE2(122.5 fold upregulation,P<0.01),and its target gene ATM was downregu-lated.[Conclusions]The miR-18a overexpression and suppression lentivirus vectors are successfully constructed.These vec-tors could alter the expression level of miR-18a in NPC cell line significantly,and provide a stable cell line for functional studies in the future.
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Objective To detect the expression of miR-18a and estrogen receptor alpha (ER alpha) in single and multiple uterine flesh tumor tissues, discuss the relationship between miR-18a and ER alpha, and their effect in single and multiple uterine fibroids.Methods The expression of miR-18a and ER alpha in single and multiple uterine fibroids tissue paraffin section were detected by in situ hybridization method and immunohistochemical method, respectively.And the correlation between the miR-18a and ER alpha were evaluated.Results The expression of ER alpha in multiple uterine fibroids group was significantly higher than that of single uterine fibroids tissues (P<0.05);while miR-18a was weaker than that of single uterine fibroids tissues(P <0.05).The correlation results showed that miR-18a expression was correlated negatively with ER alpha expression either in single(r =-0.4421) and multiple uterine fibroids(r =-0.4181).Conclusion The expression of miR-18a is low in multiple uterine fibroids, while ER alpha had high expression.miR-18a could bea new target for the treatment of multiple uterine fibroids.
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OBJECTIVE: To explore the effect of Aitongxiao granule (traditional Chinese medicines) therapy on miRNAs of replant liver cancer rats. METHODS: The 60 replant liver cancer rats were randomly divided into high dose(A), middle dose(B), lower dose(C), 5-Fu(D), distilled water(E) groups. After 14 days of experimental treatment, the expressions of miR-199a-3p,miR-199a-5p, miR-18 in liver cancer tissues were detected with qRT-PCR. RESULTS: Before experimental treatment, in comparison with E group, the level of miR-18 was higher but level of miR-199a-3p, miR-199a-5p were lower in A,B,C,D groups(P < 0.01). After 14 days of experimental treatment, in comparison with E group, the level of miR-18 was lower but level of miR-199a-3p, miR-199a-5p were higher in A,B,C,D groups(P < 0.01). The more significant differences were found in B group than that in D group. CONCLUSION: The Aitongxiao granule has the regulative effects on miRNAs of replant liver cancer rats. Its therapeutical effects relate to the miRNAs expressions of liver cellular cancer development.
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ObjectiveTo investigate the inhibitory effect of all-tram-retinoicacid (ATRA) on HCC cell growth and probe the potential molecular mechanism.MethodsHCC cell lines,HepG2 and SMMC-7721 were treated by ATRA and cell growth was analyzed by using MTT assay.The expression levels of miR-18a were evaluated in HepG2 and SMMC-7721,compared with the normal livers pool by using RealTime PCR analysis.Cell growth analysis by using MTT assay was performed on HepG2 and SMMC-7721 after transfection with anti-miR-18a.Rescued assay was designed to probe the mechanism of ATRA on cell growth by using ATRA with or without miR-18a mimic.ResultsHepG2 cell growth was suppressed about 74％ (P＜0.05,36 h),72％ (P＜0.01,48 h),and 67％ (P＜0.05,72 h) and SMMC-7721 cell growth was inhibited about 68％ (P＜0.05,48 h),and 64％ (P＜0.05,72 h) after treatment with ATRA,compared with the cells treated with Ethanol.MiR-18a expression was up-regulated in HepG2 and SMMC-7721 cell lines about 4.7- and 3.8-fold (P＜0.05),respectively.Endogenous miR-18a levels were down-regulated by ATRA about 67％ and 56％ (P＜0.05).The inhibitory effect of ATRA on HCC cell growth was reversed about 1.2-fold (P＜0.05,48 h) by overexpression of miR-18a in HepG2 cells and cell growth of SMMC-7721 was enhanced about 1.25- and 1.2-fold (P＜0.05,24 and 48 h) with ectopic expression of miR-18a.ConclusionHCC cells growth is suppressed by ATRA through miR-18a mediated network.