ABSTRACT
We investigated the inhibitory effect and mechanism of action of bruceantin (BCT) on the proliferation, invasion and migration of non-small cell lung cancer (NSCLC) cells. The cytotoxic activity of BCT was measured by MTT assay; a colony forming assay, wound healing assay, and a Transwell assay were used to investigate the anti-proliferative, anti-migration, and anti-invasion effects, respectively; immunoblotting and RT-qPCR were used to detect the expression of related proteins, miRNA, and mRNA, respectively, that were involved in cell proliferation, migration, and invasion. Two gene prediction websites were used to predict the downstream target gene of miRNA. Our results show that BCT has a potent cytotoxic effect on NSCLC cell lines, with a half maximal inhibitory concentration (IC50) of BCT against H1299, PC-9, and A549 of 0.12 ± 0.02, 0.31 ± 0.20, and 2.07 ± 0.70 μmol·L-1, respectively. When H1299 cells were treated with 0.03, 0.15, and 0.75 μmol·L-1 BCT for 24 h, the proliferation, migration, and invasive ability were inhibited in a concentration-dependent manner. It is worth noting that the expression level of miRNAs related to cell migration and invasion, such as miR-29a-3p, miR-21-3p, miR-183-5p, and miR-34b-5p increased with the concentration of BCT, especially for miR-29a-3p. Using the two gene prediction websites, we predict that integrin β1 (ITGB1) may be the target gene of miR-29a-3p; immunoblot results further show that a variety of proteins related to cell proliferation, migration, and invasion, such as various proteins of the integrin family, β-catenin, p-Src, and vascular endothelial growth factor, all decreased in a concentration-dependent manner, among which the reduction of ITGB1 protein was the most obvious. RT-qPCR results showed that there was no change in ITGB1 mRNA expression. We speculate that BCT might inhibit the expression of ITGB1 protein by up-regulating miR-29a-3p independent of its mRNA level. The in-depth mechanism needs to be further explored. This study suggests that BCT has the potential for further development in the treatment of NSCLC.
ABSTRACT
Sevoflurane (SEVO) is widely applied as an anesthetic, which exerts antitumor capacity in various cancers, including hepatocellular carcinoma (HCC). Previous studies indicated that long non-coding RNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) was upregulated, while microRNA-29a-3p (miR-29a-3p) was downregulated in HCC. Thus, we aimed to explore the roles of KCNQ1OT1 and miR-29a-3p in HCC cells exposed to SEVO. Cell proliferation, apoptosis, migration, and invasion were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and transwell assays, respectively. The levels of genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Furthermore, the interaction between miR-29a-3p and KCNQ1OT1 or chromebox protein homolog 3 (CBX3) was predicted by Starbase or Targetscan, and then confirmed by dual-luciferase reporter assay. We found that the levels of KCNQ1OT1 and CBX3 were decreased, while miR-29a-3p was increased in SEVO-treated HCC cells. KCNQ1OT1 overexpression weakened the inhibitory effects of SEVO on HCC cell proliferation, apoptosis, migration, and invasion. Interestingly, KCNQ1OT1 bound to miR-29a-3p, and miR-29a-3p targeted CBX3. KCNQ1OT1 upregulated CBX3 level by repressing miR-29a-3p expression. Furthermore, KCNQ1OT1 exerted tumor promotion in HCC cells via suppressing miR-29a-3p to regulate CBX3 expression. Collectively, our findings demonstrated that KCNQ1OT1 regulated the antitumor effects of SEVO on HCC cells through modulating the miR-29a-3p/CBX3 axis, providing a theoretical basis for the treatment of HCC.
Subject(s)
Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/drug therapy , Potassium Channels, Voltage-Gated , MicroRNAs/genetics , Liver Neoplasms/genetics , Liver Neoplasms/drug therapy , Chromosomal Proteins, Non-Histone , RNA, Long Noncoding/genetics , Sevoflurane/pharmacologyABSTRACT
The aim of this study was to investigate the effect and mechanisms of miR-29a in migration and inva-sion of human breast cancer MCF-7 cells in vitro. MCF-7 cells were treated with miR-29a mimic or miR-29a inhibitor to up-regulate/down-regulate the expression level of miR-29a. Wound-healing assay and transwell chamber were employed to determine cell migration and invasion in vitro. The target gene of miR-29a was predic-ted with the Targetscan7. 1 database and verified through luciferase reporter method. The effects of miR-29a on the expression of the potential target were detected by Western blot and real-time PCR. Results showed that in vitro migration and invasion ability of MCF-7 cells was increased significantly by miR-29a,which could target HBP1 in the 3′-UTR region. The protein expression of HBP1 was decreased by miR-29a overexpression. However, the alteration of miR-29a had no significant effect on the expression of HBP1 mRNA. The results validated that miR-29a,highly expressed in breast cancer,could down-regulate HBP1 ,which in turn promotes migration and invasion ability of breast cancer cells,thus promoting breast cancer metastasis.
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Objective To investigate the expression of miR-29s in the glioma stem cells,and explore how the members of miR-29s affect the bio-logical behaviors of glioma stem cells. Methods Eight patient specimens were used to culture glioma stem cells. Real-time PCR was adopted to test the expression of miR-29s. CCK-8 analysis was performed to test the proliferation ,Transwell was used to test cell migration and invasion ,and flow-cytometry analysis was carried out to test apoptosis. Results miR-29a,miR-29b and miR-29c were decreased in glioma stem cells. Over-ex-pression of miR-29s could inhibit the proliferation,cell migration and invasion,but promote apoptosis of glioma stem cells. Conclusion miR-29s acts as a cancer suppressor gene in the glioma stem cells ,and miR-29a plays the dominant functional role in the family.
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Objective To investigate the changes of plasma miR-29a level in patients with hepatocellular carcinoma(HCC) and its clinical significance.Methods Case-control study,30 patients with HCC,30 patients with cirrhosis of liver (LC) and 30 healthy controls (HC) were recruited from Shiyan Taihe Hospital,2016 January to June.The level of microRNA-29a (miR-29a) in plasma was detected by real time quantitative PCR,and the sensitivity and specificity of plasma miR-29a expression in the diagnosis of HCC were analyzed by receiver operating characteristic curve (ROC),and to analyze the correlation between the miR-29a and the alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma,the combination of miR-29a and AFP could improve the diagnostic efficiency of HCC.Results The relative expression of miR-29a was significantly different between HCC group (3.38±8.37),LC group (8.79±3.80) and HC group (11.98±6.64),the expression level of miR-29a in HCC group was significantly lower than that in LC group (P=0.046) and HC group (P=0.001),and that in LC group was significantly lower than that in HC group (P=0.026).The area under the ROC curve of miR-29a was 0.816 (95％ CI 0.695 to 0.938) less than AFP 0.918 (95％ CI 0.853 to 0.982),and the diagnostic efficiency of miR-29a was not as good as that of AFP.The levels of miR-29a and AFP in plasma of patients with hepatocellular carcinoma were significantly correlated (P=0.002).Conclusion The level of miR 29a in plasma of patients with HCC decreased,which may become the reference index of HCC diagnosis.
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Background:Diagnosis of liver cirrhosis in early stage with early intervention may stabilize disease progression, avoiding or delaying the occurrence of decompensation. Seeking non-invasive serum biomarkers is becoming an important topic in the diagnosis and assessment of liver cirrhosis. Aims:To study the value of serum miR-192 and miR-29a as non-invasive biomarkers for the diagnosis of liver cirrhosis. Methods:Differentially expressed serum miRNAs between patients with liver cirrhosis and healthy controls were screened through online literature retrieval and then confirmed by real-time PCR. Serum levels of two confirmed miRNAs,miR-192 and miR-29a were analyzed in 120 patients with liver cirrhosis and 76 healthy volunteers by real-time PCR. A mathematical model of combined detection of miR-192 and miR-29a for diagnosis of liver cirrhosis was established by binary logistic regression. The diagnostic performance of various non-invasive serum indicators was evaluated by ROC curve analysis. Results:Compared with healthy controls,expression level of serum miR-192 in cirrhotic patients was significantly increased and that of serum miR-29a was significantly reduced(P ﹤ 0. 001). The diagnostic performance of risk score obtained from mathematical model of combined detection of serum miR-192 and miR-29a was superior to that of single miRNA detection or other non-invasive serum indicators,such as APRI,FIB-4 and ARR,the areas under ROC curve of the above mentioned indicators were 0. 968,0. 887,0. 933,0. 796,0. 793,and 0. 571,respectively. Serum levels of miR-192,miR-29a and the risk score of their combined detection were significantly correlated with the stage of liver cirrhosis according to the Child-Pugh classification( P ﹤ 0. 05). Conclusions:Serum miR-192,miR-29a and the risk score of their combined detection might be novel non-invasive biomarkers for the diagnosis and assessment of liver cirrhosis.
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Objective To investigate the expression levels of microRNA(miR)-125b, miR-29a and miR-155-5p in ce?rebrospinal fluid (CSF) and plasma from the patients with tuberculous meningitis and their clinic significance. Methods Cerebrospinal fluid and plasma samples were collected from 20 patients with tuberculous meningitis (tuberculous meningitis group) and 20 patients suffered from primary headache (control group). The total RNAs were extracted.The levels of miR-125b, miR-29a and miR-155-5p were determined by real-time quantitative PCR (q-PCR). Results Levels of miR-29a and miR-125b in both CSF and plasma of patients in tuberculous meningitis group were significantly higher than those in pa?tients from control group with statistical significance (P<0.01). Mean time, the level of miR-155-5p in plasma but not in CSF of patients in tuberculous meningitis group was higher than those in control group with statistical significance ( P<0.01). Conclusion Expression of miR-125b, miR-29a and miR-155-5p may participate in regulating the occurrence and development of tuberculous meningitis. And miR-125b and miR-29a may be used as potential biomarkers for diagnosing tu?berculous meningitis.