ABSTRACT
Objective To investigate the effect of MRE11 on the proliferation and apoptosis of esophageal squamous cancer cells and its molecular mechanism. Methods MRE11 expression was downregulated by MRE11 siRNA transfection in esophageal squamous cancer cells. The AKT agonist SC79 (0, 0.1, 0.5, 1, 1.5, 1.8, 2 μg/ml) were used to treat cells with MRE11 inhibition for 24 h. Overexpression vector pcDNA.3.1-c-myc was constructed and co-transfected cells with MRE11 siRNA. Western blot method was used to detect the protein expressions of MRE11, p-AKT and c-myc in esophageal squamous cancer cells Ec9706 and TE-1. The Annexin-V FITC/PI kit was used to detect the apoptosis of Ec9706 and TE-1 cells; the activity of caspase-3 was detected by the Caspase-3 activity detection kit; the proliferation of Ec9706 and TE-1 cells was tested by the BrdU method. Results The protein expressions of MRE11 in Ec9706 and TE-1 cells were significantly increased, compared with human esophageal epithelial Het-1A cells. After MRE11 siRNA transfection, AKT phosphorylation and the protein expressions of MRE11 and c-myc were significantly decreased in esophageal squamous cancer cells. MRE11 inhibition significantly promoted the apoptosis and caspase-3 activity in Ec9706 and TE-1 cells, while inhibited the proliferation of Ec9706 and TE-1 cells. SC79 (1.5, 1.8 and 2 μg/ml) significantly increased AKT phosphorylation in MRE11-suppressed esophageal squamous cancer cells, and reversed the inhibitory effects of MRE11 inhibition on c-myc protein expression and cell proliferation and the promoting effect on cell apoptosis. Overexpression of c-myc inhibited the inhibitory effect of MRE11 down-regulation on cell proliferation and the promotion on caspase-3 activity. Conclusion MRE11 inhibition could effectively inhibit the proliferation of esophageal squamous cancer cells and promote cell apoptosis by regulating AKT and c-myc.
ABSTRACT
Objective To investigate the expression profiles of microRNA in early esophageal squamous cancer and normal esophageal tissues and verify the significantly different expression miRNAs , further to study the effects on proliferation of EC109. Methods The microarray assay was performed to analyze miRNA expression profiles in three pairs of early esophageal squamous cancer and the corresponding normal esophageal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was used in another 38 pairs samples to further verify the differentially expressed miRNAs . Three verified miRNAs ( miR-29a, miR-221 and miR-222) mimics were transfected into EC109 respectively and CCK8 method was used to study the effect of cell proliferation in each miRNA. Results Microarray technique selected 53 miRNAs that differentially expressed in early esophageal squamous cancer and normal esophageal tissues , 32 miRNAs were up-regulated and 21 miRNAs were down-regulated. qRT-PCR verified that miR-29a was significantly down-regulated (P < 0.05) and miR-221, miR-222 were significantly up-regulated (P < 0.05) in early esophageal squamous cancer tissue. Over-expression of miR-29a could significantly inhibit the proliferation of EC109 (P < 0.05) whereas over-expression of miR-221 or miR-222 could both significantly promote the proliferation of EC109 (P < 0.05). Conclusion There was significant difference of miRNAs expression between early esophageal squamous cancer and normal esophageal tissues, and the differentially expressed miRNAs could be used as new biomarkers for early diagnosis of esophageal squamous cancer.
ABSTRACT
Objective MiR-375 is lowly expressed in esophageal squamous cancer cells and the downstream target gene of miR-375 remains unclear .This paper discusses the role of miR-375 in regulating the expression of short stature homobox 2 ( SHOX2) in human esophageal squamous cancer cells . Methods The bioinformatics software TargetScan , miRanda, PicTar, miRTarget2, and PITA were used to predict the assumptive targets of miR-375 in SHOX2.Then, two recombinant luciferase gene report plasmids containing wild pSHOX2 3′UTR ( pSHOX2-375-WT ) and mutant pSHOX2 3′UTR ( pSHOX2-375-mut ) were constructed , sequenced , and identified.Human esophageal squamous cancer cells were co-transfected with miR-375 mimics and pSHOX2-375-WT or pSHOX2-375-mut, respectively , and divided into 7 groups: pmiR, pSHOX2-375-WT, pSHOX2-375-WT +miR-375, pSHOX2-375-WT +miR-NC, pSHOX2-375-mut, pSHOX2-375-mut+miR-375, and pSHOX2-375-mut+miR-NC, each subjected to the measurement of luciferase activity .The expressions of SHOX 2 mRNA and protein were de-termined after transfection of the esophageal squamous cancer cells with miR-375 mimics, and so were the expressions of miR-375 and SHOX2 in the esophageal squamous carcinoma tissue samples obtained postoperatively . Results Prediction with the five software showed only one conserved function site of miR-375 in SHOX2 3′UTR at 1156-1170 bp.Luciferase activity was significantly lower in the pSHOX2-375-WT+miR-375 group (0.261 ±0.036) than in the pmiR (1.818 ±0.061), pSHOX2-375-WT (1.820 ±0.086), pSHOX2-375-WT+miR-NC (1.851 ±0.094), pSHOX2-375-mut (1.861 ±0.059), pSHOX2-375-mut +miR-375 (1.896 ± 0.048), and pSHOX2-375-mut+miR-NC group (1.760 ±0.062) ( P<0.01).SHOX2 mRNA and protein expressions were sup-pressed by the overexpression of miR-375 in the EC9706 cells.The expression of miR-375 was decreased, while that of SHOX2 in-creased in the esophageal squamous carcinoma tissue as compared with the normal esophageal tissue . Conclusion MiR-375 regu-lates the expression of the SHOX 2 gen in esophageal squamous cancer cells .
ABSTRACT
Objective: To investigate the effect of black currant extract on human esophageal squamous cancer cell Eca109 line to reveal its probable mechanism. Methods: The survival cells and protein synthesis of tumor cell lines treated with 10-1, 10 -2,10-3 g/ml of the water extracts of black currant for 24 h were determined by MTT and Bradford assays, and the cell-DNA ploidy distribution and apoptotic rate were measured by flow cytometry (FCM) and morphological observation. Results: There was significant inhibition of the cell survival and protein synthesis of cancer cells at dose of 10-1 g/ml of the extracts. In the normal rat liver cells, the cell survival was not affected. An apoptosis peak appeared before diploid peak in FCM and apoptotic rate was 49.6%, and morphological change of apoptosis was observed. Conclusion: Black currant could significantly inhibit growth of human esophageal squamous cancer cell Eca109 line through inducing apoptosis.