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Objective:To evaluate the effect of circLPAR3 on the radiosensitivity of esophageal cancer cells and investigate its mechanism.Methods:The cancer tissues and and adjacent tissues of 37 patients with esophageal cancer were collected, and esophageal cancer cell lines Eca-109, EC9706 and KYSE30 and esophageal epithelial cells HET-1A were cultured in vitro. The expression levels of circLPAR3 and miR-1238 in the tissues and cells were measured by RT-qPCR. Eca-109 cells were transfected with circLPAR3 siRNA and miR-1238 mimics or co-transfected with circLPAR3 siRNA and miR-1238 inhibitor. Cell cloning experiment was conducted to evaluate the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 on the radiosensitivity of Eca-109 cells. After Eca-109 cells that silenced circLPAR3, overexpressed miR-1238 or silenced both circLPAR3 and miR-1238 were exposed to 4 Gy irradiation, CCK-8 assay (A value), flow cytometry and Western blot were employed to assess the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 combined with 4 Gy irradiation on the proliferation and apoptosis of Eca-109 cells and the expression levels of CyclinD1, p21, Bcl-2 and Bax proteins. Dual luciferase reporter gene experiment and RNA pull down experiment were performed to verify the regulatory relationship between circLPAR3 and miR-1238. Results:Compared with adjacent tissues, the expression level of circLPAR3 was up-regulated in the esophageal cancer tissues ( P<0.05), while that of miR-1238 was down-regulated ( P<0.05). Compared with HET-1A cells, the expression levels of circLPAR3 were up-regulated in the esophageal cancer cell lines Eca-109, EC9706 and KYSE30(all P<0.05), whereas those of miR-1238 were down-regulated (all P<0.05). Silencing circLPAR3 or overexpressing miR-1238 reduced the survival fraction of Eca-109 cells (all P<0.05), and the sensitization ratio was 1.21 and 1.75, respectively. Silencing circLPAR3 or overexpressing miR-1238 decreased the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins (all P<0.05), while increased the apoptosis rate of Eca-109 cells and the expression levels of p21 and Bax proteins (all P<0.05). After silencing circLPAR3 or overexpressing miR-1238 combined with 4 Gy irradiation, the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins were decreased (all P<0.05), while Eca-109 cell apoptosis rate and the expression levels of p21 and Bax proteins were increased (all P<0.05). circLPAR3 targeted and negatively regulated the expression level of miR-1238 in Eca-109 cells. After silencing miR-1238 and circLPAR3 simultaneously, the survival fraction of Eca-109 cells was higher than that when only silencing circLPAR3, and the sensitization ratio was 0.59. Silencing miR-1238 reversed the effects of silencing circLPAR3 combined with 4 Gy irradiation on the proliferation and apoptosis of Eca-109 cells. Conclusion:circLPAR3 is highly expressed in esophageal cancer tissues and cell lines, and silencing the expression of circLPAR3 can inhibit the proliferation of esophageal cancer Eca-109 cells, promote their apoptosis, and enhance cell radiosensitivity by up-regulating miR-1238.
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Objective:To examine the effect of FAM83D knockdown on proliferation, survival ability and invasion of human esophageal squamous cell carcinoma after X-ray radiation, and explore the mechanism.Methods:The expression of FAM83D, E-cadherin and vimentin in tumor tissues was detected in 69 cases of esophageal squamous cell cancer by using immunohistochemical method. The siRNA based on the sequences of the FAM83D mRNA were synthesized to transfect into the cultured ECA109 cells as FAM83D shRNA group. The effect of silencing FAM83D gene was evaluated to determine the protein levels of FAM83D in the human oesophageal squamous cell carcinoma ECA109 and KYSE30 cells using western blotting. MTS, clone formation, and Transwell assay were employed to examine the proliferation, survival ability and invasion of ECA109 and KYSE30 cells in vitro, respectively. We used flow cytometry assay to analyze distribution of cell apoptosis in different groups. Western blotting was used to examine the expression of cell metastasis-related molecules and apoptosis-related protein. Results:The strong expression rates of FAM83D, E-cadherin, and vimentin were 55%(38/69), 36%(25/69) and 61%(42/69) in the tumor tissues, respectively. FAM83D protein expression was significantly and negatively correlated with the expression of E-cadherin ( r=-0.350, P<0.01), and positively with the expression of vimentin ( r=0.470, P<0.01). Western blotting results demonstrated that silencing FAM83D gene significantly reduced the FAM83D protein expression ( P<0.01). MTS data demonstrated that FAM83D knockdown after irradiation significantly inhibited the proliferation of esophageal squamous cell carcinoma ECA109 and KYSE30 cells ( P<0.05). The data from the clone formation assay revealed that the radiosensitivity was increased after downragulation of FAM83D expression ( P<0.01). In addition, the invasive abilities of oesophageal carcinoma cells transfected with FAM83D shRNA after irradiation were significantly inhibited compared with those of the NC group ( P<0.01), followed by the downregulation of N-cadherin, vimentin, Snail, p-Akt and p-GSK-3β expression, and the upregulation of E-cadherin expression ( P<0.01). The apoptosis rate of tumor cells in FAM83D shRNA group after irradiation was markedly increased ( P<0.01), followed by a decrease of Bcl-2 and Mcl-1 expression and an increase of Cleaved caspase-3 expression ( P<0.01). Conclusions:FAM83D expressions was found to be closely related to the invasion and development of ESCC. Furthermore, siRNA interference technology inhibited the expression of FAM83D gene in oesophageal squamous cell carcinoma cells, reduced the proliferation, invasion of cells, induced cell apoptosis, and increased radiosensitivity, which may be associated with regulating the epithelial-mesenchymaltransition via Snail/Akt/GSK-3β signaling pathways.
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Objective:To clarify the role of classic Wnt signaling pathway in the radioresistance of esophageal cancer cells (ECC), and investigate the underlying mechanism, aiming to identify critical molecular targets for clinically enhancing the radiosensitivity of esophageal cancer.Methods:The radiosensitivity of four types of ECCs (EC9706, ECA109, KYSE70 and KYSE150) were assessed by colony formation assay. Western blot and RT-PCR were used to detect the activation of classical Wnt signaling pathway after irradiation. Classic Wnt signaling pathway activator (AZD2858) and inhibitor (XAV-939) were utilized to comprehensively evaluate the effect of classic Wnt signaling pathway on the radiosensitivity of ECCs. Cellular immunofluorescence staining was performed to detect the production and repair of DNA double-strand breaks (DSB), as well as the foci formation of DSB repair proteins after irradiation.Results:The results of colony formation assay showed that the radiosensitivity of four types of ECCs from high to low was EC9706, ECA109, KYSE70 and KYSE150. In KYSE150, a radioresistant cell type, the level of nuclear β-catenin and the transcription of c-Myc gene were significantly increased after irradiation (both P<0.05). However, in EC9706, a radiosensitive cell type, the level of nuclear β-catenin and c-Myc gene transcription were not affected by irradiation (both P>0.05). Moreover, EC9706 cells showed enhanced radioresistance in the presence of AZD2858( P<0.05), whereas XAV-939 treatment decreased the radioresistance in KYSE150 cells ( P<0.05). AZD2858 accelerated the DSB repair in EC9706 cells ( P<0.05), whereas XAV-939 delayed the DSB repair in KYSE150 cells ( P<0.05). Furthermore, the results of immunofluorescence staining showed that XAV-939 reduced the DSB repair capacity by inhibiting homologous recombination repair-related proteins (BRCA1 and RAD51) rather than non-homologous end junction repair-related proteins (Ku80 and XRCC4). Conclusions:The classic Wnt signaling pathway participates in the regulation of radiosensitivity in ECCs by regulating the homologous recombination repair of DSB after irradiation. Inhibition of the classic Wnt signaling pathway can counteract the radioresistance of ECCs and enhance the killing effect of irradiation on ECCs.
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Objective To evaluate the effect of GOLPH3 on the proliferation, apoptosis and radiosensitivity of OE33 esophageal cancer cell line. Methods The expression levels of GOLPH3 mRNA and protein in the esophageal cancer cells and normal esophageal epithelial cells were detected by qRT-PCR and Western blot, respectively. The OE33 esophageal cancer cells were transfected with GOLPH3 siRNA and subject to irradiation treatment simultaneously. The cell proliferation was detected by MTT assay. The cell apoptosis was detected by flow cytometry. The radiosensitivity was assessed by cell cloning test. The expression levels of cleaved Caspase-3, Bax and cleaved Caspase-9 protein levels were quantitatively measured by Western blot. Results The expression levels of GOLPH3 mRNA and protein in the esophageal cancer cells were significantly higher than those in the normal esophageal epithelial cells ( both P<0.05) . GOLPH3 siRNA could obviously down-regulate the expression levels of GOLPH3 mRNA and protein in the OE33 esophageal cancer cells. The proliferation activity of esophageal cancer cells was decreased, whereas the apoptosis rate was increased and the expression levels of cleaved Caspase-3, Bax and cleaved Caspase-9 were up-regulated after down-regulating the expression of GOLPH3 or irradiation treatment ( all P<0.05) . After down-regulating the expression of GOLPH3 in the esophageal cancer cells treated with irradiation, the cell proliferation activity was more significantly decreased, whereas the apoptosis rate was elevated and the expression levels of cleaved Caspase-3, Bax and cleaved Caspase-9 were more evidently up-regulated ( all P<0.05) . In the irradiated OE33 esophageal cancer cells after down-regulating the expression of GOLPH3, the radiosensitization ratio of the cells was 1.673. Conclusions GOLPH3 is highly expressed in the esophageal cancer cells. Down-regulating the expression of GOLPH3 can increase the radiosensitivity, induce the apoptosis and inhibit the proliferation of OE33 esophageal cancer cells.
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Objective To establish a radioresistant esophageal squamous cancer cell line,and to identify the radioresistant genes and mechanisms.Methods The radioresistant KYSE410-res cell line was established by repeated exposure of cell line KYSE410 to radiation.The proliferation and apoptosis of esophageal squamous cancer cells were evaluated before and after radiation.The changes in gene expression of the esophageal squamous cancer cells after radiation were determined by gene microarray and analyzed by group t test.The genes with significant difference in expression after radiation were validated.Results The KYSE410-res cells had significantly enhanced proliferation and anti-apoptosis than the KYSE410 cells (all P＜0.05).The result of gene microarray showed that compared with the KYSE410 cells,the KYSE410-res cells had the expression of 463 and 251 genes upregulated and downregulated by no less than 4 folds,respectively.Those genes with different expression levels after radiation were mainly responsible for cell proliferation,adhesion,signal transduction,angiogenesis,reactive oxygen metabolism,cell damage repair,and the MAPK/ERK signaling pathway.OAS2 and UBD were key proteins in the network.In the KYSE410-res cells,the expression of HLA-DQBI,MMP1,NCAM1,ZNF521,GPC6,SELENBP1,LCN15,and TFPI-2 genes measured by real-time PCR was consistent with that measured by gene microarray.Conclusions Abnormal activation of the MAPK/ERK signaling pathway,upregulated expression of OAS2 and UBD,downregulated expression of TFPI-2,and upregulated expression of MMPs may play a role in radioresistance of esophageal cancer cells.
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Objective To explore and analyze the effects of allicin prodrug on proliferation of esophageal cancer cell line Eca9706 and the expression of apoptosis gene.Methods Different concentrations of allicin prodrugs were divided into a total of four groups:A1 group(10μg/mL),A2 group(20 μg/mL),A3 group (40 μg/mL),A4 group (normal saline,0 μg/mL),and respectively applicated in esophageal carcinoma cell line Eca9706.After culturing for 1 days,2 days,3 days,cell proliferation was detected by MTT and expression of p53 at the mRNA level was detected by RT-PCR.Results The optimum concentration of proliferation inhibition of allicin prodrug on esophageal cancer cell line Eca9706 was 20μg/mL and the best inhibition time was 2 days;the gene level of esophageal cancer cell line Eca9706 was inhibited with a dose-dependent.Conclusion Allicin prodrugs could effectively inhibit the proliferation of esophageal cancer cell line Eca9706,and control the proliferation of esophageal cancer cells by regulating the expression of apoptosis associated genes,so as to eliminate tumor cells.
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Objective: To investigate the inhibition of luteoloside on the proliferation of human esophageal carcinoma cell line Eca109 and its mechanism. Methods: MTT assay was used for detecting the influence of luteoloside at different concentration on the proliferation of Eca109. The morphological changes of cells were observed under inverted microscope. The cell cycle and apoptosis were detected by flow cytometry. RT-PCR was used to detect the mRNA expression of cyclin D1, survivin, and c-myc genes. Results: The results of MTT assay showed different doses (80, 120, 160, 200, and 240 μmol/L) of luteoloside could inhibit the cell proliferation of Eca109 cells in a dose-response manner. Luteoloside could also change the morphological characteristics of cells, reduce the cell size, and separate from peripheral cells. Treated with 240 μmol/L luteoloside, the cells were sprouting and some of them developed multiple pseudopodia-like protrusions. Treated with luteoloside (160 and 240 μmol/L), the cell cycle of Eca109 cells was blocked in G2/M phase and apoptosis was induced (P < 0.05). Compared with the control group, the gene expression of cyclin D1, survivin, and c-myc was decreased after treated with 240 μmol/L luteoloside for 48 h (P < 0.05). Conclusion: Luteoloside could significantly inhibit the proliferation of Eca109 cells by changing the cell cycle and inducing the apoptosis. Moreover, it could decrease the mRNA expression of relative genes.