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@#[Abstract] Objective: To investigate the effect of long non-coding RNA DiGeorge syndrome critical region gene 5 (DGCR5) on proliferation, invasion and migration of esophageal squamous cell carcinoma (ESCC) TE1 cells and its mechanism. Methods: qPCR was used to detect the expression level of DGCR5 in ESCC cell lines (TE1, Yes-2, KYSE150 and Eca9706). TE1 cells were transfected with siRNA-DGCR5(si-DGCR5) and negative control (si-NC) plasmids, respectively. CCK-8, Wound healing and Transwell assay were used to detect the proliferation, migration and invasion of TE1 cells before and after DGCR5 knockdown. The relationship between DGCR5 expression and epidermal growth factor receptor (EGFR) in ESCC tissues was analyzed by GEPIA database. The mRNA and protein expressions of EGFR in ESCC cell line were examined by qPCR and Western blotting (WB). WB was further used to detect the expression of EGFR protein in TE1 cells before and after DGCR5 knockdown. Results: lncRNA DGCR5 was highly expressed in ESCC cell lines (all P<0.01). qPCR confirmed that the expression of DGCR5 in TE1 cells of si-DGCR5 group was significantly lower than that of si-NC group (P<0.01). The proliferation, migration and invasion ability of TE1 cells in si-DGCR5 group were significantly lower than those in si-NC group (all P<0.01). GEPIA database showed that the expression of DGCR5 was positively correlated with EGFR in ESCC tissues (P<0.01). WB showed that the protein level of EGFR in TE1 cells of si-DGCR5 group decreased significantly (P<0.01). Conclusion: lncRNA DGCR5 is highly expressed in ESCC cells, and promotes the proliferation, invasion and migration of TE1 cells possibly by up-regulating EGFR expression.
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@#[Abstract] Objective: To observe the effects of shikonin on the proliferation, apoptosis and cell cycle of human esophageal carcinoma TE-1 cells, and to explore its mechanism. Methods: TE-1 cells were treated with different concentrations of shikonin (0, 1, 5, 10 µmol/L). MTT assay was used to detect cell proliferation at different time points (24, 48 and 72 h). After treatment with shikonin for 48 h, cell apoptosis in TE-1 cells of each group was observed with Hoechst 33258 fluorescence staining. Flow cytometry was used to detect apoptosis and cell cycle. The changes in expression of TRAP1/Akt/mTOR signaling pathway related proteins were detected by Western blotting. Results: Shikonin inhibited the proliferation of TE-1 cells in a time-dose-dependent manner (P<0.05 or P<0.01). Compared with the control group, shikonin significantly promoted the apoptosis of TE-1 cells (P<0.01), induced the G0/G1 phase block of TE-1 cells (P<0.05 or P<0.01), and reduced the expression levels of TRAP1, p-Akt and p-MTOR (P<0.05 or P<0.01). The above effects were all dose-dependent. Conclusion: Shikonin can significantly inhibit the proliferation of TE-1 cells in vitro, induce G0/G1 phase arrest and promote apoptosis, which may be closely related to the inhibition of TRAP1/Akt/mTOR signaling pathway.
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Objective To analyze the regulatory of miRNA (miR)-149-5p for the expression of Aurora-B in esophageal squamous cell carcinoma (ESCC). Methods The pathologic histology paraffin blocks of 61 patients with ESCC in Shanxi Provincial People's Hospital from January 2010 to December 2017 were collected. The immunohistochemical staining and tissue section in situ hybridization method were used to observe the expressions of Aurora-B and miR-149-5p in the tumor tissues and adjacent mucosas of the patients, and their relationship with clinicopathological parameters was analyzed. miRNA was predicted by using software TargetScan and miR walk. The relationship between Aurora-B and miR-149-5p were verified by using Western blot in ESCC TE-1 cells. Results The result of immunohistochemical staining showed that in 61 patients, 37 (61％) tumor tissues showed higher expression of Aurora-B compared with adjacent mucosas, the Aurora-B expression in 15 (26％) tumor tissues were in line with benign tissues, the Aurora-B expression in 9 (13％) tumor tissues were inferior to benign tissues. The expression of Aurora-B were not correlated with age (χ2=0.008, P=0.929), gender (χ2=0.088, P=0.767), grade of differentiation (χ2=2.632, P=0.268), but correlated with TNM staging (χ2=15.153, P<0.01) and lymph node metastasis (χ2=5.979, P=0.014). The miR-149-5p was predicted to combine with Aurora-B 3'untraslated region (UTR) by using TargetScan and miRwalk software. The result of in situ hybridization showed that the miR-149-5p showed low expression in 22 (36％) tumor tissues. The expression of miR-149-5p was correlated with Aurora-B expression (χ2 = 5.622,P= 0.018), and not correlated with age (χ2= 2.617, P= 0.106), gender (χ2= 1.529, P= 0.216), grade of differentiation (χ2 = 2.854, P= 0.240), TNM staging (χ2 = 0.870, P= 0.351) and lymph node metastasis (χ2= 0.128, P= 0.351). The Western blot results of TE-1 cells showed that the expression of Aurora-B in simultaneous over-expression of miR-149-5p and Aurora-B group was higher than that in over-expression of miR-149-5p group, and lower than that in over-expression of Aurora-B group. Conclusion The miR-149-5p can be involved in ESCC progression through regulating the expressions of Aurora-B.
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@#Objective: To investigate the expression of lncRNA01296 in esophageal cancer (EC) tissues and its effect on the proliferation and migration of EC TE-2 cells. Methods:Atotal of 36 pairs of esophageal cancer tissues and corresponding para-cancerous tissues were collected from EC patients admitted to the Department of Thoracic Surgery,Affiliated Hospital of Chengde Medical College from January 2017 to September 2018. The human normal esophageal epithelial (HEEC) cells and human esophageal cancer cell lines ECA109, TE-1 and TE-2 were cultured. qPCR was used to detect the mRNAexpressions of lincRNA01296, SNRPA(small nuclear ribonucleoproteinA) and NGF (nerve growth factor) in EC tissues and cells. Recombinant lentiviral interference vectoror control vector were used to transfect EC cell lines, as sh-lncRNA01296#1,#2 and Mock groups. WB was used to detect the protein expressions of SNRPAand NGF in transfected cells. MTS assay was used to detect cell proliferation, and Transwell assays were used to detect cell invasion and migration of TE-2 cells after transfection. Results: The mRNAexpressions of lncRNA01296, SNRPAand NGF were significantly increased in esophageal cancer tissues and cell lines (all P<0.01), and these expressions in poorly differentiated TE-2 cells were higher than those in highly differentiated ECA109 and TE-1 cells (all P<0.05). The mRNAexpressions of lncRNA01296 and NGF in sh-lncRNA01296#1 and sh-lncRNA01296#2 groups were significantly lower than those in Mock group (all P<0.01), while the mRNAexpression of SNRPAshowed no statistical difference among three groups (P>0.05). The protein expressions of lncRNA01296 and NGF in sh-lncRNA01296#1 and sh-lncRNA01296#2 groups were significantly lower than those in Mock group (all P<0.01). The relative proliferation ability of cells in sh-lncRNA01296#1 and shlncRNA01296#2 groups was significantly lower than that of Mock group at 48 and 72 h after transfection (P<0.05 or P<0.01). The number of invasive cells was (72.0±6.3), (36.6±4.3) and (33.9±3.7) in Mock, sh-lncRNA01296#1 and sh-lncRNA01296#2 groups, respectively; and the number of migrated cells was (85.2±9.9), (47.5±8.1) and (43.8±6.5), respectively, indicating that the numbers of invasive and migrated cells in sh-lncRNA01296#1 and sh-lncRNA01296#2 groups were significantly less than those in Mock group(all P<0.01). Conclusion: lncRNA01296 can up-regulate SNRPAexpression to promote NGF-mediated proliferation and metastasis of EC cells, which may provide new target for the diagnosis and treatment of esophagealcancer.
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@#Objective: To investigate the expression of ciRS-7 in esophageal squamous cell carcinoma (ESCC) and its effect on the cellular proliferation, migration and invasion. Methods: The cancer tissues and paired adjacent normal tissues from 60 ESCC patients treated in the Fourth Hospital of Hebei Medical University between May, 2016 andApril, 2017 were selected for this study. The expressions of ciRS-7 were detected by qRT-PCR. After over-expressing or silencing of ciRS-7, the proliferation of ESCC cell line TE1 was measured by CCK-8 assay; and the migration and invasion were tested by wound healing assay and Transwell invasion assay，respectively. Finally, the effect was validated via animal experiment. Results: CiRS-7 was highly expressed in ESCC tissues (P<0.05), and its expression level was closely related to pathological grade and lymph node metastasis (P<0.05). Over-expression of ciRS-7 significantly increased the proliferation, migration and invasion (all P<0.05) of TE1 cells; while silencing of ciRS-7 remarkably suppressed the proliferation, migration and invasion (all P<0.05). Conclusion: CiRS-7 was up-regulated in ESCC and could enhance ESCC cell proliferation, migration and invasion, suggesting that ciRS-7 could be used as a potential target for the diagnosis and treatment of ESCC.
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Objective To explore the radiosensitization effect of thalidomide combined with X-ray on esophageal carcinoma TE1 cells.Methods Cell scratch assay Was used to detect the inhibition ability of different concentration of Thalidomide on cell invasion and metastasis.H3-TdR incorporation assay Was used to investigate the inhibition of DNA synthesis in TE1 cells by treated with Thalidomide singly or combination with X-rays.The colony formation assay Was used to analyze the radiosensitization of Thalidomide effect on TE1 cells.Results Thalidomide had obvious inhibition effect on TE1 cell metastasis.DNA synthesis and colony formation,which were correlated with drug concentration.The values D0,Dq and SF2 in TE1 cells were gradually decreased with thalidomide concentration increased.When the concentration of thalidomide was 100μg/ml,the SERD0 and SERDq were(1.4±0.2)and(1.5±0.1),respectively,While the concentration of thalidomide Was 1 50μg/ml,the SERD0 and SERDq were metastasis,DNA synthesis,and significantly enhance the radiosensitizing effect on esophageal carcinoma TE1 cells.
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Objective To investigate the effects of 2-(3carboxy-1-oxoprogy1)amino-2-deoxy-D-glucose(COPADG) on the proliferation and cell cycle of human esophageal cancer cells Eca-109 and TE-1.Methods In vitro experiments,MTT colorimetric assay was performed to determine the growth inhibitory rates of Eca-109 and TE-1 cells treated by COPADG.Flow cytometry was applied to observe the change of cell proliferation cycle and apoptosis index.Cell morphological change was observed by transmission electron microscopy.Results COPADG inhibited effectively the growth of esophageal cancer cells Eca-109 and TE-1 in a time-and dose-dependent manner(P