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Objective To explore the expression of circ_0006692 in NSCLC and its relation with clinicopathological characteristics and related mechanism. Methods We collected 50 pairs of NSCLC tissues and adjacent tissues. The expression of circ_0006692 was detected by qRT-PCR and its relation with clinicopathological features was analyzed. We constructed lung cancer A549 cell line with circ_0006692 overexpression and knockdown. Cell proliferation, migration and invasion were detected by MTS, colony forming, wound healing and Transwell invasion assays. qRT-PCR and Western blot were used to detect the effect of circ_0006692 expression change on EMT-related gene expression. Results The expression of circ_0006692 in NSCLC was significantly higher than that in adjacent tissues (P < 0.05). The expression level of circ_0006692 was closely related to tumor size, TNM stage and pulmonary membrane invasion (P < 0.05). circ_0006692 knockdown inhibited cell proliferation, invasion and metastasis, while its overexpression promoted cell proliferation, invasion and metastasis. circ_0006692 knockdown inhibited the expression of EMT-related genes CDH2 and MMP7 in A549 cells and promoted the expression of CDH1. Conclusion The upregulated expression of circ_0006692 in NSCLC is closely related to tumor size, TNM stage and pulmonary membrane invasion. circ_0006692 expression could regulate the proliferation, invasion, metastasis and EMT progression of lung cancer A549 cells.
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Objective To investigate the effect of piR-9994 on the biological behavior of gastric cancer cells and its possible mechanism. Methods The expression of piR-9994 in gastric cancer cell lines (MGC803 and AGS) and normal gastric epithelial cells (GES-1) were detected by qRT-PCR. MGC803 cell line with piR-9994 overexpression and knockdown were constructed. The effects of piR-9994 expression changes on cell proliferation were detected by MTT and clone formation assay. The scratch wound healing assay and Transwell invasion assay were used to detect cell migration and invasion abilities. qRT-PCR and Western blot were used to detect cell proliferation and EMT-related genes expression. Results The expression level of piR-9994 in MGC803 cells was significantly higher than that in normal gastric epithelial cell line GES-1 (P < 0.05). The overexpression of piR-9994 promoted the proliferation, invasion and metastasis of gastric cancer cells, while piR-9994 knockdown had the opposite effect. piR-9994 expression was closely related to cell cycle, especially in S phase. The overexpression of piR-9994 promoted the expression of proliferation-related genes PCNA, CCND1 and Bcl-2, inhibited the expression of apoptosis gene C-PARP, and promoted the expression of EMT-related genes N-cadherin, MMP7, Twist and Vimentin; while piR-9994 knockdown had the opposite effect. Conclusion Abnormal expression of piR-9994 affects the proliferation, invasion, metastasis and EMT process of gastric cancer cells. piR-9994 may be a new biomarker and therapeutic target for gastric cancer.
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Objective To investigate the effect of small interfering RNA (siRNA) interference in the expression of epidermal growth factor receptor (EGFR) on the radiosensitivity of esophageal squamous carcinoma (Eca-109) and esophageal adenocarcinoma (OE-19) cell lines.Methods Human Eca-109 and OE-19 cell lines were selected as study subjects.Various EGFR-siRNA and negative siRNA were synthesized chemically through lipofection.Reverse transcription-polymerase chain reaction and Western blot were applied to measure the expression of EGFR before and after transfection,and the CCK8 assay was applied to analyze the influence of transfection on cell proliferation.Blank control groups of Eca-109 and OE-19 cells (O1 and O2 groups),simple irradiation groups (R1 and R2 groups),and EGFR-siRNA irradiation groups (E-R1 and E-R2 groups) were set,and the doses for single irradiation were 0,2,4,6,and 8 Gy.The colony-forming assay was applied to calculate survival fraction (SF) and sensitization enhancement ratio (SERD0 ratio),and flow cytometry was applied to evaluate the influence of EGFR-siRNA combined with radiotherapy on cell cycle distribution and apoptosis rate,and the dose for single irradiation was 6 Gy.Results The expression of EGFR in both cell lines was significantly down-regulated by EGFR-siRNA,and the inhibition rate of cell proliferation by transfection was＜5％ (4.9％ and 4.5％,respectively).The results of colony-forming assay showed that the cells in the E-R1 and E-R2 groups had a lower SF than those in the O1 and O2 groups,with an SERD0ratio of 1.40 and 1.01,respectively.The results from flow cytometry showed that compared with the E-R2 group,the E-R1 group had a higher proportion of cells in G2/M phase and a lower proportion of cells in S phase after irradiation (P=0.016 and 0.028),as well as a higher apoptosis rate (P=0.007).Conclusions Compared with the cell line OE-19,the cell line Eca-109 has a significantly increased radiosensitivity when treated with siRNA interference in EGFR expression.
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Objective To investigate the effect of silencing Annexin A2 gene expression by small interfering RNA (siRNA) on the radiosensitivity of nasopharyngeal carcinoma cells CNE-2 (R743).Methods siRNA targeting the Annexin A2 gene was chemically synthesized and transfected into R743 cells by HiPerFect.The mRNA and protein levels of Annexin A2 before and after transfection were measured by RT-PCR and Western blot,respectively.The change in radiosensitivity of R743 cells was analyzed by colonyforming assay.Cell cycle distribution and apoptosis after X-ray irradiation were analyzed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay,respectively.Results The results from RT-PCR and Western blot showed that the expression of Annexin A2 was down-regulated after transfection.The colony-forming assay indicated that the D0,Dq,and SF2 in transfected cells were significantly lower than those in untransfected cells with radiation alone and in cells transfected with control siRNA.The sensitization enhancement ratios (D0 ratios) of transfected cells relative to untransfected and control siRNA transfected cells were 1.30 and 1.27,respectively.After X-ray irradiation,the proportion of cells in G2/M phase was significantly higher in the transfected cells thin in untransfected and control siRNA transfected cells (32.46％ vs.9.17％ and 9.42％,respectively;P =0.000 and 0.000).The apoptosis rate was also significantly higher in the transfected cells than in the untransfected and control siRNA transfected cells (35.20％ vs.10.87％ and 11.33％,respectively;P=0.000 and 0.000).Conclusions Silencing Annexin A2 gene expression by siRNA can increase the radiosensitivity of R743 cells,which may be associated with DNA damage repair and change in cell cycle distribution.
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Objective To investigate the proteins which were associated with radiosensitivity of nasopharyngeal carcinoma (NPC) cells and could be used to predict the radiosensitivity.Methods A radioresistant subclone cell line CNE-2 (R743) derived from NPC cell line CNE-2 was established.Radiosensitivity and cell cycle characteristics of CNE-2 and CNE-2 ( R743 ) were examined and compared by clonogenic survival assay and flow cytometry.The total proteins from the two cell lines were extracted and separated by two-dimensional gel electrophoresis,and the images were analyzed by Image Master 7.0analysis software.Differentially expressed proteins in the two cell lines were identified through MALDITOF/TOF peptide mass fingerprint and searched in the protein sequence database.The protein expressions were confirmed by RT-PCR and Western blot.Results Totally seven differentially expressed proteins were identified,six of which were upregulated and one downregulated in the radioresistant CNE-2 (R743),compared with those of CNE-2.Three out of the seven,Annexin A2,Tropomyosin 4 and GRP78 were upregulated in the CNE-2 ( R743 ),which were confirmed by Western blot and RT-PCR ( t =24.22,24.20,29.19,P ＜ 0.05).Conclusions Differentially expressed proteins might be involved in different radiosensitivities of nasopharyngeal carcinoma cell lines,among which Annexin A2,Tropomyosin 4 and GRP78 could be the candidate biomarkers for predicting radiosensitivity of nasopharyngeal carcinoma cells.