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Aim To study the expression of microRNA(miR-34c-5p)in gastric cancer tissues and the mechanism of regulating the invasion and migration of gastric cancer cells. Methods The expression of miR-34c-5p mRNA in human gastric cancer tissues and cells was detected by quantitative real-time polymerase chain reaction(RT-qPCR). An overexpression model of miR-34c-5p was constructed in human gastric cancer cells. The transfection of lentivirus was observed under inverted fluorescence microscope, and the transfection efficiency was verified by RT-qPCR. Scratch test and Transwell assay were used to detect the effects of overexpression of miR-34c-5p on invasion and migration of gastric cancer cells. Western blot was used to detect the expression of MAP2K1, E-Cadherin and Vimentin in gastric cancer cells of each group. Dual-luciferase reporter assay was employed to detect whether MAP2K1 was a target gene of miR-34c-5p. Results The expression of miR-34c-5p mRNA in 10 human gastric cancer tissues was significantly lower than that in adjacent tissues(P<0.05). The expression of miR-34c-5p mRNA in gastric cancer HGC-27 cell was significantly lower than that in normal gastric mucosa GES-1 cell(P<0.01). Compared with blank control group and miR-34c-5p-NC group, miR-34c-5pmimics group significantly increased its miR-34c-5p mRNA expression level(P<0.05). The results of scratch test and Transwell experiment showed that overexpression of miR-34c-5p significantly inhibited the migration and invasion ability of gastric cancer cells(P<0.05, P<0.01). Western blot results showed that the protein expression levels of MAP2K1 and Vimentin significantly decreased and E-Cadherin markedly increased in gastric cancer HGC-27 cells after overexpression of miR-34c-5p(P<0.05). In addition, compared with mimic-NC+miR-34c-5p-mimics group, the lucifase activity of pmiR-MAP2K1-WT plasmid decreased(P<0.01), while the lucifase activity of pmiR-MAP2K1-MUT plasmid was not changed. Conclusions The low expression of miR-34c-5p in gastric cancer tissues and cells may inhibit the invasion and migration of gastric cancer cells through targeted regulation of MAP2K1 expression.
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Objective To investigate the effect of LncRNA OIP5-AS1 on radiosensitivity of non-small cell lung cancer (NSCLC) cells and its mechanism.Methods The radiation-resistant cell A549R was established by using A549 cells irradiated by X-ray 6Gy in 5 fractions.The expression levels of OIP5-AS1 and miR-34c-5p in A549 and A549R cells were detected by qRT-PCR.OIP5-AS1 inhibitor or miR-34c-5p mimetic was transfected into A549R cells,or OIP5-AS1 overexpression plasmid was transfected into A549 cells.Cell apoptosis was detected by flow cytometry.Cell radiosensitivity was analyzed by colony formation assay.The expression levels of p-Chk2 and p-ATM proteins were measured by Western blot.Dual luciferase assay was adopted to verify the relationship between OIP5-AS1 and miR-34c-5p.Results Compared with A549 cells,the expression of OIP5-AS1 was significantly up-regulated in A549R cells (1.97±0.11 vs.1.01±0.05,P＜0.05),whereas the expression of miR-34c-5p was remarkably down-regulated (0.43±0.02 vs.1.02±0.06,P＜0.05).The expression levels of p-Chk2 and p-ATM proteins in A549R cells in the silencing OIP5-AS1 +6Gy group were significantly lower (0.43±0.03 vs.1.39±0.15,0.51 ±0.0 5 vs.1.21 ± 0.11,both P＜0.05),whereas the apoptotic rate was significantly higher than those in the silencing control + 6Gy group [(13.29± 1.25)％ vs.(28.47± 2.31)％,P＜0.05)].The expression levels of p-Chk2 and p-ATM proteins in A549 cells in overexpressing OIP5-AS1+6 Gy group were significantly higher than those in overexpression control+6 Gy group (1.23±0.13 vs.0.75±0.06,1.08±0.11 vs.0.59± 0.04,both P＜0.05).Inhibiting miR-34c-5p expression reversed the effect of silencing OIP5-AS1 on survival fraction of A549R cells (SER =1.42).OIP5-AS1 negatively regulated the expression of miR-34c-5p.Conclusion Silencing OIP5-AS1 enhances the radiosensitivity of radiation-resistant A549 cells by up-regulating the expression of miR-34c-5p,providing a potential target for radiotherapy of NSCLC cells.
ABSTRACT
Objective@#To investigate the effect of LncRNA OIP5-AS1 on radiosensitivity of non-small cell lung cancer (NSCLC) cells and its mechanism.@*Methods@#The radiation-resistant cell A549R was established by using A549 cells irradiated by X-ray 6Gy in 5 fractions. The expression levels of OIP5-AS1 and miR-34c-5p in A549 and A549R cells were detected by qRT-PCR. OIP5-AS1 inhibitor or miR-34c-5p mimetic was transfected into A549R cells, or OIP5-AS1 overexpression plasmid was transfected into A549 cells. Cell apoptosis was detected by flow cytometry. Cell radiosensitivity was analyzed by colony formation assay. The expression levels of p-Chk2 and p-ATM proteins were measured by Western blot. Dual luciferase assay was adopted to verify the relationship between OIP5-AS1 and miR-34c-5p.@*Results@#Compared with A549 cells, the expression of OIP5-AS1 was significantly up-regulated in A549R cells (1.97±0.11 vs.1.01±0.05, P<0.05), whereas the expression of miR-34c-5p was remarkably down-regulated (0.43±0.02 vs.1.02±0.06, P<0.05). The expression levels of p-Chk2 and p-ATM proteins in A549R cells in the silencing OIP5-AS1+ 6Gy group were significantly lower (0.43±0.03 vs.1.39±0.15, 0.51±0.0 5 vs.1.21± 0.11, both P<0.05), whereas the apoptotic rate was significantly higher than those in the silencing control+ 6Gy group [(13.29±1.25)% vs. (28.47±2.31)%, P<0.05)]. The expression levels of p-Chk2 and p-ATM proteins in A549 cells in overexpressing OIP5-AS1+ 6Gy group were significantly higher than those in overexpression control+ 6Gy group (1.23±0.13 vs.0.75±0.06, 1.08±0.11 vs.0.59±0.04, both P<0.05). Inhibiting miR-34c-5p expression reversed the effect of silencing OIP5-AS1 on survival fraction of A549R cells (SER=1.42). OIP5-AS1 negatively regulated the expression of miR-34c-5p.@*Conclusion@#Silencing OIP5-AS1 enhances the radiosensitivity of radiation-resistant A549 cells by up-regulating the expression of miR-34c-5p, providing a potential target for radiotherapy of NSCLC cells.
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OBJECTIVE@#To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells.@*METHODS@#Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist.@*RESULTS@#VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells.@*CONCLUSIONS@#VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.
Subject(s)
Humans , Autophagy , Autophagy-Related Proteins , Genetics , Metabolism , Cell Line , Cysteine Endopeptidases , Genetics , Metabolism , Dactinomycin , Pharmacology , Down-Regulation , Genes, Reporter , MicroRNAs , Metabolism , Microtubule-Associated Proteins , Metabolism , RNA, Messenger , Metabolism , Signal Transduction , Transfection , Valproic Acid , PharmacologyABSTRACT
Objective To investigate the effect of up-regulating miR-34c-5p on cloning formation and migration of nasopharyngeal carcinoma cell line HONE-1 in vitro. Methods Expression levels of miR-34c-5p in nasopharyngeal cell line NP69 and nasopharyngeal carcinoma cell line HONE-1 were investigated by real time quantitative PCR;Cloning Forma?tion Test and Transwell Migration Assay were used to explore the effect of up-regulating miR-34c-5p on proliferation and migration of nasopharyngeal carcinoma cell line HONE-1. Results The expression of miR-34c-5p in nasopharyngeal car?cinoma cell line HONE-1 was lower than that in normal nasopharyngeal cell line NP69. Up-regulating miR-34c-5p signifi?cantly suppressed the cloning formation and migration capacity, compared to those of NP69 cell line and HONE-1 with nor?mal level of miR-34c-5p(P<0.05). Conclusion Down-regulating miR-34c-5p involve in proliferation and migration of nasopharyngeal carcinoma and may be a used as a new therapeutic target for nasopharyngeal carcinoma.