mir-3168 targeted inhibition of TP53 promotes malignant transformation and cisplatin resistance of AGS and AGS/DDP gastric cancer cells

Objective:To investigate the effect of mir-3168 on the malignant transformation and cisplatin resistance of AGS and AGS/DDP gastric cancer cells,and to verify its target gene.Methods:The expression of mir-3168 in AGS and AGS/DDP gastric cancer cells was detected by qPCR,and mir-3168 mimic,inhibitor and negative control were synthesized.They were transfected into AGS and AGS/DDP gastric cancer cells,respectively.The expression of mir-3168 and TP53 mRNA was detected by qPCR.Cell viability was detected by CCK8 under gradient cisplatin treatment and non treatment,apoptosis was detected by flow cytometry,cell invasion was detected by Transwell,and TP53 protein expression was detected by western blot,The database predicted the binding sites of mir-3168 and TP53.According to the binding sites,the double luciferase experiment was used to verify the binding of mir-3168 and TP53.Results:Compared with cisplatin sensitive gastric cancer cell AGS,mir-3168 was significantly overexpressed in cisplatin resistant gastric cancer cell AGS/DDP;mir-3168 mimic promotes cisplatin resistance,proliferation and invasion of AGS and AGS/DDP gastric cancer cells,and inhibits apoptosis of AGS and AGS/DDP gastric cancer cells;mir-3168 inhibitor inhibits cisplatin resistance,proliferation and invasion of AGS and AGS/DDP gastric cancer cells,and promotes apoptosis of AGS and AGS/DDP gastric cancer cells;mir-3168 mimic inhibits the expression of TP53 mRNA and protein,and mir-3168 inhibitor promotes the expression of TP53 mRNA and protein;Targetscan database predicted that there was a binding point between mir-3168 and TP53,and the double luciferase experiment suggested that mir-3168 was bound to TP53 through the predicted binding site.Conclusion:mir-3168 may promote the malignant transformation of AGS and AGS/DDP gastric cancer cells and cisplatin resistance by targeting TP53.

miR-3168靶向PDCD5调控A549细胞凋亡

目的:探讨miR-3168通过调节PDCD5对非小细胞肺癌A549细胞凋亡的影响。方法:运用TargetScan在线分析miR-3168与PDCD5的相关性;将PDCD5的3′端非编码区克隆进pmiR-RB-Repor Luciferase载体,利用双荧光素酶报告系统检测miR-3168是否靶向调控PDCD5基因;之后用Xfect RNA Transfection Reagent转染miR-3168 mimics或者miR-3168 inhibitor进A549细胞后,用Etoposide处理,通过Western blot检测PDCD5蛋白表达量和A549细胞凋亡标志蛋白的表达量和P53定位情况;同时采用Annexin V染色法测定细胞凋亡水平。结果:通过TargetScan分析,miR-3168仅在一个区域与PDCD5具有较高匹配度;通过双荧光素酶报告系统发现,miR-3168靶向结合PDCD5的3′端非编码区;过表达miR-3168时,PDCD5的表达量下降,细胞凋亡标志蛋白PDCD5的表达量显著下降(P<0.05),细胞凋亡相关蛋白P21和BAK的表达量显著下降(P<0.05),而BCL-2的表达量显著上升(P<0.05),P53入核受到抑制(P<0.05);而用转染miR-3168 inhibitor进A549细胞时,PDCD5的表达量显著上升(P<0.05),细胞凋亡相关蛋白P21和BAK的表达量显著上升(P<0.05),而BCL-2的表达量显著下降(P<0.05),并促进P53入核(P<0.05)。结论:miR-3168通过抑制PDCD5表达量来抑制非小细胞肺癌细胞A549的凋亡。

mir⁃3168靶向抑制TP53促进AGS与AGS/DDP胃癌细胞恶性转化与顺铂耐药

目的:探讨mir‑3168对AGS与AGS/DDP胃癌细胞恶性转化与顺铂耐药影响,并验证其靶基因。方法:qPCR检测胃癌顺铂敏感细胞AGS与胃癌顺铂耐药细胞AGS/DDP细胞中mir‑3168表达,合成mir‑3168 mimic、inhibitor与阴性对照,分别转染AGS与AGS/DDP胃癌细胞,qPCR检测mir‑3168与TP53 mRNA表达,梯度浓度顺铂处理与非处理下采用CCK8检测细胞活力,流式检测细胞凋亡,Transwell检测细胞侵袭,Western blot检测TP53蛋白表达,数据库预测mir‑3168与TP53结合位点,根据结合位点采用双荧光素酶实验验证mir‑3168与TP53结合。结果:相对胃癌顺铂敏感细胞AGS,mir‑3168在胃癌顺铂耐药细胞AGS/DDP细胞中的表达水平明显提高;mir‑3168 mimic促进AGS与AGS/DDP胃癌细胞顺铂抵抗、增殖与侵袭,抑制AGS与AGS/DDP胃癌细胞凋亡;mir‑3168 inhibitor抑制AGS与AGS/DDP胃癌细胞顺铂抵抗、增殖与侵袭,促进AGS与AGS/DDP胃癌细胞凋亡;mir‑3168 mimic抑制TP53 mRNA与蛋白表达,mir‑3168 inhibitor促进TP53 mRNA与蛋白表达;Targetscan数据库预测mir‑3168与TP53存在结合点,双荧光素酶实验提示mir‑3168与TP53结合,且通过预测的结合位点结合。结论:mir‑3168可能通过靶向抑制TP53促进AGS与AGS/DDP胃癌细胞恶性转化与顺铂耐药。