长链非编码RNA肿瘤蛋白53靶基因1在皮肤基底癌细胞中的表达及其与增殖的关系

Expression of long chain non coding RNA tumor protein 53 target gene 1 in skin basal cancer cells and its relationship with proliferation

目的探讨长链非编码RNA肿瘤蛋白53靶基因1(lncRNA TP53TG1)在人皮肤基底癌细胞中的表达及其与增殖的关系。方法人正常皮肤细胞(HaCaT)和人皮肤基底细胞癌TE 354.T和A431细胞系采用实时荧光定量聚合酶链反应(PCR)技术分析lncRNA TP53TG1表达水平。选取高表达的皮肤基底细胞癌TE 354.T细胞作为研究对象,采用慢病毒感染TE 354.T细胞,建立lncRNA TP53TG1过表达细胞系和对照细胞,分别命名为对照组和TP53TG1组。采用细胞计数试剂盒(CCK-8)和克隆形成实验分析对照组和TP53TG1组细胞的增殖能力;采用蛋白质免疫印迹分析两组细胞增殖指标细胞核增殖抗原(Ki-67)和增殖细胞核抗原(PCNA)的表达水平;采用7-氨基放线菌素D(7-AAD)试剂盒测定两组细胞的增殖活性;在裸鼠建立体外移植瘤模型,分析两组细胞形成肿瘤的体积和重量;采用实时荧光定量PCR分析lncRNA TP53TG1下游与细胞增殖相关的基因水平。组间计量数据比较采用t检验。结果人正常皮肤细胞(HaCaT)lncRNA TP53TG1表达水平(0.83±0.34)明显低于人皮肤基底细胞癌TE 354.T和A431细胞系表达水平(1.65±0.20、1.58±0.13),差异统计学意义(t=5.579、5.503,P<0.05)。TP53TG1组细胞24 h吸光度值(2.06±0.12)明显高于对照组细胞(1.50±0.08),差异有统计学意义(t=10.070,P<0.05)。TP53TG1组细胞14 d克隆形成率[(81.86±8.71)%]明显高于对照组细胞[(54.85±6.79)%],差异有统计学意义(t=6.469,P<0.05)。TP53TG1组细胞7-AAD阳性率[(65.57±5.62)%]明显高于对照组细胞[(37.43±4.50)%],差异有统计学意义(t=10.340,P<0.05)。TP53TG1组细胞Ki-67和PCNA蛋白表达水平(1.31±0.14、1.38±0.15)明显高于对照组细胞(0.70±0.08、0.72±0.08),差异有统计学意义(t=10.240、10.150,P<0.05)。TP53TG1组细胞小鼠皮下形成肿瘤的体积和质量[(986.71±81.64)mm^(3)、(3.45±0.36)g]明显高于对照组细胞[(699.29±53.62)mm^(3)、(2.15±0.26)g],差异有统计学意义(t=7.786、7.709,P<0.05)。TP53TG1组细胞微小RNA(miR)-33a-5p和miR-33b(0.72±0.10、0.65±0.06)明显高于对照组细胞(0.99±0.30、1.03±0.12),差异有统计学意义(t=2.227、7.589,P<0.05)。结论lncRNA TP53TG1在人皮肤基底癌细胞中的表达显著下降。过表达lncRNA TP53TG1可显著抑制人皮肤基质癌细胞的增殖,可能与miR-33a-5p和miR-33b表达有关。

Objective To investigate the expression of long non-coding RNA TP53 target gene 1(lncRNA TP53TG1)in human skin basal carcinoma cells and its relationship with proliferation.MethodsThe expression levels of lncRNA TP53TG1 were analyzed using real-time fluorescence quantitative polymerase chain reaction(PCR)in normal human skin cells(HaCaT)and human basal cell carcinoma TE 354.T and A431 cell lines.The highly expressed skin basal cell carcinoma TE 354.T cells were selected as the research objects,and infected with lentivirus to establish lncRNA TP53TG1 overexpressing cell lines and control cells,named as the control group and TP53TG1 group,respectively.The proliferation ability of cells in the control group and TP53TG1 group was analyzed using cell counting kit-8(CCK-8)and clone formation experiments.The expression levels of proliferation cell nuclear antigen(Ki-67)and proliferating cell nuclear antigen(PCNA)in two groups were analyzed by Western blotting.The proliferation activity of two groups was measured using the 7-aminoactinomycin D(7-AAD)assay kit.The in vitro tumor transplantation model was prepared in nude mice.The tumor volume and weight of control group and TP53TG1 group were analyzed.The gene levels of lncRNA TP53TG1 downstream related to cell proliferation were analyzed by real time fluorescence quantitative PCR.The comparison of inter group measurement data was done by t-test.ResultsThe expression level of TP53TG1 in normal human skin cells(HaCaT)(0.83±0.34)was significantly lower than that in TE 354.T and A431 cell lines(1.65±0.20,1.58±0.13,t=5.579,5.503,P<0.05).The 24-h absorbance value in the TP53TG1 group(2.06±0.12)was significantly higher than that in the control group(1.50±0.08,t=10.070,P<0.05).The 14-day clone formation rate in the TP53TG1 group[(81.86±8.71)%]was significantly higher than that in the control group[(54.85±6.79)%,t=6.469,P<0.05].The positive rate of 7-AAD in the TP53TG1 group[(65.57±5.62)%]was significantly higher than that in the control group[(37.43±4.50)%,t=10.340,P<0.05].The expression levels of Ki-67 and PCNA proteins in the TP53TG1 group(1.31±0.14,1.38±0.15)were significantly higher than those in the control group(0.70±0.08,0.72±0.08,t=10.240,10.150,P<0.05).The volume and mass of tumors formed subcutaneously in the TP53TG1 group[(986.71±81.64)mm^(3),(3.45±0.36)g]were significantly increased as compared with those in the control group[(699.29±53.62)mm^(3),(2.15±0.26)g,t=7.786,7.709,P<0.05].The levels of microRNA(miR)-33a-5p and miR-33b in the TP53TG1 group(0.72±0.10,0.65±0.06)were significantly higher than those in the control group(0.99±0.30,1.03±0.12,t=2.227,7.589,P<0.05).ConclusionThe expression of lncRNA TP53TG1 in human skin basal cancer cells significantly decreased.Overexpression of lncRNA TP53TG1 can significantly inhibit the proliferation of human skin stromal cancer cells,which may be related with miR-33a-5p and miR-33b.