LncRNA P5848表达对食管鳞癌细胞生物学行为影响

Effect of lncRNA P5848 on biological behavior of esophageal squamous cell carcinoma

目的近年来研究显示,长链非编码RNA(long non-coding RNA,lncRNA)与食管鳞癌的发生、发展密切相关,为探索食管鳞癌的诊治提供了新靶点。本研究旨在阐明lncRNA P5848在食管鳞癌组织及细胞中的表达,并深入探讨其对细胞增殖的影响。方法利用实时荧光定量反转录-聚合酶链反应(real-time quantitative reverse transcription polymerase chain reaction,qRT-PCR)检测16例食管鳞癌组织(食管癌组)和配对癌旁组织(癌旁组织组)、5例食管活检黏膜上皮组织(正常对照组)以及人食管上皮细胞系HEEC、食管鳞癌细胞系EC-109、EC-9706和KYSE-150中P5848的表达水平;采用质粒转染使P5848在食管鳞癌细胞系中过表达;同时应用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)比色法及磺酰罗丹明B(sulforhodamine B,SRB)比色法检测细胞增殖率;碘化丙啶细胞周期检测试剂盒检测细胞周期变化情况;Caspase-3活性试剂盒检测细胞凋亡情况;应用qRT-PCR及蛋白质印迹法检测Bcl-2、p53及Caspase-3的mRNA与蛋白质的表达变化。结果食管鳞癌组织中P5848表达量为0.567(0.483,0.667),低于癌旁组织的0.975(0.529,1.317)和正常对照组的1.007(0.772,1.150),差异均有统计学意义(Z=-9.688,P=0.011;Z=-13.4,P=0.015)。EC-109、EC-9706和KYSE-150细胞中P5848的表达水平分别为0.562±0.056、0.509±0.095和0.629±0.118,与HEEC细胞(1.054±0.200)相比降低,差异有统计学意义(F=4.605,P=0.005;F=5.111,P=0.002;F=3.963,P=0.007)。质粒转染过表达P5848可上调食管鳞癌细胞中P5848的表达水平,过表达P5848后,MTT和SRB检测结果显示,3种细胞的增殖能力降低(F值分别为9.546、13.260、7.394和3.838、9.705、4.678,均P<0.01)。过表达P5848降低了S期细胞比率(F值分别为4.673、3.189和4.146,均P<0.05),同时增加G1期细胞比率(F值分别为8.217、6.944和5.074,均P<0.01),提示P5848可能通过抑制细胞从G1期向S期转换,从而抑制食管鳞癌细胞增殖能力。此外,P5848过表达可使细胞凋亡蛋白Caspase-3和p53的表达增加,抗凋亡蛋白Bcl-2表达减少,均P<0.05。结论过表达P5848能够引起食管鳞癌细胞生物学行为的改变,抑制细胞增殖诱导其凋亡,从而在一定程度上起到抑制肿瘤发生、发展的作用。

OBJECTIVE Recent studies have shown that long non-coding RNA(lncRNA)is closely related with the occurrence and development of esophageal squamous cell carcinoma(ESCC),which provides new target for the exploration of the diagnosis and treatment.The present study is aimed to examine the expression of lncRNA P5848 in ESCC tissues and cells,and explore the effect and mechanism of lncRNAP5848 on cell proliferation.METHODS Firstly,the expression of P5848 in 16 cases of ESCC(ESCC tissues and adjacent normal esophageal tissues),5 cases of esophageal biopsy mucosal epithelial tissues(normal control group),human esophageal epithelial cell line HEEC and esophageal squamous cell line EC-109,EC-9706 and KYSE-150 were detected by real-time quantitative PCR(qRT-PCR).The over-expression of P5848 in ESCC cell lines was regulated by plasmid transfection.Meanwhile,the cell proliferation rate was detected by MTT and SRB methods.The change of cell cycle was detected by propidium iodide cell cycle detection kit,and apoptosis was detected by caspase-3 activity kit.The change of expression in Bcl-2,p53 and caspase-3 mRNA and protein were detected by qRT-PCR and Western blot.RESULTS The expression of P5848 in ESCC tissues was 0.567(0.483,0.667),significantly lower than that in adjacent tissues[0.975(0.529,1.317)]and normal tissues[1.007(0.772,1.150)],and there was significant difference(Z=-9.688,P=0.011;Z=-13.4,P=0.015).Compared with HEEC cells(1.054±0.200),the expression of P5848 in EC-109,EC-9706,and KYSE-150 cells was 0.562±0.056,0.509±0.095 and 0.629±0.118 respectively,and there was significant difference(F=4.605,P=0.005;F=5.111,P=0.002;F=3.963,P=0.007).Overexpression of P5848 by plasmid transfection significantly upregulated the expression of P5848 mRNA in ESCC,and its overexpression significantly inhibited the proliferation status of esophageal cancer cells(Fvalue was 9.546,13.260,7.394 and3.838,9.705,4.678 respectively,P<0.01).The S-phase cell ratio was reduced(Fvalue was4.673,3.189,4.146;P<0.05),while the G1 phase cell ratio increased(Fvalue was 8.217,6.944,5.074;P<0.01),which suggested that P5848 may inhibit the proliferation of esophageal cancer cells by inhibiting the cells from transitioning from G1 to S phase.In addition,P5848 overexpression can significantly increase apoptosis(Bcl-2 mRNA and protein expression decreasing,Caspase-3 and p53 mRNA and protein expression increasing).CONCLUSIONS Overexpression of P5848 can lead to the change of biological behavior of ESCC,inhibit its proliferation and increase its apoptosis.Therefore,it plays a role in inhibiting the occurrence and development of tumors to a certain extent.