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口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)属于头颈部常见恶性肿瘤之一[1]。临床主要采用手术与化疗等手段治疗OSCC,但由于缺乏早期诊断OSCC的有效标志物导致大部分病人确诊时已处于中晚期,目前关于OSCC发生及转移机制尚未完全阐明。长链非编码RNA(long non-coding RNA,LncRNA)可调控基因转录、翻译等生物学过程,LncRNA表达异常与多种肿瘤发生发展密切相关[2-3]。研究[4]表明长链非编码RNA PCBP1-AS1(long non-coding RNA PCBP1-AS1,LncRNA PCBP1-AS1)可抑制外阴鳞癌细胞增殖、侵袭及迁移并促进其凋亡。近来报道指出PCBP1-AS1在口腔鳞癌中呈低表达,但关于其具体作用机制尚未阐明[5]。Janus激酶2(JAK2)/信号转导与转录激活因子3(STAT3)信号通路可参与细胞增殖、凋亡等多种生物学过程,JAK2可促使STAT3活化从而促进肿瘤发生发展[6-7]。但PCBP1-AS1是否可通过调控JAK2/STAT3信号通路而参与OSCC发生发展进程尚需进一步研究。本研究主要探讨PCBP1-AS1在OSCC细胞中的表达状态及其对细胞增殖、侵袭及凋亡的影响,初步探究其对JAK2/STAT3信号通路的调控作用,旨在为揭示OSCC发生发展机制奠定理论基础。
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与正常口腔上皮细胞HOK相比,口腔鳞癌细胞CAL-27、Tca8113、KB中PCBP1-AS1的表达水平显著降低(P < 0.01),PCBP1-AS1在口腔鳞癌CAL-27细胞中的表达水平相对降低,因而选用口腔鳞癌CAL-27细胞进行后续研究(见表 1)。
分组 PCBP1-AS1表达量 HOK 1.00±0.15 CAL-27 0.28±0.04** Tca8113 0.31±0.05** KB 0.33±0.03** F 52.63 P < 0.01 MS组内 0.007 LSD-t检验:与正常口腔上皮细胞HOK比较**P < 0.01 表 1 qRT-PCR检测口腔鳞癌细胞株中PCBP1-AS1的表达(x±s;ni=3)
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pcDNA-control组与pcDNA-PCBP1-AS1组细胞中PCBP1-AS1表达量分别为1.00±0.13和3.05±0.38,差异有统计学意义(P < 0.01)。相较于pcDNA-control组,pcDNA-PCBP1-AS1组口腔鳞癌CAL-27细胞增殖活力显著降低(P < 0.01)(见表 2)。
分组 PCBP1-AS1表达量 细胞增殖(OD值) F P MS组内 24 h 48 h 72 h pcDNA-control 1.00±0.13 0.50±0.06 0.74±0.07 1.05±0.12 19.51 < 0.01 0.010 pcDNA-PCBP1-AS1 3.05±0.38 0.36±0.04** 0.42±0.06** 0.53±0.05** 135.10 < 0.01 0.038 t 8.84 3.36 6.01 6.93 — — — P < 0.01 < 0.05 < 0.01 < 0.01 — — — LSD-t检验:与PCBP1-AS1表达量比较**P < 0.01 表 2 PCBP1-AS1过表达对CAL-27细胞增殖的影响(x±s;ni=3)
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实验结果显示,与pcDNA-control组相比,pcDNA-PCBP1-AS1组口腔鳞癌CAL-27细胞侵袭数显著减少(P < 0.01)(见图 1、表 3)。
分组 细胞侵袭数/个 pcDNA-control 115.67±12.36 pcDNA-PCBP1-AS1 61.53±6.59 t 6.69 P < 0.01 表 3 PCBP1-AS1过表达对CAL-27细胞侵袭的影响(x±s;ni=3)
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实验结果显示,相较于pcDNA-control组,pcDNA-PCBP1-AS1组口腔鳞癌CAL-27细胞凋亡率显著增加(P < 0.01)(见图 2、表 4)。
分组 凋亡率 pcDNA-control 3.15±0.29 pcDNA-PCBP1-AS1 38.62±3.57 t 17.15 P < 0.01 表 4 PCBP1-AS1过表达对CAL-27细胞凋亡的影响(x±s;ni=3)
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与pcDNA-control组相比,pcDNA-PCBP1-AS1组口腔鳞癌CAL-27细胞中CDK1、MMP-2、Bcl2蛋白表达水平显著降低(P < 0.01),Bax蛋白表达水平显著升高(P < 0.01)(见图 3、表 5)。
分组 CDK1 MMP-2 Bax Bcl2 pcDNA-control 1.00±0.12 1.00±0.15 1.00±0.11 1.00±0.16 pcDNA-PCBP1-AS1 0.31±0.05 0.28±0.03 3.56±0.38 0.30±0.03 t 9.19 8.15 11.21 7.45 P < 0.01 < 0.01 < 0.01 < 0.01 表 5 PCBP1-AS1过表达对CAL-27细胞增殖、侵袭和凋亡相关蛋白表达的影(x±s;ni=3)
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实验结果显示,相比于pcDNA-control组,pcDNA-PCBP1-AS1组口腔鳞癌CAL-27细胞中p-JAK2、p-STAT3蛋白表达水平显著降低(P < 0.01),JAK2、STAT3蛋白表达水平无明显变化(P>0.05)(见图 4、表 6)。
分组 p-JAK2 JAK2 p-STAT3 STAT3 pcDNA-control 1.00±0.13 1.00±0.15 1.00±0.12 1.00±0.18 pcDNA-PCBP1-AS1 0.31±0.04 0.96±0.12 0.28±0.03 1.04±0.11 t 8.79 0.36 10.08 0.33 P < 0.01 >0.05 < 0.01 >0.05 表 6 PCBP1-AS1对CAL-27细胞JAK2/STAT3信号通路相关蛋白表达的影响(x±s;ni=3)
LncRNA PCBP1-AS1对口腔鳞癌细胞增殖、侵袭和凋亡的影响
Effect of LncRNA PCBP1-AS1 on the proliferation, invasion and apoptosis of oral squamous carcinoma cells
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摘要:
目的探讨长链非编码RNA PCBP1-AS1(LncRNA PCBP1-AS1)对口腔鳞状细胞癌(OSCC)细胞增殖、侵袭及凋亡的影响及其可能机制。 方法采用实时荧光定量聚合酶链反应检测OSCC细胞中PCBP1-AS1的表达水平;采用Lipofectamine 2000将pcDNA-PCBP1-AS1及pcDNA-control转染入口腔鳞癌CAL-27细胞;甲基噻唑基四唑实验检测CAL-27细胞的增殖能力;Transwell实验检测细胞的侵袭能力;流式细胞术检测细胞的凋亡率;蛋白免疫印迹法检测细胞周期依赖蛋白激酶1(CDK1)、基质金属蛋白酶-2(MMP-2)、B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)、Janus激酶2(JAK2)/信号转导与转录激活因子3(STAT3)信号通路相关蛋白表达。 结果与正常口腔上皮细胞比较,OSCC细胞CAL-27、Tca8113、KB中PCBP1-AS1的表达水平显著降低(P < 0.01);PCBP1-AS1过表达可显著抑制CAL-27细胞的增殖、侵袭(P < 0.01),促进细胞凋亡(P < 0.01);PCBP1-AS1过表达可显著抑制CDK1、MMP-2、Bcl2、p-JAK2、p-STAT3的蛋白表达(P < 0.01),而促进Bax的蛋白表达(P < 0.01)。 结论PCBP1-AS1过表达可抑制OSCC细胞增殖、侵袭,诱导细胞凋亡,其作用可能与抑制JAK2/STAT3信号通路激活有关。 -
关键词:
- 口腔鳞状细胞癌 /
- LncRNA PCBP1-AS1 /
- 增殖 /
- 侵袭 /
- 凋亡 /
- JAK2/STAT3信号通路
Abstract:ObjectiveTo investigate the effects of long-chain non-coding RNA PCBP1-AS1(LncRNA PCBP1-AS1)on the proliferation, invasion and apoptosis of oral squamous carcinoma cells(OSCC)cells, and its possible mechanism. MethodsThe expression level of PCBP1-AS1 in OSCC cells was detected using Real-time quantitative polymerase chain reaction(qRT-PCR).The pcDNA-PCBP1-AS and pcDNA-control were transfected into the luminal squamous cell carcinoma CAL-27 cells using Lipofectamine 2000.The proliferation ability, invasive ability and apoptotic rate of CAL-27 cells were detected using MTT assay, Transwell experiments and flow cytometry, respectively. ResultsCompared with the normal oral epithelial cells, the expression levels of PCBP1-AS1 in oral squamous cell carcinoma cells CAL-27, Tca8113, and KB significantly decreased(P < 0.01).The overexpression of PCBP1-AS1 could significantly inhibit the proliferation and invasion of CAL-27 cells(P < 0.01), and promote apoptosis of cells(P < 0.01).The overexpression of PCBP1-AS1 could significantly inhibit the protein expression levels of CDK1, MMP-2, Bcl2, p-JAK2 and p-STAT3(P < 0.01), and promote the protein expression of Bax(P < 0.01). ConclusionsThe overexpression of PCBP1-AS1 can inhibit the proliferation and invasion of OSCC cells, and induce its apoptosis, and the mechanism of which may be related to the inhibition of JAK2/STAT3 signaling pathway activation. -
表 1 qRT-PCR检测口腔鳞癌细胞株中PCBP1-AS1的表达(x±s;ni=3)
分组 PCBP1-AS1表达量 HOK 1.00±0.15 CAL-27 0.28±0.04** Tca8113 0.31±0.05** KB 0.33±0.03** F 52.63 P < 0.01 MS组内 0.007 LSD-t检验:与正常口腔上皮细胞HOK比较**P < 0.01 表 2 PCBP1-AS1过表达对CAL-27细胞增殖的影响(x±s;ni=3)
分组 PCBP1-AS1表达量 细胞增殖(OD值) F P MS组内 24 h 48 h 72 h pcDNA-control 1.00±0.13 0.50±0.06 0.74±0.07 1.05±0.12 19.51 < 0.01 0.010 pcDNA-PCBP1-AS1 3.05±0.38 0.36±0.04** 0.42±0.06** 0.53±0.05** 135.10 < 0.01 0.038 t 8.84 3.36 6.01 6.93 — — — P < 0.01 < 0.05 < 0.01 < 0.01 — — — LSD-t检验:与PCBP1-AS1表达量比较**P < 0.01 表 3 PCBP1-AS1过表达对CAL-27细胞侵袭的影响(x±s;ni=3)
分组 细胞侵袭数/个 pcDNA-control 115.67±12.36 pcDNA-PCBP1-AS1 61.53±6.59 t 6.69 P < 0.01 表 4 PCBP1-AS1过表达对CAL-27细胞凋亡的影响(x±s;ni=3)
分组 凋亡率 pcDNA-control 3.15±0.29 pcDNA-PCBP1-AS1 38.62±3.57 t 17.15 P < 0.01 表 5 PCBP1-AS1过表达对CAL-27细胞增殖、侵袭和凋亡相关蛋白表达的影(x±s;ni=3)
分组 CDK1 MMP-2 Bax Bcl2 pcDNA-control 1.00±0.12 1.00±0.15 1.00±0.11 1.00±0.16 pcDNA-PCBP1-AS1 0.31±0.05 0.28±0.03 3.56±0.38 0.30±0.03 t 9.19 8.15 11.21 7.45 P < 0.01 < 0.01 < 0.01 < 0.01 表 6 PCBP1-AS1对CAL-27细胞JAK2/STAT3信号通路相关蛋白表达的影响(x±s;ni=3)
分组 p-JAK2 JAK2 p-STAT3 STAT3 pcDNA-control 1.00±0.13 1.00±0.15 1.00±0.12 1.00±0.18 pcDNA-PCBP1-AS1 0.31±0.04 0.96±0.12 0.28±0.03 1.04±0.11 t 8.79 0.36 10.08 0.33 P < 0.01 >0.05 < 0.01 >0.05 -
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