沉默HIF-1α调控MAPK/ERK信号通路抑制NSCLC转移的分析

doi:10.3877/cma.j.issn.1674-6902.2021.05.006

目的

探讨HIF-1α在体内及体外对非小细胞肺癌(NSCLC)细胞转移的影响及其作用机制。

方法

采用实时荧光定量PCR(RT-qPCR)检测HIF-1α在癌旁正常组织、肺鳞癌组织(LUSC)、肺腺癌组织(LUAD)、A549细胞和HEB细胞中的表达量。上调HIF-1α表达后，通过MTT、细胞侵袭和Western blot实验检测过表达HIF-1α对A549细胞增殖、侵袭和EMT的影响。下调HIF-1α表达后，Western blot检测A549细胞ERK和p-ERK蛋白的表达量。THBQ激活MAPK/ERK通路后，分析A549细胞增殖、侵袭和EMT能力。将细胞株植入裸鼠体内，构建移植瘤模型，最后测量裸鼠移植瘤的体积和重量。

结果

HIF-1α在NSCLC组织的表达水平明显高于癌旁正常组织(P<0.05)。A549细胞中HIF-1α表达水平明显高于HEB细胞(P<0.01)，过能够促进A549细胞的增殖和侵袭，N-cadherin蛋白表达量明显上升，E-cadherin蛋白表达量明显下降。下调HIF-1α能够抑制A549细胞内MAPK/ERK通路，且抑制了A549细胞的增殖、侵袭和EMT。下调HIF-1α使裸鼠移植瘤的重量和体积明显减小。

结论

沉默HIF-1α通过MAPK/ERK信号通路在体内及体外抑制NSCLC转移。

关键词: HIF-1α, MAPK/ERK, 转移, 非小细胞肺癌

Objective

To investigate the effects of HIF-1α on metastasis of non-small cell lung cancer and its mechanism in vitro and in vivo.

Methods

Quantitative real-time PCR (RT-qPCR) was used to detect the expression of HIF-1αin normal paracancer tissues (Normal), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), A549 celland HEB cell. HIF-1α expression was upregulated, and the effects of overexpression of HIF-1α on the proliferation, invasion, and EMT of A549 cells were detected by MTT, cell invasion, and Western blot. HIF-1α expression was down-regulated, ERK and p-ERK protein expressions in A549 cells were detected by Western blot. After THBQactivated the MAPK/ERK pathway, the proliferation, invasion and EMT abilities of A549 cells were analyzed. Cell lines were implanted into nude mice to construct a xenograft model. And the volume and weight of the xenograft tumor were measured.

Results

HIF-1α expression in non-small cell lung cancer tissues was significantly higher than that in adjacent normal tissues(P<0.05). The expression level of HIF-1α in A549 cells was significantly higher than that in HEB cells(P<0.01). Overexpression of HIF-1α promoted the proliferation and invasion of A549 cells, and the expression level of N-cadherin protein is significantly increased, while that of E-cadherin is significantly decreased. Down-regulation of HIF-1α inhibited the MAPK/ERK pathway in A549 cells and inhibited the proliferation, invasion and EMT of A549 cells. Downregulation of HIF-1α significantly reduced the weight and volume of transplanted tumors in nude mice.

Conclusion

Silencing HIF-1α inhibited metastasis of non-small cell lung cancer in vivo and in vitro via the MAPK/ERK signaling pathway.

Key words: HIF-1α, MAPK/ERK, Metastasis, Non-small cell lung cancer

表1 引物序列

引　物	序列(5′-3′)
HIF-1α-F	AGAAACCACCTATGACCTGC
HIF-1α-R	GTCGTGCTGAATAATACCACTC
GAPDH-F	GCACCGTCAAGGCTGAGAAC
GAPDH-R	TGGTGAAGACGCCAGTGGA

表1 引物序列

引　物	序列(5′-3′)
HIF-1α-F	AGAAACCACCTATGACCTGC
HIF-1α-R	GTCGTGCTGAATAATACCACTC
GAPDH-F	GCACCGTCAAGGCTGAGAAC
GAPDH-R	TGGTGAAGACGCCAGTGGA

图1 HIF-1α在NSCLC组织和细胞中表达上调；注：A：RT-qPCR检测HIF-1α在NSCLC组织和癌旁正常组织中的表达量；B：RT-qPCR检测HIF-1α在HEB和A549细胞中的表达量

图2 过表达HIF-1α促进A549细胞增殖、侵袭和EMT；注：A：RT-qPCR检测过表达HIF-1α后A549细胞中HIF-1α的表达量；B：MTT检测过表达HIF-1α对A549细胞增殖的影响；C：A549细胞的侵袭数目；D：细胞侵袭实验检测过表达HIF-1α对A549细胞侵袭的影响(×200)；E：过表达HIF-1α对A549细胞EMT的影响；F：E-cadherin、N-cadherin蛋白的相对表达量

图3 下调HIF-1α抑制了A549细胞内MAPK/ERK信号通路；注：A：RT-qPCR检测下调HIF-1α后A549细胞中HIF-1α的表达量；B：Western blot检测下调HIF-1α对A549细胞MAPK/ERK信号通路的影响；C：p-ERK/ERK比值

图4 下调HIF-1α通过MAPK/ERK信号通路抑制A549细胞增殖、侵袭和EMT；注：A：Western blot检测TBHQ作用A549细胞后对MAPK/ERK信号通路的影响；B：MTT检测下调HIF-1α通过MAPK/ERK信号通路对A549细胞增殖的影响；C：A549细胞侵袭数目；D：细胞侵袭实验检测下调HIF-1α通过MAPK/ERK信号通路对A549细胞侵袭的影响(×200)；E：Western blot检测下调HIF-1α通过MAPK/ERK信号通路对A549细胞EMT的影响；F：E-cadherin、N-cadherin蛋白的相对表达量

表2 移植瘤体积、重量比较(±s)

组　别	数量(只)	体积(cm³)	重量(g)
Control组	20	0.71±0.83	1.15±0.14
siRNA NC组	20	0.68±0.92	1.18±0.28
HIF-1αsiRNA组	20	0.26±0.74	0.37±0.19
HIF-1αsiRNA+THBQ组	20	0.67±0.57	1.09±0.32

表2 移植瘤体积、重量比较(±s)

组　别	数量(只)	体积(cm³)	重量(g)
Control组	20	0.71±0.83	1.15±0.14
siRNA NC组	20	0.68±0.92	1.18±0.28
HIF-1αsiRNA组	20	0.26±0.74	0.37±0.19
HIF-1αsiRNA+THBQ组	20	0.67±0.57	1.09±0.32

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Silencing HIF-1α regulates MAPK/ERK pathway and inhibits metastasis of NSCLC

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Silencing HIF-1α regulates MAPK/ERK pathway and inhibits metastasis of NSCLC