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

1	钱桂生. 肺癌不同病理类型发病率的变化情况及其原因[J/CD]. 中华肺部疾病杂志(电子版), 2011, 4(1): 1-5.
2	Lu H, Zhang H, Wei Y, et al. Ambient mass spectrometry for molecular diagnosis of lung cancer[J]. Analyst, 2019, 145(2): 313-320.
3	Zhong Y, Gao T, Yuan X, et al. Targeting BRD4 proteins suppresses the growth of NSCLC through downregulation of eIF4E expression[J]. Cancer Biol Therapy, 2018, 19(5): 407-415.
4	刘蕾，魏素菊. KRAS突变的非小细胞肺癌的研究进展[J]. 中国肺癌杂志，2018, 21(5): 419-424.
5	侯宛昕，李和根，朱丽华. 中医药防治非小细胞肺癌术后复发转移概况[J]. 湖南中医杂志，2014, 30(4): 183-186.
6	Rangel D, Rojas , Irmgard , et al. Hypoxia-inducible factor 1α protects peripheral sensory neurons from diabetic peripheral neuropathy by suppressing accumulation of reactive oxygen species.[J]. J Molecul Med, 2018, 96(12): 1395-1405.
7	刘瑞清. 缺氧诱导因子-1α参与肝纤维化形成机制研究进展[J]. 实用肝脏病杂志，2021, 24(1): 145-148.
8	Khuc T, Hsu CWA, Sakamuru S, et al. Using β-lactamase and nanoLuc luciferase reporter gene assays to identify inhibitors of the HIF-1 signaling pathway[J]. Other, 2016, 1473: 23-31.
9	Lin MC, Lin JJ, Hsu CL, et al. GATA3 interacts with and stabilizes HIF-1α to enhance cancer cell invasiveness[J]. Oncogene, 2017, 36(30): 4243-4252.
10	Zhao Y, Wang XX, Wu W, et al. EZH2 regulates PD-L1 expression via HIF-1α in non-small cell lung cancer cells[J]. Biochem Biophys Res Commun, 2019, 517(2): 201-209.
11	Hou P, Shi P, Jiang T, et al. DKC1 enhances angiogenesis by promoting HIF-1α transcription and facilitates metastasis in colorectal cancer[J]. Br J Cancer, 2020, 122(5): 668-679.
12	赵志成，庄莉，贾长库，等. EGCG抑制肝癌细胞株HepG2增殖及HIF-1α/VEGF的表达[J]. 中国病理生理杂志，2010, 26(4): 713-720.
13	唐静，贾昱娴，农丽，等. MALAT-1和HIF-1α在三阴型乳腺癌中表达及临床意义[J]. 重庆医学，2019, 48(10): 28-31.
14	Wenming F, Tao X, Sanxiong H, et al. HIF-1α promotes the migration and invasion of hepatocellular carcinoma cells via the IL-8-NF-κB axis[J]. Cell Molecul Biol Lett, 2018, 23(1): 26-36.
15	Lai HH, Li JN, Wang MY, et al. HIF-1α promotes autophagic proteolysis of Dicer and enhances tumor metastasis[J]. J Clin Invest, 2018, 128(2): 625-643.
16	焦言，杨文锋. 缺氧诱导因子-1与恶性肿瘤的相关性研究进展[J]. 中国实用医刊，2018, 45(15): 125-128.
17	马苑，付秀华，王立红. 肿瘤缺氧微环境的研究进展[J]. 癌症进展，2020, 18(2): 7-10+45.
18	Xu M, Zheng YL, Xie XY, et al. Sorafenib blocks the HIF-1α/VEGFA pathway, inhibits tumor invasion, and induces apoptosis in hepatoma cells[J]. DNA Cell Biol, 2014, 33(5): 275-281.
19	Zhang K, Han ES, Dellinger TH, et al. Cinnamon extract reduces VEGF expression via suppressing HIF-1α gene expression and inhibits tumor growth in mice[J]. Molecul Carcinogen, 2016, 56(2): 436-446.
20	Xiao H, Tong R, Ding C, et al. γ-H2AX promotes hepatocellular carcinoma angiogenesis via EGFR/HIF-1α/VEGF pathways under hypoxic condition[J]. Oncotarget, 2015, 6(4): 2180-2192.
21	Sun QY, Ding LW, Johnson K, et al. SOX7 regulates MAPK/ERK-BIM mediated apoptosis in cancer cells[J]. Oncogen, 2019, 38(34): 6196-6210.
22	Su X, Shen Z, Yang Q, et al. Vitamin C kills thyroid cancer cells through ROS-dependent inhibition of MAPK/ERK and PI3K/AKT pathways via distinct mechanisms[J]. Theranost, 2019, 9(15): 4461-4473.
23	Jiang XL, Gao JC, Jiang L, et al. [Expression and significance of MAPK/ERK in the specimens and cells of epithelial ovarian cancer][J]. Zhonghua Fu Chan Ke Za Zhi, 2019, 54(8): 541-547.
24	Costa RLB, Han HS, Gradishar WJ. Targeting the PI3K/AKT/mTOR pathway in triple-negative breast cancer: a review[J]. Breast Cancer Res Treat, 2018, 169(3): 397-406.
25	Lin YT, Wang HC, Hsu YC, et al. Capsaicin induces autophagy and apoptosis in human nasopharyngeal carcinoma cells by downregulating the PI3K/AKT/mTOR pathway[J]. Int J Molecul Ences, 2017, 18(7): 1343-1355.
26	Dong P, Hao F, Dai S, et al. Combination therapy Eve and Pac to induce apoptosis in cervical cancer cells by targeting PI3K/AKT/mTOR pathways[J]. J Recept Sign Transd, 2018, 38(1): 83-88.

[1]	周荷妹, 金杰, 叶建东, 夏之一, 王进进, 丁宁. 罕见成人肋骨郎格汉斯细胞组织细胞增生症被误诊为乳腺癌术后骨转移一例[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 380-383.
[2]	周世振, 朱兴亚, 袁庆港, 刘理想, 王凯, 缪骥, 丁超, 汪灏, 管文贤. 吲哚菁绿荧光成像技术在腹腔镜直肠癌侧方淋巴结清扫中的应用效果分析[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 44-47.
[3]	高杰红, 黎平平, 齐婧, 代引海. ETFA和CD34在乳腺癌中的表达及与临床病理参数和预后的关系研究[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 64-67.
[4]	贺斌, 马晋峰. 胃癌脾门淋巴结转移危险因素[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 694-699.
[5]	张志兆, 王睿, 郜苹苹, 王成方, 王成, 齐晓伟. DNMT3B与乳腺癌预后的关系及其生物学机制[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 624-629.
[6]	韩婧, 郝少龙, 康骅. 北京市单中心甲状腺癌患者临床特征的回顾分析[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(05): 490-493.
[7]	林逸, 钟文龙, 李锴文, 何旺, 林天歆. 广东省医学会泌尿外科疑难病例多学科会诊（第15期）——转移性膀胱癌的综合治疗[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 648-652.
[8]	杨慧, 郭丽娟, 冯晓丹, 李静, 黄成谋, 蔡兴锐, 覃英娇, 王远礼. 非小细胞肺癌铂类药物耐药mi RNA表达特征及预测分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 719-724.
[9]	赖淼, 景鑫, 李桂珍, 李怡. 非小细胞肺癌EGFR 突变亚型的临床病理和预后意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 731-737.
[10]	梁丽斯, 李洁, 贺帅, 来艳君, 刘铭, 张琳. MMP-9、MMP-2 及TLR4、HE4对非小细胞肺癌早期诊断意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 756-761.
[11]	刘敏思, 李荣, 李媚. 基于GGT与Plt比值的模型在HBV相关肝细胞癌诊断中的作用[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(06): 831-835.
[12]	陆镜明, 韩大为, 任耀星, 黄天笑, 向俊西, 张谞丰, 吕毅, 王傅民. 基于术前影像组学的肝内胆管细胞癌淋巴结转移预测的系统性分析[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(06): 852-858.
[13]	张润锦, 阳盼, 林燕斯, 刘尊龙, 刘建平, 金小岩. EB病毒相关胆管癌伴多发转移一例及国内文献复习[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(06): 865-869.
[14]	刘郁, 段绍斌, 丁志翔, 史志涛. miR-34a-5p 在结肠癌患者的表达及其与临床特征及预后的相关性研究[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 485-490.
[15]	王曦娅, 尹弘青, 丁伟, 徐滨, 于海源, 马东升, 邵军. 桥本背景下甲状腺乳头状癌多参数分析预测大容量淋巴结转移[J/OL]. 中华临床医师杂志(电子版), 2024, 18(06): 548-554.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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