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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (1/2): 58-71    DOI: 10.13523/j.cb.2107052
研究报告     
UPF1在乳腺癌细胞中的表达与作用的研究*
胡凯,胡静,孙子久,刘施妍,廖德宇,余伙梅,张彦()
重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆 400016
Effects of UPF1 on the Proliferation, Migration and Invasion of Breast Cancer Strains MDA-MB-231 and MCF-7
HU Kai,HU Jing,SUN Zi-jiu,LIU Shi-yan,LIAO De-yu,YU Huo-mei,ZHANG Yan()
Key Laboratory of Clinical Laboratory Diagnosis, Ministry of Education, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
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摘要：

目的:观察UPF1在乳腺癌中的表达,对人乳腺癌细胞MDA-MB-231增殖、迁移和侵袭的影响,及其可能的作用机制。方法:使用生物信息学方法分析UPF1在乳腺癌组织中的表达及作用,构建UPF1小干扰RNA(siRNA)并转染乳腺癌MDA-MB-231和MCF-7细胞株,构建外源性的UPF1低表达的重组细胞,通过实时荧光定量PCR法和蛋白质印迹法检测重组细胞中UPF1的表达水平;CCK-8法检测细胞的增殖;划痕愈合实验及Transwell小室法分别检测细胞横向和纵向迁移以及侵袭能力;实时荧光定量PCR法检测基质金属蛋白酶9(matrix metalloproteinase,MMP9)以及上皮-间质转化(epithelial-mesenchymal transition,EMT)标志物E-钙黏着蛋白(E-cadherin)、波形蛋白(vimentin)mRNA表达量的变化;蛋白质印迹法检测MMP2、E-cadherin、Vimentin蛋白表达量的变化。结果:生物信息学分析表明UPF1在乳腺癌肿瘤组织中高表达,与免疫细胞浸润相关,并与抑癌基因表达呈正相关,mRNA水平进一步验证UPF1在乳腺癌细胞中高表达,敲低UPF1后,乳腺癌细胞MDA-MB-231和MCF-7中UPF1的mRNA和蛋白质表达水平均显著下降(P<0.05,P<0.05),MDA-MB-231和MCF-7 细胞的体外增殖能力显著增强(P<0.0001,P<0.05),迁移(P<0.05,P<0.001)与侵袭能力(P<0.01,P<0.01)也显著提升,实时荧光定量PCR、Western blot 结果显示UPF1可抑制 EMT 相关通路。结论:UPF1在乳腺癌中高表达,但是UPF1在乳腺癌中可能起抑癌作用,UPF1可能通过抑制 EMT 通路抑制乳腺癌 MDA-MB-231和MCF-7 细胞的增殖、迁移与侵袭。
关键词： 乳腺癌;  UPF1;  增殖;  迁移;  侵袭;  EMT    
Abstract:

Objective: To investigate the effects of UPF1 on the proliferation, migration and invasion of human breast cancer cells MDA-MB-231 and its possible mechanism. Methods: The role of UPF1 in breast cancer and the expression level of UPF1 in pan-cancer and breast cancer was evaluated by bioinformatics analysis. Breast cancer cells MDA-MB-231 and MCF-7 were transfected with siRNA. The experiment was divided into two groups: control group (transfected with siRNA negative control) and treatment group (transfected with siUPF1). The mRNA and protein levels of UPF1, MMP9, EMT-related makers were measured by qRT-PCR and Western blot; the proliferation of MDA-MB-231 and MCF-7 cells was detected by CCK-8 assay; the lateral migration ability was studied by wound healing assay; the longitudinal migration and invasion abilities were evaluated by Transwell migration and invasion assays. Results: Bioinformatics analysis showed that UPF1 was highly expressed in breast cancer and associated with immune cell infiltration processes and positively correlated with tumor suppressor genes. UPF1 was also highly expressed in breast cancer cells. After knockdown of UPF1, the mRNA and protein expression levels of UPF1 in MDA-MB-231 and MCF-7 breast cancer cells were significantly decreased (P<0.05, P<0.05). Furthermore, the proliferation, migration and invasion abilities of MDA-MB-231 and MCF-7 cells were significantly enhanced. The mRNA and protein levels of MMP9 and Vimentin were increased, but E-cadherin decreased. Conclusion: UPF1 is highly expressed but plays a cancer-inhibiting role in breast cancer. UPF1 may inhibit the proliferation, migration and invasion of breast cancer MDA-MB-231 and MCF-7 cells by inhibiting the EMT signaling pathway.
Key words: Breast cancer    UPF1    Proliferation    Migration    Invasion    EMT
收稿日期: 2021-07-23 出版日期: 2022-03-03
ZTFLH:  Q813  
基金资助: * 国家自然科学基金资助项目(81974449)
通讯作者: 张彦     E-mail: yanzhang@cqmu.edu.cn
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胡凯
胡静
孙子久
刘施妍
廖德宇
余伙梅
张彦

引用本文:

胡凯,胡静,孙子久,刘施妍,廖德宇,余伙梅,张彦. UPF1在乳腺癌细胞中的表达与作用的研究*[J]. 中国生物工程杂志, 2022, 42(1/2): 58-71.

HU Kai,HU Jing,SUN Zi-jiu,LIU Shi-yan,LIAO De-yu,YU Huo-mei,ZHANG Yan. Effects of UPF1 on the Proliferation, Migration and Invasion of Breast Cancer Strains MDA-MB-231 and MCF-7. China Biotechnology, 2022, 42(1/2): 58-71.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2107052    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I1/2/58

Name Sequence
siRNA negative control (siNC) Forward primer(5'→3'): UUCUCCGAACGUGUCACGUdTdT
Reverse primer(5'→3'): ACGUGACACGUUCGGAGAATTdTdT
siUPF1 Forward primer(5'→3'): GCAAGAAGUGGUUCUGCAAdTdT
Reverse primer(5'→3'): UUGCAGAACCACUUCUUGCdTdT
E-cadherin Forward primer(5'→3'): TTAATCCGGACACTGGTGCC
Reverse primer(5'→3'): AGATGAGGCCCCCTTACCAT
Vimentin Forward primer(5'→3'): CAGGAAATCCAGGAGCTGCA
Reverse primer(5'→3'): ACCATTCTTCTGCCTCCTGC
MMP9 Forward primer(5'→3'): CCCTTGTGCTCTTCCCTGGA
Reverse primer(5'→3'): TCTGCCACCCGAGTGTAACC
GAPDH Forward primer(5'→3'): CAGCGACACCCACTCCTC
Reverse primer(5'→3'): TGAGGTCCACCACCCTGT
表1  实时荧光定量PCR引物序列
图1  UPF1在乳腺癌组织中表达上调
图2  UPF1与免疫浸润相关
图3  UPF1与NMD相关分子及抑癌基因呈正相关
图4  UPF1在乳腺癌MDA-MB-231和MCF-7细胞中成功敲低
图5  UPF1抑制乳腺癌细胞的增殖
图6  UPF1抑制乳腺癌细胞的迁移
图7  UPF1抑制乳腺癌细胞的侵袭
图8  UPF1抑制EMT信号通路
图9  UPF1调控EMT的潜在途径
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