HLA-E基因表达沉默对人乳腺癌细胞生物学行为的影响

doi:10.3969/j.issn.1004-616x.2023.01.003

摘要/Abstract

摘要： 目的: 探讨HLA-E基因表达对人乳腺癌细胞增殖、凋亡、侵袭、迁移等生物学行为的影响，分析靶向HLA-E基因的小分子RNA干扰(siRNA)技术应用于乳腺癌治疗的可行性。方法: 设计并合成靶向HLA-E基因的特异性siRNA序列，转染至人乳腺癌细胞MDA-MB-231，同时设立空白对照组、阴性对照组及脂质体组。实时荧光定量PCR(qPCR)和Western blot法分别检测各组乳腺癌细胞HLA-E mRNA及蛋白的表达；CCK-8法和流式细胞术检测各组乳腺癌细胞增殖率和凋亡率；Transwell实验检测各组乳腺癌细胞侵袭和迁移能力。结果: 乳腺癌细胞转染HLA-E siRNA后，HLA-E mRNA及蛋白表达水平明显降低。与阴性对照组相比，HLA-E siRNA转染组乳腺癌细胞增殖率降低(P<0.01)；凋亡率增加(P<0.01)；侵袭迁移能力降低(P<0.01)。结论: 靶向人乳腺癌细胞HLA-E基因的siRNA能有效抑制其基因表达，进而抑制细胞增殖，诱导细胞凋亡，降低乳腺癌细胞侵袭和迁移能力。

关键词: HLA-E基因, 乳腺癌, 小分子RNA干扰技术, 基因治疗

Abstract: OBJECTIVE: To investigate the expression of HLA-E gene in human breast cancer cells and its effect on rates of proliferation，apoptosis，invasion and migration of breast cancer cells，and analyze the feasibility of siRNA (small interfering RNA) silencing of HLA-E as gene therapy. METHODS: Specific siRNA sequences of HLA-E gene were designed and synthesized，and the sequences were transfected into human breast cancer cells MDA-MB-231 with liposome Vigofect. For comparisons，blank control，negative control and liposome groups were established. Quantitative real-time PCR and Western blot were used to detect HLA-E mRNA and protein expression. CCK-8 and flow cytometry were used to detect proliferation and apoptosis rates of the cancer cells. Transwell assay was used to detect the invasion and migration. RESULTS: After transfection with HLA-E siRNA，gene and protein expressions of HLA-E in the cancer cells were reduced significantly compared with the control groups. In addition， proliferation rates of the transfected cancer cells were significantly decreased (P<0.01)，apoptosis rates were significantly increased (P<0.01) while invasion and migration were significantly decreased (P<0.01). CONCLUSION: siRNA targeting of HLA-E effectively suppressed its expression in the breast cancer cells together with inhibition of their proliferation， induction of apoptosis， and reduction of migration and invasion.

Key words: HLA-E gene, breast cancer, small interfering RNA, gene therapy

中图分类号:

吴振村, 周艳. HLA-E基因表达沉默对人乳腺癌细胞生物学行为的影响[J]. 癌变·畸变·突变, 2023, 35(1): 15-20.

WU Zhencun, ZHOU Yan. Effect of HLA-E gene silencing on biological behavior of breast cancer cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2023, 35(1): 15-20.

参考文献

[1] EMENS L A. Breast cancer immunotherapy:facts and hopes[J]. Clin Cancer Res, 2018, 24(3):511-520.
[2] BASU A, RAMAMOORTHI G, JIA Y, et al. Immunotherapy in breast cancer:current status and future directions[J]. Adv Cancer Res, 2019, 143:295-349.
[3] SHEIKH-HOSSEINI M, LARIJANI B, GHOLIPOOR KAKROODI Z, et al. Gene therapy as an emerging therapeutic approach to breast cancer:new developments and challenges[J]. Hum Gene Ther, 2021, 32(21/22):1330-1345.
[4] 郭智慧, 周庆, 蒋安科, 等. 小干扰RNA技术对沉默乳腺细胞中DEK基因的表达及乳腺癌细胞增殖与凋亡的影响[J]. 中国妇幼保健, 2018, 33(14):3307-3310.
[5] 管海涛, 薛兴欢, 王西京, 等. 靶向survivin的siRNA抑制乳腺癌MCF-7细胞增殖并诱导其凋亡[J]. 中华肿瘤杂志, 2006, 28(5):326-330.
[6] 周艳, 吴振村, 唐玉红, 等. HLA-E基因多态性及血浆可溶性HLA-E水平与乳腺癌遗传易患性的关系[J]. 细胞与分子免疫学杂志, 2015, 31(4):524-527.
[7] 周艳, 吴振村, 程健君, 等. 人类白细胞抗原-E基因多态性与乳腺癌遗传易感性的关系[J]. 肿瘤防治研究, 2015, 42(2):190-193.
[8] SZEKELY B, BOSSUYT V, LI X, et al. Immunological differences between primary and metastatic breast cancer[J]. Ann Oncol, 2018, 29(11):2232-2239.
[9] KANEVSKIY L, EROKHINA S, KOBYZEVA P, et al. Dimorphism of HLA-E and its disease association[J]. Int J Mol Sci, 2019, 20(21):5496.
[10] SHARPE H R, BOWYER G, BRACKENRIDGE S, et al. HLA-E:exploiting pathogen-host interactions for vaccine development[J]. Clin Exp Immunol, 2019, 196(2):167-177.
[11] DA SILVA, SILVA T G A, DUARTE R A, et al. Expression of the classical and nonclassical HLA molecules in breast cancer[J]. Int J Breast Cancer, 2013, 2013:250435.
[12] DE KRUIJF E M, SAJET A, VAN NES J G H, et al. HLA-E and HLA-G expression in classical HLA class I-negative tumors is of prognostic value for clinical outcome of early breast cancer patients[J]. J Immunol, 2010, 185(12):7452-7459.
[13] TALEBIAN YAZDI M, VAN RIET S, VAN SCHADEWIJK A, et al. The positive prognostic effect of stromal CD8⁺ tumor-infiltrating T cells is restrained by the expression of HLA-E in non-small cell lung carcinoma[J]. Oncotarget, 2016, 7(3):3477-3488.
[14] HIRAOKA N, INO Y, HORI S, et al. Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients[J]. Cancer Sci, 2020, 111(8):3057-3070.
[15] HUANG R X, ZHANG D Y, LI F, et al. Loss of Fas expression and high expression of HLA-E promoting the immune escape of early colorectal cancer cells[J]. Oncol Lett, 2017, 13(5):3379-3386.
[16] ÖZGÜL ÖZDEMIR R B, OZDEMIR A T, OLTULU F, et al. A comparison of cancer stem cell markers and nonclassical major histocompatibility complex antigens in colorectal tumor and noncancerous tissues[J]. Ann Diagn Pathol, 2016, 25:60-63.
[17] SINGH A, TRIVEDI P, JAIN N K. Advances in siRNA delivery in cancer therapy[J]. Artif Cells Nanomed Biotechnol, 2018, 46(2):274-283.
[18] LI C F, CAO S, LIU Z, et al. RNAi-mediated downregulation of uPAR synergizes with targeting of HER2 through the ERK pathway in breast cancer cells[J]. Int J Cancer, 2010, 127(7):1507-1516.
[19] CHEN Y C, STAMATOYANNOPOULOS G, SONG C Z. Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro[J]. Cancer Res, 2003, 63(16):4801-4804.
[20] LAPTEVA N, YANG A G, SANDERS D E, et al. CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo[J]. Cancer Gene Ther, 2005, 12(1):84-89.
[21] YHEE J Y, SONG S Y, LEE S J. et al. Cancer-targeted MDR-1 siRNA delivery using self-cross-linked glycol chitosan nanoparticles to overcome drug resistance[J]. J Control Release, 2015, 198:1-9.