HHLA2过表达胃癌细胞株构建及细胞功能的初步研究

doi:10.3877/cma.j.issn.2095-2015.2023.06.002

中华消化病与影像杂志(电子版) ›› 2023, Vol. 13 ›› Issue (06) : 373 -377. doi: 10.3877/cma.j.issn.2095-2015.2023.06.002

论著

HHLA2过表达胃癌细胞株构建及细胞功能的初步研究

韩晓娟, 徐佳倩, 朱玉兰, 王莹, 李源, 冯珺, 邵东^†(

)

213003　江苏常州，苏州大学附属第三医院消化内科
213003　江苏常州，苏州大学附属第三医院肿瘤科
213003　江苏常州，苏州大学附属第三医院肿瘤生物诊疗中心

收稿日期:2023-05-18 出版日期:2023-12-01
通信作者: 邵东
基金资助:
常州市应用基础研究计划(CJ20210074); 常州市卫生健康委青年人才科技项目(QN202015)

Construction and cell function study of HHLA2-overexpressing gastric cancer cells

Xiaojuan Han, Jiaqian Xu, Yulan Zhu, Ying Wang, Yuan Li, Jun Feng, Dong Shao^†()

Department of Gastroenterology, the Third Affiliated Hospital of Soochow University, Changzhou 213003, China
Department of Oncology, the Third Affiliated Hospital of Soochow University, Changzhou 213003, China
Cancer Biological Diagnosis and Treatment Center, the Third Affiliated Hospital of Soochow University, Changzhou 213003, China

Received:2023-05-18 Published:2023-12-01
Corresponding author: Dong Shao

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目的

使用慢病毒载体构建稳定过表达人内源性逆转录病毒H-长末端重复关联蛋白2(HHLA2)的胃癌细胞系，探讨细胞发生的生物学功能改变。

方法

由目的基因以及Lipo2000转染试剂构建重组质粒，将该质粒转染293T细胞，浓缩其细胞上清液并测定浓度，再将浓缩液转染胃癌细胞株BGC-823和MGC80-3，利用流式细胞术分选并验证GFP＋稳定过表达的细胞株。开展细胞功能实验(含迁移试验和增殖试验等)以分析过表达HHLA2胃癌细胞株的生物学功能。

结果

酶切及测序结果示，重组慢病毒载体成功构建。流式分选及验证示，HHLA2过表达胃癌稳转株。过表达HHLA2可使胃癌细胞株MGC80-3和BGC-823加快增殖，其24 h、48 h、72 h吸光度值均较对照细胞株升高(P<0.05)。过表达HHLA2可增强胃癌细胞株BGC-823的迁移能力，其24 h、48 h迁移率均较对照细胞株升高(P<0.05)；MGC80-3细胞迁移距离未明显变长(P>0.05)。

结论

HHLA2过表达能够提高胃癌细胞的增殖和迁移能力。

关键词: 胃癌, 人内源性逆转录病毒-H长末端重复关联蛋白2, 慢病毒载体, 流式细胞术

Objective

To construct gastric cancer cell lines overexpressing human endogenous retrovirus subfamily H long terminal repeat associating protein 2(HHLA2)by lentivirus vector and verify their biological functions.

Methods

The target gene and Lipo2000 reagent were used to construct recombinant plasmid, which was then applied to transfect 293T cell.We collect The cell supernatant was concentrated and the concentration was determined.The concentrated liquid was then transfected into gastric cancer cell lines BGC-823 and MGC80-3.The flow cytometry was used to sort and verify the GFP+ stable overexpressing cell lines.Cell function experiments(including migration experiment and proliferation experiment)were applied to explore the biological functions of overexpressing HHLA2 gastric cancer cell lines.

Results

The results of restriction enzyme digestion and sequencing showed that the recombinant lentivirus vector was successfully constructed.Flow sorting and verification showed that HHLA2 overexpressed gastric cancer stably transformed strains.Overexpression of HHLA2 could accelerate the proliferation of gastric cancer cell lines MGC80-3 and BGC-823, and their absorbance values at 24 h, 48 h and 72 h were higher than those of the control cell lines(P<0.05). Overexpression of HHLA2 could enhance the migration ability of gastric cancer cell line BGC-823, and its migration rate at 24 h and 48 h was higher than that of the control cell line(P<0.05). However, the migration distance of MGC80-3 cells did not increase significantly(P>0.05).

Conclusion

Overexpression of HHLA2 can promote the proliferation and migration ability of gastric cancer.

Key words: Gastric cancer, Human endogenous retrovirus subfamily H long terminal repeat associating protein 2, Lentiviral vector, Flow cytometry

图1 质粒转染293T细胞

图2 过表达HHLA2慢病毒载体图片

图3 过表达HHLA2胃癌细胞株GFP荧光图片

图4 过表达HHLA2对胃癌细胞株增殖能力的影响注：^*P<0.05，^**P<0.01，^***P<0.001，^****P<0.0001

图5 过表达HHLA2对胃癌细胞株迁移能力的影响

图6 过表达HHLA2对胃癌细胞株迁移能力的影响

1	Bolandi N，Derakhshani A，Hemmat N，et al.The Positive and Negative Immunoregulatory Role of B7 Family：Promising Novel Targets in Gastric Cancer Treatment[J]．Int J Mol Sci，2021，22(19)：10719.
2	Janakiram M，Chinai JM，Fineberg S，et al.Expression，Clinical Significance，and Receptor Identification of the Newest B7 Family Member HHLA2 Protein[J]．Clin Cancer Res，2015，21(10)：2359-2366.
3	Wei L，Tang L，Chang H，et al.HHLA2 overexpression is a novel biomarker of malignant status and poor prognosis in gastric cancer[J]．Hum Cell，2020，33(1)：116-122.
4	Shimonosono M，Arigami T，Yanagita S，et al.The association of human endogenous retrovirus-H long terminal repeat-associating protein 2(HHLA2)expression with gastric cancer prognosis[J]．Oncotarget，2018，9(31)：22069-22078.
5	曹维维，袁成良．人类白细胞抗原-G5过表达慢病毒R-HLA-G5稳转细胞株的建立[J]．中国临床药理学杂志，2021，37(4)：2495-2498.
6	Sung H，Ferlay J，Siegel RL，et al.Global Cancer Statistics 2020：GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]．CA Cancer J Clin，2021，71(3)：209-249.
7	Rieder SA，Wang J，White N，et al.B7-H7(HHLA2)inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling[J]．Cell Mol Immunol，2021，18(6)：1503-1511.
8	Cheng H，Borczuk A，Janakiram M，et al.Wide Expression and Significance of Alternative Immune Checkpoint Molecules，B7x and HHLA2，in PD-L1-Negative Human Lung Cancers[J]．Clin Cancer Res，2018，24(8)：1954-1964.
9	Xiao Y，Li H，Yang L，et al.The Expression Patterns and Associated Clinical Parameters of Human Endogenous Retrovirus-H Long Terminal Repeat-Associating Protein 2 and Transmembrane and Immunoglobulin Domain Containing 2 in Oral Squamous Cell Carcinoma[J].2019，2019 5421985.
10	Xu Y，Huang Z，Yu X，et al.HHLA2 Expression is Associated with Poor Survival in Patients with Hepatocellular Carcinoma[J]．Biologics，2021，15 329-341.
11	Chen L，Zhu D，Feng J，et al.Overexpression of HHLA2 in human clear cell renal cell carcinoma is significantly associated with poor survival of the patients[J]．Cancer Cell Int，2019，19：101.
12	Zhou QH，Li KW，Chen X，et al.HHLA2 and PD-L1 co-expression predicts poor prognosis in patients with clear cell renal cell carcinoma[J]．J Immunother Cancer，2020，8(1)：e000157.
13	Boor PPC，Sideras K，Biermann K，et al.HHLA2 is expressed in pancreatic and ampullary cancers and increased expression is associated with better post-surgical prognosis[J]．Br J Cancer，2020，122(8)：1211-1218.
14	Janakiram M，Chinai JM，Zhao A，et al.HHLA2 and TMIGD2：new immunotherapeutic targets of the B7 and CD28 families[J]．Oncoimmunology，2015，4(8)：e1026534.
15	Bhatt RS，Berjis A，Konge JC，et al.KIR3DL3 Is an Inhibitory Receptor for HHLA2 that Mediates an Alternative Immunoinhibitory Pathway to PD1[J]．Cancer Immunol Res，2021，9(2)：156-169.
16	Sun W，Li S，Tang G，et al.HHLA2 deficiency inhibits non-small cell lung cancer progression and THP-1 macrophage M2 polarization[J]．Cancer Med，2021，10(15)：5256-5269.

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