SAMHD1抑制肺腺癌细胞中PD-L1表达的研究

doi:10.3760/cma.j.cn371439-20220427-00130

摘要/Abstract

摘要：

目的探讨在肺腺癌中含SAM域和HD域蛋白1（SAMHD1）与程序性死亡配体-1（PD-L1）表达的相关性。方法通过在线数据库GEPIA和Kaplan-Meier Plotter分析SAMHD1在肺腺癌中的表达及对预后的影响。通过实时荧光定量PCR（qPCR）和蛋白质印迹法检测SAMHD1在多个肺腺癌细胞株中的表达。利用小干扰RNA转染及慢病毒感染技术分别对H1975、H1299及LLC细胞进行SAMHD1基因沉默，通过qPCR、蛋白质印迹法检测对照组、siSAMHD1-1组和siSAMHD1-2组肺腺癌细胞中PD-L1 mRNA及蛋白表达水平，用流式细胞术检测细胞膜PD-L1的表达水平。构建小鼠肺腺癌移植瘤模型，用免疫组织化学法检测对照组和shSAMHD1组移植瘤组织中PD-L1的表达。用CCK-8检测对照组、siSAMHD1-1组和siSAMHD1-2组肺腺癌细胞增殖活力。结果 GEPIA数据库结果表明，SAMHD1 mRNA在肺腺癌中的表达较肺正常组织低（4.81±0.90 vs. 5.99±0.76，t=20.67，P<0.001）。SAMHD1高表达患者中位总生存期明显长于低表达患者（109.0个月 vs. 87.7个月，χ²=26.83，P=0.002）。A549、PC9、H1299和H1975细胞中SAMHD1 mRNA相对表达量分别为1.00±0.02、0.75±0.05、3.49±0.19和7.25±0.38（F=589.00，P<0.001），蛋白相对表达量分别为1.00±0.06、0.34±0.07、1.67±0.22和2.11±0.63（F=15.79，P=0.001）。H1975细胞中，对照组、siSAMHD1-1组和siSAMHD1-2组的PD-L1 mRNA相对表达水平分别为1.00±0.00、1.54±0.26、2.89±0.13（F=102.30，P<0.001），蛋白相对表达水平分别为1.00±0.01、1.50±0.10和1.52±0.33（F=6.65，P=0.030）；H1299细胞中，3组的PD-L1 mRNA相对表达水平分别为1.00±0.08、1.63±0.03和2.14±0.03（F=368.80，P<0.001），蛋白相对表达水平分别为1.00±0.07、1.88±0.35和2.05±0.38（F=10.66，P=0.011），siSAMHD1-1组和siSAMHD1-2组PD-L1表达水平均高于对照组（均P<0.05）。流式细胞术结果表明，H1975细胞中，对照组、siSAMHD1-1组和siSAMHD1-2组膜PD-L1荧光强度分别为246.83±27.59、325.60±8.00和308.93±7.60（F=17.56，P=0.003）；H1299细胞中，3组的荧光强度分别为959.00±6.25、1 084.33±7.64和1 085.33±21.22（F=86.74，P<0.001），siSAMHD1-1组和siSAMHD1-2组荧光强度均高于对照组（均P<0.05）。在小鼠移植瘤模型中，shSAMHD1组PD-L1的H-SCORE评分高于对照组（7.99±1.10 vs. 4.49±0.43，t=5.13，P=0.007）。H1975细胞中，对照组、siSAMHD1-1组和siSAMHD1-2组72 h细胞增殖活力分别为0.50±0.02、0.75±0.05和0.73±0.06（F=25.01，P=0.001）；H1299细胞中，3组72 h细胞增殖活力分别为0.80±0.01、1.00±0.04和0.93±0.07（F=13.90，P=0.006），siSAMHD1-1组和siSAMHD1-2组细胞增殖活力均高于对照组（均P<0.05）。结论在肺腺癌中，沉默SAMHD1可以提高PD-L1的表达。

关键词: 肺腺癌, B7-H1抗原, 含SAM域和HD域蛋白1

Abstract:

Objective To explore the correlation between SAM domain and HD domain-containing protein 1（SAMHD1） and programmed death-ligand 1（PD-L1） expression in lung adenocarcinoma. Methods The expression of SAMHD1 in lung adenocarcinoma and its effect on prognosis were analyzed by online database GEPIA and Kaplan-Meier Plotter. The expression of SAMHD1 in lung adenocarcinoma cell lines was detected by quantitative real-time PCR （qPCR） and Western blotting. SAMHD1 gene was silenced in H1975， H1299 and LLC cells by small interfering RNA transfection and lentivirus infection， respectively. The mRNA and protein expression levels of PD-L1 in lung adenocarcinoma cells of control group， siSAMHD1-1 group and siSAMHD1-2 group were detected by qPCR and Western blotting. The membrane PD-L1 level was detected by flow cytometry. A mouse lung adenocarcinoma xenograft model was constructed. The PD-L1 levels in the tumor tissues of control group and shSAMHD1 group were detected by immunohistochemistry. Cell proliferation activities of the control， siSAMHD1-1 and siSAMHD1-2 groups were detected by CCK-8 assays. Results The GEPIA database results showed that the mRNA expression of SAMHD1 in lung adenocarcinoma was lower than that in normal lung tissue （4.81±0.90 vs. 5.99±0.76， t=20.67， P<0.001）. The median overall survival time of patients with high SAMHD1 expression was significantly longer than that of patients with low SAMHD1 expression （109.0 months vs. 87.7 months， χ²=26.83， P=0.002）. The relative mRNA expression levels of SAMHD1 in A549， PC9， H1299 and H1975 cells were 1.00±0.02， 0.75±0.05， 3.49±0.19 and 7.25±0.38 （F=589.00， P<0.001）， and the relative protein expression levels were 1.00±0.06， 0.34±0.07， 1.67±0.22 and 2.11±0.63 （F=15.79， P=0.001）. In H1975 cells， the relative mRNA levels of PD-L1 in the control， siSAMHD1-1 and siSAMHD1-2 groups were 1.00±0.00， 1.54±0.26 and 2.89±0.13 （F=102.30， P<0.001）， and the relative protein expression levels were 1.00±0.01， 1.50±0.10 and 1.52±0.33 （F=6.65， P=0.030）. In H1299 cells， the relative mRNA levels of PD-L1 in the three groups were 1.00±0.08， 1.63±0.03 and 2.14±0.03 （F=368.80， P<0.001）， and the relative protein levels of PD-L1 were 1.00±0.07， 1.88±0.35 and 2.05±0.38 （F=10.66， P=0.011）. The expression level of PD-L1 in the siSAMHD1-1 and siSAMHD1-2 groups was higher than that in the control group （all P<0.05）. Flow cytometry results showed that in H1975 cells， the fluorescence intensity of membrane PD-L1 in the control， siSAMHD1-1 and siSAMHD1-2 groups were 246.83±27.59， 325.60±8.00 and 308.93±7.60 （F=17.56， P=0.003）， and in H1299 cells， the fluorescence intensity of membrane PD-L1 in the three groups were 959.00±6.25， 1 084.33±7.64 and 1 085.33±21.22 （F=86.74， P<0.001）. The fluorescence intensity of PD-L1 in the siSAMHD1-1 group and siSAMHD1-2 group was higher than that in the control group （all P<0.05）. In xenograft mouse model， the H-SCORE of PD-L1 in the shSAMHD1 group was higher than that in the control group （7.99±1.10 vs. 4.49±0.43， t=5.13， P=0.007）. The proliferative activities of H1975 cells in the control group， siSAMHD1-1 group and siSAMHD1-2 group at 72 h were 0.50±0.02， 0.75±0.05 and 0.73±0.06 （F=25.01， P=0.001）. The proliferative activities of H1299 cells in the three groups at 72 h were 0.80±0.01， 1.00±0.04 and 0.93±0.07 （F=13.90， P=0.006）. The cell proliferation activity in the siSAMHD1-1 group and siSAMHD1-2 group was higher than that in the control group （all P<0.05）. Conclusion SAMHD1 silencing induces PD-L1 expression in lung adenocarcinoma.

Key words: Adenocarcinoma of lung, B7-H1 antigen, SAM and HD domain-containing protein 1

李杨仪, 龚龑, 谢丛华. SAMHD1抑制肺腺癌细胞中PD-L1表达的研究[J]. 国际肿瘤学杂志, 2022, 49(11): 658-664.

Li Yangyi, Gong Yan, Xie Conghua. SAMHD1 inhibits PD-L1 expression in lung adenocarcinoma cells[J]. Journal of International Oncology, 2022, 49(11): 658-664.

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基因	引物
GAPDH	正向：5'-ACAACTTTGGTATCGTGGAAGG-3'
GAPDH	反向：5'-GCCATCACGCCACAGTTTC-3'
SAMHD1	正向：5'-CCCAAAGTATTGCTAGACGTGA-3'
SAMHD1	反向：5'-TGCATTCCATAATCCATGTTG-3'
PD-L1	正向：5'-GGACAAGCATGACATCAAG-3'
PD-L1	反向：5'-CCCAGAATTACCAAGTGAGTCCT-3'

组别	SAMHD1 mRNA		SAMHD1蛋白
组别	H1975	H1299	H1975	H1299
对照组	1.00±0.01	1.00±0.02	1.00±0.29	1.00±0.07
siSAMHD1-1组	0.17±0.02^a	0.23±0.00^a	0.37±0.07^a	0.36±0.13^a
siSAMHD1-2组	0.21±0.00^a	0.08±0.00^a	0.24±0.06^a	0.46±0.10^a
F值	3 062.00	3 664.00	15.43	34.07
P值	<0.001	<0.001	0.004	<0.001

组别	0 h	24 h	48 h	72 h
对照组	0.15±0.01	0.19±0.01	0.43±0.01	0.50±0.02
siSAMHD1-1组	0.16±0.01	0.25±0.03^a	0.49±0.01^a	0.75±0.05^a
siSAMHD1-2组	0.16±0.00	0.21±0.01	0.50±0.03^a	0.73±0.06^a
F值	0.21	6.31	9.77	25.01
P值	0.811	0.033	0.013	0.001

组别	0 h	24 h	48 h	72 h
对照组	0.19±0.03	0.30±0.04	0.44±0.01	0.80±0.01
siSAMHD1-1组	0.20±0.02	0.37±0.06	0.62±0.03^a	1.00±0.04^a
siSAMHD1-2组	0.20±0.01	0.34±0.07	0.54±0.04^a	0.93±0.07^a
F值	0.18	1.19	30.73	13.90
P值	0.838	0.366	<0.001	0.006