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