关键词: 免疫监视, PD-L1, 肺癌

Abstract:  Background and purpose: Programmed death ligand-1 (PD-L1) plays an important role in sheltering tumor cell from surveillance of immune system. While the potential modulation is regarded as being performed at MAPK, PI3K-AKT and STAT3 pathway in most cases, the key control point has never been explicitly reported. This study aimed to probe on this mechanism of lung carcinoma via PI3K/AKT pathway based on the A549 and H460 cell lines. Methods: By using shRNA technology, we created several selectively ‘silent’ mutations of A 549 and H460 cell lines, involving A549 ^HEB- , A549 ^HTF4- , A549 ^Nrf2- A549 ^FOXO3a- , H460 ^HEB- , H460 ^HTF4- , H460 ^Nrf2- and H460 ^FOXO3a- . 3’UTR region of PD-L1 was constructed into pGL3-basic vector in order to create a dual luciferase reporter gene system which was able to be used for monitoring the transcriptional activation of PD-L1. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to detect the transcriptional level of PD-L1, and the Western blot assay was used for monitoring the total expression of PD-L1; peripheral blood mononuclear cells (PBMCs) co-culturing with A549 and H460 cell lines were utilized to check the function of Nrf2 in tumor immune resistance. Results: Phosphorylation level of AKT and expression level of PD-L1 in A549 and H460 cell lines were simultaneously enhanced after treatment with 10 μg/mL insulin (P<0.001), and the same phenomenon was observed in their mutations with defect in HEB, HTF4 and FOXO3a respectively, however, the plot reversal occurred in A549 ^Nrf2- and H460 ^Nrf2- cell lines (P>0.001). Isoproterenol could boost both transcriptional activation and expression level in A549 and H460 cell lines, and this auto-action was able to be relieved by NAC. Results above further certified the transcriptional regulation of Nrf2 on PD-L1 gene. Wortmannin could change over the function of insulin to raise the expression of PD-L1, by prohibiting the increasing AKT phosphorylation level. Furthermore, the relationship between the phosphorylation level of Nrf2 and AKT was also analyzed by using Wortmannin and insulin, and the results confirmed that the two parties correlated to each other positively, for the correlation coefficient r was 0.86 and 0.93 in A549 and H460 cell lines respectively. At last, A549 ^Nrf2- and h460 ^Nrf2-cell lines became more sensitive to the attack of PBMCs than that of their wild type owing to Nrf2-deficiency, for increased apoptosis was observed in co-culture experiment. Conclusion: PI3K/AKT pathway plays a vital role in regulating PD-L1 expression in A549 and H460 cell lines, and this action is potentially realized via phosphorylating Nrf2.

Key words: Immune surveillance, PD-L1, Lung carcinoma