Immunomodulatory functions of the circ_001678/miRNA-326/ZEB1 axis in non-small cell lung cancer via the regulation of PD-1/PD-L1 pathway

Abstract

High-throughput circular RNA (circRNA) sequencing identified circRNA_001678 (circ_001678) as an upregulated circRNA in non-small cell lung cancer (NSCLC) tissues. Hence, the current study sought to investigate the function and the underlying mechanism of circRNA_001678 in immune escape of NSCLC. Briefly, commercially purchased NSCLC cell lines were adopted for in vitro experiment to evaluate the effects of circ_001678 over-expression or knockdown on cell biological functions, including proliferation, migration and invasive abilities. In addition, the effects of circ_001678 on the in vivo tumorigenicity ability were evaluated for verification. Accordingly, we uncovered that circ_001678 over-expression augmented NSCLC progression in vitro and enhanced tumorigenicity ability in vivo. The interaction between circ_001678 and miR-326 predicted online was verified by means of luciferase and RNA pull-down assays. Furthermore, circ_001678 could sponge miR-326 to up-regulate ZEB1. On the other hand, the tumor-promoting effects of circ_001678 could be inhibited by anti-PD-L1/PD-1 treatment. Mechanistically, circ_001678 led to the activation of the PD-1/PD-L1 pathway to promote CD8+ T cell apoptosis, thereby inducing NSCLC cell immune escape via regulation of the miR-326/ZEB1 axis. To conclude, our findings revealed that circ_001678 sponges miR-326 to up-regulate ZEB1 expression and induce the PD-1/PD-L1 pathway-dependent immune escape, thereby promoting the malignant progression of NSCLC.

Introduction

Non-small cell lung cancer (NSCLC) represents the most frequently occurring subtype of lung cancer, accounting for 85% of all lung malignancies, while the prognosis for patients with advanced NSCLC remains poor (1). Immune escape, which is featured in the development of NSCLC, participates in the tumor invasion and leads to resistance to therapy via an immune checkpoint blockade (2). Adding to the clinical challenges, the vast majority of patients with advanced NSCLC do not respond to immunotherapy due to the development of chemoresistance (3–5). In lieu of the same, it is imperative to advance the search for novel and more efficacious immunotherapeutic agents to tackle NSCLC.

Figure 1

Differentially expressed circRNAs in NSCLC. (A) circRNA high-throughput sequencing heat map of differentially expressed circRNAs between 10 NSCLC tissues and 10 adjacent normal tissue samples. (B) Differential expression of 10 circRNAs in 10 NSCLC tissues and their corresponding adjacent normal tissues verified using RT-qPCR (the ordinate indicates the expression, and the abscissa indicates the name of the circRNAs).

Circular RNAs (circRNAs) are well-established as closed RNAs that enable splicing of potential gene regulation by a single pre-messenger, and confer important functions in various human diseases, including cancers (6). What is noteworthy is the host of evidence that have stressed the significance of circRNAs in relation to NSCLC. For instance, circ_0067934 is highly-expressed in NSCLC tissues and cells, while down-regulation of circ_0067934 can delay the progression of NSCLC by affecting the behaviors of NSCLC cells (7). Similarly, a newly identified circRNA circ_001678 was predicted to be elevated in NSCLC by our high-throughput circRNA sequencing analysis, yet its potential actions in NSCLC are yet to be elucidated. Meanwhile, online prediction with the help of the StarBase database highlighted that microRNA (miR)-326 could be the downstream target of circ_001678. The latter is particularly noteworthy as similar interactions between circRNAs and miRNAs have been previously revealed to be implicated in the growth and development of NSCLC. Moreover, a prior study indicated that circ_0003028 could sponge miR-1298-5p to hinder progression of NSCLC (8). On the other hand, circ_0008003 can expedite the growth of NSCLC by mediating the tumor-suppressive function of miR-488 (9). Moreover, circ_0003998 is capable of enhancing cell proliferation and invasion via suppression of miR-326 in NSCLC (10). Intriguingly, further online prediction in our study suggested that zinc finger E-box binding homeobox 1 (ZEB1), a hub modulator of epithelial–mesenchymal transition (11), could be a target of miR-326, while also being up-regulated in NSCLC as described in a prior study (12). Additionally, over-expression of ZEB1 was previously associated with enhanced proliferation activity and diminished apoptosis of A549 cells (13). Of note, ZEB1 can augment the expression of programmed cell death ligand-1 (PD-L1), leading to the immune escape of cancer cells (14). Recently, targeted immunotherapies such as antibodies and inhibitors targeting programmed cell death protein-1 (PD-1)/PD-L1 have made significant progress in the treatment of NSCLC (15,16). Therefore, the current study set out to investigate the potential molecular mechanism of circ_001678/miR-326/ZEB1 affecting the PD-1/PD-L1 pathway to activate CD8+ T cell activity and participate in the occurrence of NSCLC immune escape.

Results

circ_001678 is up-regulated in NSCLC tissues

Given the key roles of circRNAs in the progression of diverse cancers, including NSCLC (17), we initially screened and identified the differentially expressed circRNAs in NSCLC by means of high-throughput sequencing in paired NSCLC tissues and adjacent nontumor tissues obtained from 10 patients (Fig. 1A). The top 10 circRNAs (namely, circ_001678, circ_0018046, circ_0039868, circ_0089160, circ_0048972, circ_0018658, circ_0067857, circ_0019137, circ_0063162 and circ_0087609) were selected for further analyses. Subsequent relative quantification of these 10 circRNAs using reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that circ_001678 exhibited the highest expression in the NSCLC tissues (Fig. 1B). Together, these findings revealed that circ_001678 was highly-expressed and may serve as a key molecule in the initiation and development of NSCLC.

Highly-expressed circ_001678 aggravates the proliferation, invasiveness and migration of NSCLC cells

To explore whether circ_001678 was associated with the progression of NSCLC, in vitro function experiments were carried out. First of all, findings of RT-qPCR illustrated the presence of markedly higher expression of circ_001678 in NSCLC cells (NCI-H358, NCI-H1299 and A549) relative to normal HBE1 cells, among which A549 cells presented with the highest expression, and were selected for subsequent experimentation (Fig. 2A).

Figure 2

The promoting or suppressing effect of overexpressed or downregulated circ_001678 on NSCLC cell proliferation, invasion and migration. (A) The expression of circ_001678 in 3 NSCLC cell lines (NCI-H358, NCI-H1299 and A549) and human normal bronchial epithelial cell line HBE1 determined using RT-qPCR. ^*P < 0.05 vs. HBE1 cells. (B) The transfection efficiency of overexpression or downregulation of circ_001678 in A549 cells examined by RT-qPCR. (C) The proliferation ability of A549 cells with OD values at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h determined using CCK-8 assay. (D) The invasion and migration ability of A549 cells assessed by Transwell assay, with the number of invaded and migrated cells counted. ^*P < 0.05 vs. oe-NC transfected A549 cells. #P < 0.05 vs. sh-NC transfected A549 cells. The cell experiment was repeated three times.

We further over-expressed or silenced circ_001678 expression in A549 cells with the help of over-expression (oe)-circ_001678 or short hairpin RNA (sh)-circ_001678, with the results of RT-qPCR validated successful transfection (Fig. 2B). Subsequent cell counting kit-8 (CCK-8) and Transwell assays demonstrated that the proliferation rate and the number of invasive and migratory cells were all enhanced in oe-circ_001678-transfected A549 cells versus the oe-negative control (NC)-transfected cells. Meanwhile, intervention with sh-circ_001678 led to opposing trends, as evidenced by decreased cell proliferation and reduced migratory and invasive cells (Fig. 2C, D).

circ_001678 serves as a sponge of miR-326

To further elucidate the downstream targets of circ_001678 regulating the progression of NSCLC, the StarBase database (http://StarBase.sysu.edu.cn/) was utilized to predict the miRNAs interacting with circ_001678, which revealed that 5 miRNAs (namely, miR-1296-5p, miR-328-3p, miR-326, miR-330-5p and miR-1306-5p) may bind to circ_001678.

Accordingly, circ_001678 specific probes were adopted to perform RNA precipitation in A549 cells to testify the key miRNAs that might interact with circ_001678 in NSCLC cells. Subsequent results of RT-qPCR illustrated that circ_001678 was significantly enriched by miR-326, while the other miRNAs were not significantly enriched (Fig. 3A). Furthermore, an AGO2 antibody was utilized to conduct RIP test in A549 cells, which revealed that AGO2 antibody could precipitate both circ_001678 and miR-326 when compared with the IgG group (Fig. 3B). Together, these findings validated that circ_001678 could bind to miR-326.

Figure 3

The binding relationship between circ_001678 and miR-326 in NSCLC. (A) circ_001678 probes and NC probes adopted to precipitate the miRNA that interacted with circ_001678 in A549 cells, and the degree of miRNA enrichment detected by RT-qPCR. ^*P < 0.05 vs. NC probe group. (B) AGO2 and control IgG antibody utilized to conduct RIP experiment to test the interaction between circ_001678 and miR-326. ^*P < 0.05 vs. IgG group. (C) The binding sites of circ_001678 and miR-326 predicted on the StarBase database. (D) The binding of circ_001678 to miR-326 verified using dual-luciferase reporter assay. ^*P < 0.05 vs. NC mimic group. (E) After the cells were transfected with biotinylated miR-326 or NC, the level of circ_001678 in the lysate of NSCLC cells captured by streptavidin was detected by RNA pull down. ^*P < 0.05 vs. NC group. (F) The co-localization of circ_001678 and miR-326 in NSCLC cells measured using RNA-FISH. (G) The expression of miR-326 in 10 cases of NSCLC tissues and their corresponding adjacent normal tissues determined using RT-qPCR. (H) The correlation between the expression of circ_001678 and miR-326 in NSCLC tissues analyzed by Pearson’s correlation analysis. (I) The expression of miR-326 in 3 NSCLC cell lines (NCI-H358, NCI-H1299 and A549) and human normal bronchial epithelial cell line HBE1 examined by RT-qPCR. ^*P < 0.05 vs. HBE1 cells. (J) The expression of miR-326 after overexpression or knockdown of circ_001678 detected by RT-qPCR. ^*P < 0.05 vs. oe-NC transfected cells. #P < 0.05 vs. sh-NC transfected cells. The experiment was repeated three times.

Thereafter, we mutated the putative binding site between circ_001678 and miR-326 to carry out a dual-luciferase reporter experiment. Subsequent findings illustrated that over-expression of miR-326 indeed reduced the luciferase activity of circ_001678 WT, but did not alter that of circ_001678 MUT (Fig. 3C, D). Next, biotinylated miR-326 was transfected into A549 cells for pull-down assay, and the results of which demonstrated that circ_001678 was significantly enriched in the biotinylated miR-326 transfection group (Fig. 3E). Moreover, results of RNA-fluorescence in situ hybridization (FISH) validated that circ_001678 and miR-326 were co-localized in NSCLC cells (Fig. 3F).

Furthermore, RT-qPCR data exhibited that miR-326 was poorly-expressed in NSCLC tissues (Fig. 3G). In addition, an inverse correlation was found between circ_001678 and miR-326 (Fig. 3H). In vitro experiments further evidenced the significant down-regulation of miR-326 in NSCLC cells (Fig. 3I). Similarly, over-expression of circ_001678 led to miR-326 deficiency in A549 cells, whereas down-regulation of circ_001678 caused restoration of miR-326 expression (Fig. 3J).

circ_001678 interacts with miR-326 to stimulate immune escape in NSCLC by activating the PD-1/PD-L1 pathway

Recent evidence has shown that PD-1/PD-L1 pathway affects the anti-tumor immunity of the body in malignant tumors. Among them, PD-1/PD-L1 antibody exhibits a high value in the treatment of advanced NSCLC (15). Additionally, various studies have documented the interaction between tumor cells and CD8+ T cells within the tumor microenvironment, which can modulate their cytotoxic function (18,19). Therefore, we hypothesized that circ_001678 may confer an immunosuppressive role by modulating CD8+ T cells, thereby inducing immune escape of NSCLC and promoting disease progression.

To validate the above hypothesis, we examined the infiltration of CD8+ T cells in 10 paired NSCLC tissues and non-tumor tissues. Immunohistochemical staining illustrated a significant reduction in the positive rate of CD8+ T cells in NSCLC tissues (Fig. 4A). Meanwhile, the results of scatter diagram analysis further highlighted an inverse relation between circ_001678 expression in NSCLC tissues and the CD8+ T cell number, as well as a positive relation between miR-326 expression and the number of CD8+ T cells (Fig. 4B).

Figure 4

The modulatory effect of circ_001678/miR-326 axis on CD8+ T cell apoptosis and immune escape of NSCLC cells by mediating the PD-1/PD-L1 pathway. (A) The infiltration of CD8+ T cells in NSCLC tissues and corresponding adjacent normal tissues detected by immunohistochemical staining. N = 10. (B) The correlation between the expression of circ_001678 and miR-326 in NSCLC tissues and the number of CD8+ T cells shown in the dot plots. (C) The overexpression or downregulation transfection efficiency of miR-326 in A549 cells detected by RT-qPCR. ^*P < 0.05 vs. NC mimic group. #P < 0.05 vs. NC inhibitor group. (D) The expression of PD-L1 after up-regulating circ_001678 or miR-326 expression in A549 cells determined by RT-qPCR. ^*P < 0.05 vs. oe-NC + NC mimic group. #P < 0.05 vs. oe-circ_001678 + NC mimic group. (E) The percentage of CD8+ T cells in the co-culture system determined by flow cytometry. (F) The apoptotic rate of CD8+ T cells in the co-culture system detected by flow cytometry. (E, F) ^*P < 0.05 vs. oe-NC group. #P < 0.05 vs. oe-circ_001678 group. $P < 0.05 vs. NC inhibitor group. %P < 0.05 vs. miR-326 inhibitor group. The cell experiment was repeated three times.

Thereafter, we focused our efforts on investigating whether the circ_001678/miR-326 axis could change the PD-1/PD-L1 immune checkpoint by affecting CD8+ T cells. We subsequently altered the expression of miR-326 in A549 cells by transfection of simulant or inhibitor, and corresponding RT-qPCR results confirmed successful transfection (Fig. 4C). Moreover, over-expression of circ_001678 enhanced the expression of PD-L1, while elevation of miR-326 induced silencing of PD-L1 expression. On the other hand, oe-circ_001678-induced increase of PD-L1 was offset by miR-326 mimic (Fig. 4D). Together, these findings indicated that circ_001678 activated the PD-1/PD-L1 pathway by sponging miR-326.

In order to simulate the tumor microenvironment, we co-incubated A549 cells with CD8+ T cells, followed by treatment with the PD-1/PD-L1 antibody in the combined culture system. Results of flow cytometric analyses demonstrated that over-expression of circ_001678 or down-regulation of miR-326 led to a reduction in the percentage of CD8+ T cells, while raising their apoptosis. On this basis, the transfected cells were further treated with PD-1 or PD-L1 antibody, which contributed to enhanced proportion of CD8+ T cells, in addition to attenuation of apoptosis (Fig. 4E, F). Altogether, the aforementioned findings highlighted the involvement of the circ_001678/miR-326 axis in the PD-1/PD-L1 pathway-mediated immunity in NSCLC.

circ_001678 elevates ZEB1 expression via sponging miR-326

Subsequently, we focused our efforts on the downstream targets of miR-326. Namely, ABCC1, ZEB1, BSDC1, ALAD, RIT1, NSL1, ROR1 and NXF1, were indicated as the potential target genes of miR-326 through the StarBase, miRDB and TargetScan databases (Fig. 5A). Based on subsequent RT-qPCR results, among the eight target genes, only ZEB1 was significantly highly-expressed in NSCLC tissues (Fig. 5B). Literature review further revealed that ZEB1 expression was up-regulated in NSCLC (12). Accordingly, ZEB1 was selected for further experimentation.

Figure 5

Positive regulatory effect of circ_001678 on ZEB1 expression by sponging miR-326. (A) The intersection of miR-326 target genes among StarBase, miRdbmiRDB and TargetScan websites shown in the Venn diagram. (B) The differential expression of eight overlapping genes in 10 NSCLC tissues and the corresponding adjacent normal tissues detected by RT-qPCR. The ordinate indicates the multiple of difference, and the abscissa indicates the name of the gene. ^*P < 0.05 vs. the normal group. (C) The expression of ZEB1 in three NSCLC cell lines (NCI-H358, NCI-H1299, A549) and normal bronchial epithelial cells HBE1 determined by RT-qPCR. ^*P < 0.05 vs. the HBE1 group. (D) The binding sites of ZEB1 and miR-326 predicted in StarBase database. (E) The binding relation between ZEB1 and miR-326 confirmed by dual-luciferase reporter gene assay. ^*P < 0.05 vs. NC mimic group. (F) The regulatory effect of circ_001678/miR-326 axis on ZEB1 expression examined by RT-qPCR. ^*P < 0.05 vs. cells co-transfected with oe-NC and NC mimic. #P < 0.05 vs. cells co-transfected with oe-circ_001678 and NC mimic. The cell experiment was repeated three times.

Furthermore, RT-qPCR results showed a consistent finding that ZEB1 expression in NSCLC cells was remarkably high (Fig. 5C). Thereafter, we mutated the predicted binding site between ZEB1 and miR-326; results of luciferase assay confirmed that miR-326 gain-of-function decreased the luciferase activity of ZEB1 WT without altering that of ZEB1 MUT, indicating that miR-326 could bind to ZEB1 (Fig. 5D, E). Next, we up-regulated the expression of circ_001678 or miR-326 in A549 cells. Subsequent results illustrated that circ_001678 and ZEB1 expression was elevated, while that of miR-326 was diminished in A549 cells co-transfected with oe-circ_001678 and NC mimic as compared to those co-transfected with oe-NC and NC mimic. Reversely, miR-326 mimic augmented miR-326 expression and induced a reduction in the ZEB1 expression. Meanwhile, additional miR-326 mimic could rescue the miR-326 expression and counteract the oe-circ_001678-induced elevation of ZEB1 (Fig. 5F). Together, these findings indicated that circ_001678 could enhance the expression of ZEB1 by sponging miR-326 in NSCLC cells.

circ_001678 elevates the expression of ZEB1 to accelerate the immune escape in NSCLC

Existing evidence indicates that ZEB1 boosts the expression of PD-L1 and enhances the immune evasion of tumor cells (14). Accordingly, we explored the effect of circ_001678-mediated regulation of ZEB1 on the PD-1/PD-L1 pathway-mediated immunity. Briefly, A549 cells were co-cultured with CD8+ T cells. Loss of circ_001678 expression resulted in increased CD8+ T cells and decreased cell apoptosis. On the other hand, the percentage of CD8+ T cells was depleted and cell apoptosis was enhanced following re-expression of ZEB1. However, the administration of anti-PD-1/anti-PD-L1 led to increased percentage and reduced apoptosis of CD8+ T cells co-incubated with A549 cells treated with the sh-circ_001678 and oe-ZEB1 (Fig. 6A). Overall, the aforementioned results suggested that circ_001678 promoted the apoptosis of CD8+ T cells by up-regulating ZEB1.

Figure 6

The regulatory effect of circ_001678-mediated ZEB1 expression on CD8+ T cell activity and NSCLC immune escape. (A) The apoptotic rate and percentage of CD8+ T cells in the co-culture system determined by flow cytometry. (B) The proliferating capacity of A549 cells with OD values at 0, 12, 24, 36, 48 and 60 h examined using CCK-8 assay. (C) The representative images showing invasion and migration of A549 cells by means of Transwell assay. (D) The number of invasive and migratory cells. ^*P < 0.05 vs. sh-NC-treated cells, #P < 0.05 vs. cells co-treated with sh-circ_001678 and vector. %P < 0.05 vs. cells co-treated with sh-circ_001678 and ZEB1. The cell experiment was repeated three times.

Next, we further investigated the effect of circ_001678 on the progression of NSCLC by regulating the activity of CD8+ T cells via mediation of ZEB1. Subsequent results illustrated that circ_001678 knockdown in A549 cells led to decreased cell proliferation and reduced number of invasive and migratory cells. On this basis, we over-expressed ZEB1, which brought about accelerated cell proliferation and elevated migratory and invasive cell numbers. However, the manipulation of anti-PD-1/anti-PD-L1 impeded the cell proliferation and reduced the invasive and migratory cell numbers (Fig. 6B-D). Together, these findings suggested that circ_001678 promoted immune escape in NSCLC by up-regulating the expression of ZEB1 and inhibiting CD8+ T cell activity.

circ_001678 promotes the growth of NSCLC in nude mice by activating the PD-1/PD-L1 pathway

Lastly, we examined the influence of circ_001678 on tumorigenesis in nude mice by means of animal experiment in vivo. Briefly, A549 cells treated with oe-circ_001678 or untreated were injected into the back of nude mice to establish xenograft tumors. The volume and weight of tumors found to be markedly increased in mice intervened with oe-circ_001678 and IgG as compared to those in mice treated with oe-NC and anti-IgG. Moreover, combined treatment of oe-NC and anti-PD1/anti-PD-L1 led to decreased weight and volume of tumors in comparison with those treated with anti-IgG and oe-NC. Meanwhile, in the mice over-expressing circ_001678, anti-PD1/anti-PD-L1 treatment induced a reduction in the weight and volume of tumors (Fig. 7A-C).

Figure 7

The regulatory effect of circ_001678-mediated the PD-1/PD-L1 pathway on the growth of NSCLC cells in nude mice. (A) Representative tumor tissue images of nude mice. (B) Line graph of tumor volume changes in nude mice, measured every 7 days. (C) Comparison of tumor weight in nude mice. (D) The proportion of CD8+ T cells in tumor tissues of nude mice detected using flow cytometry. (E) The protein expression of ZEB1, Ki67 and PCNA in the tumor tissues of nude mice detected by immunohistochemical staining. ^*P < 0.05 vs. treatment with oe-NC and IgG. #P < 0.05 vs. treatment with circ_001678 and IgG. n = 5.

The proportion of CD8+ T cells in tumor tissues of nude mice were further found to be lowered upon circ_001678 over-expression, while being elevated after treatment with anti-PD1/anti-PD-L1. Moreover, anti-PD1/anti-PD-L1 elevated this proportion in the nude mice over-expressing circ_001678 (Fig. 7D).

As evidenced by the results of immunohistochemical staining, the levels of ZEB1 and tumor proliferation markers (Ki67 and PCNA) were all enhanced following circ_001678 over-expression. Meanwhile, the levels of ZEB1, Ki67 and PCNA were diminished after administration of anti-PD1/anti-PD-L1. Likewise, the increases of ZEB1, Ki67 and PCNA levels triggered by circ_001678 were inhibited by anti-PD1/anti-PD-L1 relative to anti-IgG (Fig. 7E).

To sum up, the aforementioned findings indicated that circ_001678 accelerates the growth of NSCLC through the PD-1/PD-L1 pathway activation, while anti-PD1/anti-PD-L1 could block the cancer-promoting activity of circ_001678.

Discussion

circRNAs represent a new type of non-coding RNAs that exert a plethora of key functions in the progression of cancer (20). To name a few, the ectopic expression of circ_ciRs-7 is associated with advanced disease and poor prognosis in NSCLC patients, whereas inhibition of circ_ciRs-7 can inhibit tumor cell proliferation and enhance their apoptosis (21). Herein, the current study sought to profile the role of a circRNA–miRNA–mRNA axis in the occurrence and progression of NSCLC from the aspect of immune escape. Consequently, the obtained findings revealed that circ_001678 promoted CD8+ T cell apoptosis to induce immune escape in NSCLC by modulating miR-326/ZEB1 and activating the PD-1/PD-L1 pathway.

Firstly, experimental findings in our study illustrated that circ_001678 was highly-expressed in NSCLC tissues, and the up-regulation of circ_001678 could expedite the proliferating, migrating and invading processes of A549 cells in NSCLC. Our study is not the first of its kind to uncover the involvement of various circRNA members in NSCLC. For instance, circ_0043278 is markedly enhanced in NSCLC, and further promotes NSCLC cell proliferative and migratory abilities (22). Moreover, the expression of circ_000984 is also up-regulated in NSCLC tissues and cell lines, whereas, knockdown of circ_000984 leads to the suppression of in vitro malignant progression of NSCLC (23). Further in line with our findings, circ_0026134 is over-expressed in NSCLC; whereas the down-regulation of circ_0026134 was previously associated with attenuation of NSCLC cell proliferation and metastatic properties while boosting their apoptosis (24). Further experimentation in our study revealed that circ_001678 could function as a sponge of miR-326. Interestingly, there is prior evidence to suggest that miR-326 is poorly-expressed in NSCLC (25). In addition, previous studies have highlighted miR-326 as a direct target of circ_0005397, such that circ_0005397 sponges miR-326 to facilitate cancer progression in hepatocellular carcinoma (26). Besides, circ_POLA2 strengthens lung cancer cell stemness and stimulates cancer progression similarly via sponging and inhibiting the expression of miR-326 (27). Altogether, the aforementioned evidence in conjunction with our findings support the conclusion that circ_001678 sponges miR-326 in NSCLC cells, thereby accelerating cancer progression in vitro.

Additional experimentation in our study demonstrated that the circ_001678/miR-326 axis could activate the PD-1/PD-L1 pathway and promote the apoptosis of CD8+ T cells, thereby augmenting the immune escape of NSCLC cells. What is more, a recent systematic review and meta-analysis indicated that PD-1/PD-L1 checkpoint blockade immunotherapy for NSCLC patients contributes to a markedly higher objective response rate when applied in first-line setting relative to treatment with chemotherapy (28). Meanwhile, PD-1/PD-L1 inhibitors are further regarded as an effective strategy for PD-L1-positive advanced NSCLC patients owing to its superior safety to chemotherapy (29). In addition, prior studies have shown that miR-145 directly-targets the 3'-UTR region of PD-L1, and circ_0003288 acts as a sponge of miR-145 to activate PD-L1 expression, whereby contributing to immune evasion. Similarly, elevation of circ_0003288 was previously shown to enhance PD-L1 levels in L02 cells and promote the EMT, migration and invasiveness in hepatocellular carcinoma (30). On the other hand, re-expression of miR-326 can significantly limit the proliferation and invasiveness of NSCLC cells (31), which underscores the antitumor role of miR-326 consistent with our findings. Recent investigations have uncovered that miR-326 gain-of-function restrains the EMT and invasiveness of NSCLC cells (32), whereas miR-326 depletion exerts an inhibitory effect on the growth of NSCLC suppressed by Propofol (33). Furthermore, our findings evidenced an additional mechanism showing that circ_001678 promoted the expression of ZEB1 by sponging miR-326 during the immune escape of NSCLC. Supported by the hard-done work of our peers, ZEB1 is established as the direct target of miR-326, and up-regulation of miR-326 leads to ZEB1 down-regulation; meanwhile, miR-326-targeted suppression of ZEB1 contributes to preventing the proliferation, invasiveness and migration of lung adenocarcinoma cells, as well as overcoming the resistance to apoptosis (34). Furthermore, we also determined that ZEB1 levels were enhanced in NSCLC cells. In accordance with our data, a prior study documented that ZEB1 is highly-expressed in NSCLC cells and tissues, while its post-transcriptional suppression driven by miR-5702 is associated with significant inhibition of NSCLC development (35). Besides, we further revealed that circ_001678 elevated the expression of ZEB1, and promoted CD8+ T cell apoptosis to facilitate immune evasion in NSCLC. In partially agreement with the latter, a prior study highlighted that ZEB1 attenuates miR-200-mediated inhibition of PD-L1 on tumor cells, resulting in immunosuppression of CD8+ T cells and metastasis (14). Several studies have shed a light on the association between ZEB1 and PD-1-mediated immune evasion in other cancers, such as melanoma (36) and colorectal cancer (37). It is also noteworthy that latest investigations have elucidated that ZEB1 up-regulation stimulates the transcription of PD-L1, thereby leading to the immune evasion of NSCLC (38). Lastly, we uncovered that circ_001678 promoted the growth of NSCLC in nude mice by activating the PD-1/PD-L1 pathway, while anti-PD-1/anti-PD-L1 could block the cancer-promoting effect of circ_001678, as evidenced by reduced tumor volume and weight as well as decreased levels of ZEB1, Ki67 and PCNA. Conforming to our results, the study performed by Song et al. (39) indicated that PD-L1 silencing decreases the gene expression of Ki67 in A549 cells and its suppression inhibits the proliferation of tumor cells.

Based on the aforementioned findings and evidence, it would be plausible to conclude that the circ_001678/miR-326/ZEB1 axis alters CD8+ T cell apoptosis to modulate immune escape in NSCLC by mediating the PD-1/PD-L1 pathway (Fig. 8). Our discoveries may pave the way for a novel theoretical basis for clinical targets in the immunotherapy of NSCLC.

Figure 8

Molecular mechanism diagram of circ_001678/miR-326/ZEB1 crosstalk network involved in immune escape of NSCLC cells. circ_001678 elevates ZEB1 expression by sponging miR-326, activates PD-1/PD-L1 pathway and promotes CD8+ T cell apoptosis, thereby enhancing NSCLC immune escape and NSCLC progression.

Materials and Methods

Ethics statement

The current study was approved by the Ethics Committee of the First Hospital of Qinhuangdao, and performed in strict accordance with the Declaration of Helsinki. Signed informed consents were obtained from all participants prior to specimen collection. Extensive efforts were undertaken to minimize both the number and respective suffering of the experimental animals.

Collection of clinical samples

A total of 10 cases of NSCLC and adjacent normal tissue samples were resected by thoracic surgery at the First Hospital of Qinhuangdao during 2019–2020 and included in the current study. After surgery, the obtained samples were independently diagnosed and confirmed as NSCLC by two doctors from the Department of Pathology according to morphology and immunohistochemical staining results. None of the enrolled patients received any treatment prior to sample collection. The obtained tissue samples were immediately frozen in liquid nitrogen and preserved at −80°C for further experimentation.

High-throughput circRNA sequencing analysis

Total RNA content was isolated from 10 NSCLC tissues and adjacent normal tissues using total RNA isolation kits (Life Technologies, Carlsbad, California), and quantified with the help of a Nanodrop ND-1000 spectrophotometer (ThermoFisher, Waltham, Massachusetts). The RNA integrity was assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, California). Next, high-quality total RNA was reverse-transcribed into cDNA to construct circRNA library, and the NextSeq CN500 from Illumina (San Diego, California) was adopted for sequencing. The generated original image data were converted into raw reads by base calling. To control the quality of raw reads, the Cutadapt command-line tool was utilized to remove the sequencing adapter sequence of raw reads and eliminate low-quality sequences. The remaining reads were regarded the ‘clean reads.’ The sequence was aligned to the human reference genome employing Hisat2 tool, and subsequently the gene expression was quantified using the R software package to produce the expression matrix. Afterwards, the R software package ‘DESeq2’ was adopted to analyze the differential expression, and the thresholds were set as |log2FC| > 1 and P < 0.05.

Cell culture and reagents

Three human NSCLC cell lines (namely, NCI-H358, NCI-H1299 and A549) and human normal bronchial epithelial cell line (HBE1) were procured from Mingzhou Biotechnology (Ningbo, China; MZ-0240, MZ-0218 and MZ-0015). The obtained NCI-H358, NCI-H1299 and HBE1 cells were cultured with RPMI-1640 medium (HyClone, Logan, UT) containing 10% fetal bovine serum (FBS, Gibco, Carlsbad, California), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Meanwhile, the A549 cells were cultured with Ham’s F-12 K medium (HyClone) containing 10% FBS (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco) in a 37°C thermostatic incubator with 5% CO₂ in air.

Recombinant fully humanized anti-PD-1 monoclonal antibody Tyvyt (Sintilimab) was purchased from Innovent Biologics (Suzhou, China), while the recombinant fully humanized anti-PD-1 monoclonal antibody Tecentriq (Atezolizumab) was procured from Roche (Basel, Switzerland).

Cell transfection

A549 cells were manipulated with oe-NC, circ_001678, sh-circ_001678, sh-NC, NC mimic, miR-326 mimic, NC inhibitor and miR-326 inhibitor. Briefly, 1 × 10⁵ cells were seeded in each well of a 6-well plate. Upon reaching 60–70% confluence, 750 μL optiMEM was added to each well. Next, 125 μL optiMEM was mixed with 5 μL Lipo3000 (L3000001, Invitrogen) and plasmid, respectively, and then the two mixtures were mixed together. Subsequently, 250 μL solution was added to the well and shaken slightly and evenly. Lastly, the plate was placed in an incubator, and the medium was changed after 16 h. Afterwards, cellular RNA content was extracted 48 h later and cellular protein content was extracted 72 h later.

Reverse transcription quantitative polymerase chain reaction

Total RNA content was extracted from cells using the TRIzol reagent (15596026, Invitrogen). To obtain mRNA, cDNA was reverse-transcribed from 1 μg total RNA with First Strand cDNA Synthesis kits (K1622, Fermentas, Hanover, MD). Meanwhile, to obtain miRNA, TaqManTM MicroRNA Reverse Transcription kits (4366597, Applied Biosystems, Waltham, Massachusetts) were adopted for reverse transcription into cDNA. To obtain circRNA, reverse-transcription was performed using the circRNA fluorescence quantitative PCR assay reagent (GS0201-2, Guangzhou Geneseed Biotech Co., Ltd, China). PCR was subsequently performed with the help of Fast SYBR Green PCR kits (Applied Biosystems) and the ABI Prism 7300 RT-PCR system (Applied Biosystems), with three duplicate wells set for each sample. U6 was adopted as internal reference for circRNA and miRNA, while GAPDH was regarded as internal reference for other genes. The 2^-ΔΔCT method was utilized for relative quantification of the target gene expression level. All abovementioned primers were purchased from Sangon Biotech (Shanghai) Co., Ltd and primer sequences are listed in Supplementary Material, Table S1. The reverse universal primer was CTCAACTGGTGTCGTGGA.

Cell counting kit-8 assay

A549 cell proliferation was examined with the help of CCK-8 kits (CA1210, Solarbio, Beijing, China). Briefly, cells were detached and centrifuged, and the cell suspension was added to a 96-well plate, with 5 × 10³ cells added to each well and 5 duplicate wells set for each group. Next, the CCK-8 reagent (10 μL) was supplemented at the 0, 24, 48 and 72 h time intervals, respectively. Later, the optical density value was measured at 450 nm using a microplate reader, and cell viability was calculated with the GraphPad Prism7 software.

Transwell assay for detection of cell migration and invasion

In the invasion experiment, the chamber was pre-coated with Matrigel (356 235, BD-Biocoat). After 24 h of culture in serum-free DMEM, the A549 cells were detached and dispersed into a cell suspension (1 × 10⁵ cells/mL). Next, DMEM containing 10% FBS was introduced to the basolateral chamber, and 200 μL cell suspension was added to the apical chamber. After 48 h, the non-invaded cells and Matrigel were carefully removed with cotton swabs. Cells that had migrated to, or invaded through the basolateral chamber were fixed with 4% paraformaldehyde for 10 min, and stained using 0.5% crystal violet. Five visual fields were randomly chosen under an inverted microscope for observation, photographing and cell counting. No Matrigel was required for migration assay, and the remaining procedures were the same as those in the invasion experiment.

In vitro co-culture system

The co-culture system contained the Transwell chambers (8 μM pore size, Millipore Corporation, Billerica, Massachusetts). Briefly, A549 cells were seeded in the apical compartment and CD8+ T cells in the basolateral compartment, allowing direct contact of A549 cells with CD8+ T cells. Next, the Ficoll density gradient method was adopted to isolate peripheral blood mononuclear cells (PMBCs) from the peripheral blood samples of healthy volunteers. The obtained single cell suspension was incubated with the CD8 fluorescent antibody in a 4°C refrigerator for 45 min in conditions void of light, diluted in serum-free medium, and finally sorted on machine.

Flow cytometry

Each well was seeded with 1 × 10⁶ A549 cells in a 6-well plate. After 24 h, the cells were detached and subjected to centrifugation. In accordance with the instructions of the Annexin V fluorescein isothiocyanate (FITC)/prodium lodide (PI) double staining apoptosis detection kits (Bestbio, Shanghai, China), the cell precipitate was resuspended with 400 μL Annexin V binding solution. Subsequently, 10 μL of PI and 5 μL of Annexin V-FITC staining were added to cells for incubation in conditions void of light at 4°C for 10 min. Afterwards, a FACSCalibur flow cytometer (BD Biosciences, San Jose, California) was utilized to determine the percentage and apoptosis of CD8+ T cells.

RNA pull-down and RNA immunoprecipitation assays

Biotin-labeled circ_001678 and NC probes were synthesized by Shanghai Genechem Co., Ltd (China). Briefly, A549 cells were fixed with 1% formaldehyde and then lysed in co-immunoprecipitation (Co-IP) buffer, followed by sonication and centrifugation. Next, incubation was performed at 30°C with the supernatant cultured together with M280 streptavidin Dynabeads (Invitrogen) for 12 h. Subsequently, lysis buffer and proteinase K were added for reaction, followed by the TRIzol extraction (Invitrogen) of RNA. The expression of various miRNAs precipitated by circRNA was detected by means of RT-qPCR.

With the help of biotin-labeled miR-326 and NC probes (Shanghai Genechem Co., Ltd), procedures were performed as stated above. The expression patterns of circ_001678 was measured to verify the interaction between miR-326 and circ_001678.

A RNA immunoprecipitation (RIP) assay was carried out in accordance with the instructions of the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore). In short, cell lysates were incubated with magnetic beads pre-coated with AGO2 antibody (Millipore) or IgG antibody (Millipore) at 4°C for 12 h, followed by extraction of total RNA content and RT-qPCR detection of enriched circ_001678 and miR-326.

Dual-luciferase reporter gene assay

The binding sites of miR-326 with circ_001678 and ZEB1 were predicted with the help of the StarBase database. Next, circ_001678 or ZEB1 3’untranslated region (UTR) sequences harboring the miR-326 binding site were fused into psiCHECK-2 luciferase vectors (Promega Corporation, Madison, Wisconsin) to establish circ_001678-WT (CCCAGAG) or ZEB1-WT (CCAGAG) sequences. Mutations were introduced into the circ_001678 or ZEB1 3’UTR sequences conforming to manufacturer’s instructions of the SiteDirected mutagenesis kit (Transgene Biotech, Beijing, China) and fused into the psiCHECK-2 luciferase reporter vector to establish circ_001678-MUT (GGUCUC) or ZEB1-MUT (GGUCUC) sequences. Cells on the 24-well plates were subsequently introduced with WT or MUT sequences together with miR-326 mimic or NC mimic following 24 h culture. After a 24 h period of transfection, luciferase activity was assessed using a dual luciferase assay system (E1910, Promega Corporation).

Fluorescence in situ hybridization

Cy3-tagged circ_001678 probe and fam-tagged miR-326 probes were procured from Genepharma (Shanghai, China). Next, FISH assay was carried out in accordance with the manufacturer’s instructions of a Fluorescent In Situ Hybridization Kit (Genepharma). Signals were photographed using a Leica TCS SP8 Laser Scanning Confocal Microscope (Leica Microsystems, Mannheim, Germany).

In vivo tumor growth

A total of 30 male BALB/c nude mice (aged 4–6 weeks, weighing 17–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and placed in a specific-pathogen-free environment of 24–26°C, with 50% humidity for 1 week as the acclimatization period. The nude mice were randomly divided into six groups (oe-NC + IgG, oe-NC + anti-PD-L1, oe-NC + anti-PD-1, circ_001678 + IgG, circ_001678 + anti-PD-L1 and circ_001678 + anti-PD-1), with five mice in each group. Next, 2 × 10⁶ A549 cells stably transfected with oe-NC or circ_001678 were implanted subcutaneously into the left dorsal portion of each mouse. When the tumor volume reached approximately 100 mm³, the mice were administered with 100 μg anti-PD1 or anti-PD-L1 (Hengrui Medicine Company, Jiangsu, China) or its mouse isotype control IgG via the tail vein, 3 times per week, for a duration of 2 weeks. Tumor volume (volume = maximum diameter × minimum diameter² × 0.5) was measured every 7 days from the first treatment of the nude mice. When the maximum tumor volume reached 1000 mm³, the nude mice were euthanized.

Immunohistochemical staining

Transplanted tumor tissues from nude mice or clinical tissues were fixed with 10% formalin solution, followed by dewaxing with xylene and hydration with gradient ethanol. Next, the samples were incubated with H₂O₂ for 10 min and added with 0.01 mol/L citrate, followed by undergoing microwave repair for 20 min. Subsequently, the samples were incubated with one drop of normal goat serum for 5 min, and then added with rabbit primary antibodies against CD8 (1: 100, ab237709, Abcam), ZEB1 (1: 100, ab203829, Abcam), PCNA (1: 200, ab92552, Abcam) and Ki67 (1: 200, ab15580, Abcam) at 4°C overnight. The following day, biotinylated goat anti-rabbit IgG (1: 500, ab150077, Abcam) serving as the secondary antibody was added for another 30-min incubation at 37°C. Following DAB color development (DA1015, Solarbio) for 1–2 min, the samples were counterstained with hematoxylin (G1080, Solarbio) for 1 min, dehydrated, cleared and sealed with neutral gum. Afterwards, the samples were observed under a light microscope (BX50; Olympus, Tokyo, Japan) in five randomly-chosen representative high-power visual fields. Brown chromatin was indicative of positive immune response.

Statistical analysis

Statistical analyses were performed using the SPSS 21.0 software (IBM, Armonk, New York). Measurement data were presented as mean ± standard deviation. Paired t test was conducted for comparisons of paired data that conformed to normal distribution and homogeneity of variance, and unpaired t test was utilized for unpaired data. Additionally, one-way analysis of variance (ANOVA) was performed for multiple group comparison, followed by Tukey's post-hoc test. Time-based data were compared by means of repeated measurement ANOVA. If data did not conform to normal distribution or equal variance, the rank sum test was performed. A value of P < 0.05 was regarded statistically significant.

Funding

Key Project of Medical Science Research of Ministry of Health of Hebei Province (No. 20130289); Science and Technology Plan of Qinhuangdao Science and Technology Bureau (No. 201601B045) and Natural Science Foundation of Hebei Province (No. H2021107002).

Conflict of Interest Statement. The authors declare no conflicts of interest.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors’ Contributions

Conceived and designed the experiments: Q.T., T.W. Performed experimental: X.D.Z., K.X., X.B.Y. Performed experimental validation: X.J.W., S.S.S., P.W. Analyzed the data and conceived figures: L.M.G., S.F.X., X.Y.L. Wrote the paper: Q.T., T.W. Contributed to the writing of the manuscript: L.M.G., S.F.X., X.Y.L. All authors revised and approved the final version of the manuscript.

Author notes

Qi Tian, Tong Wu are regarded as co-first authors.