CircRUNX1 was upregulated in LUAD tissues
Recently, a circular RNA profiling analysis has reported that circRUNX1 (hsa_circ_0002360) was upregulated in human LUAD tissues [18]. However, the biological functions of circRUNX1 on LUAD remains unclear. To further understand the clinical characteristics and significance of the circRUNX1 expression in LUAD, qRT-PCR was used to detect the expression level of circRUNX1 in 110 tumor tissues and paired adjacent non-tumor tissues of LUAD patients. Results indicate that the levels of circRUNX1 was significantly increased in tumor tissue compared with that in paired adjacent normal tissues (Fig. 1a). Next, we analyzed the relationship between circRUNX1 expression and the clinicopathological characteristics of 110 LUAD patients, as listed in Table 1. The results demonstrate that the expression levels of circRUNX1 in TNM stage III was significantly higher than that in TNM stage I-II (Fig. 1b). Additionally, tumor size bigger than 5 cm had higher circRUNX1 expression than those tumor size smaller than 5 cm (Fig. 1c). Subsequently, we investigated the prognostic implications of circRUNX1 expression in LUAD patients. Our results demonstrated that patients with higher circRUNX1 expression had a significantly poor prognosis than those with low levels of circRUNX1 (Fig. 1d and e). Multivariate analysis results indicated that circRUNX1 expression is an independent predictor for decreased overall survival (OS) rate and enhanced postoperative relapse rate (Tables 2 and 3). Taken together, our results indicate that circRUNX1 was significantly forced in LUAD tissues and correlated with bad prognosis of LUAD patients.
Table 1
Correlation between circRUNX1 and clinical characteristics in 110 LUAD patients.
Variables
|
circRUNX1 expression level
|
P value
|
Low
|
High
|
Age
|
|
|
|
60 <
|
28
|
24
|
0.567
|
60 ≥
|
27
|
31
|
Gender
|
|
|
|
Male
|
40
|
36
|
0.536
|
Female
|
15
|
19
|
Smoking status
|
|
|
|
Smokers
|
33
|
43
|
0.659
|
Nonsmokers
|
22
|
12
|
Tumor stage
|
|
|
|
I–II
|
46
|
30
|
0.002
|
III
|
9
|
25
|
Lymph node metastasis
|
|
|
|
Yes
|
34
|
29
|
0.441
|
No
|
21
|
26
|
Tumor size
|
|
|
|
≤ 5 cm
|
45
|
18
|
0.000
|
> 5 cm
|
10
|
37
|
Differentiation
|
|
|
|
Well and moderate
|
38
|
41
|
0.672
|
Poor
|
17
|
14
|
Table 2
Univariate and Multivariate Analyses of Factors Associated with Overall Survival
|
OS
|
|
|
Multivariate
|
Factors
|
Univariate, P
|
HR
|
95% CI
|
P value
|
Sex (Female vs. Male)
|
0.622
|
|
|
NA
|
Age (years) (≤ 60 vs. > 60)
|
0.481
|
|
|
NA
|
Smoking status (Smokers vs. Nonsmokers)
|
0.217
|
|
|
NA
|
Lymph node metastasis (Yes vs. No)
|
0.035
|
|
|
NS
|
Differentiation (Well and moderate vs. Poor)
|
0.042
|
|
|
NS
|
Tumor size (diameter, cm) (> 5 vs. ≤ 5)
|
0.015
|
1.375
|
1.194–2.492
|
0.039
|
TNM (III vs. I-II)
|
0.162
|
|
|
NA
|
CircRUNX1 expression (High vs. Low)
|
0.004
|
1.403
|
0.837–2.204
|
0.027
|
OS, overall survival; NA, not adopted; NS, not significantly; 95%CI, 95% confidence interval; HR, hazard ratio; Cox proportional hazards regression model. |
Table 3
Univariate and Multivariate Analyses of Factors Associated with Cumulative Recurrence
|
Cumulative Recurrence
|
|
|
Multivariate
|
Factors
|
Univariate, P
|
HR
|
95% CI
|
P value
|
Sex (Female vs. Male)
|
0.301
|
|
|
NA
|
Age (years) (≤ 60 vs. > 60)
|
0.582
|
|
|
NA
|
Smoking status (Smokers vs. Nonsmokers)
|
0.641
|
|
|
NA
|
Lymph node metastasis (Yes vs. No)
|
0.037
|
|
|
NS
|
Differentiation (Well and moderate vs. Poor)
|
0.212
|
|
|
NA
|
Tumor size (diameter, cm) (> 3 vs. ≤ 3)
|
0.046
|
|
|
NS
|
TNM (III-IV vs. I-II)
|
0.032
|
|
|
NS
|
CircFGFR1 expression (High vs. Low)
|
0.019
|
1.627
|
1.293–2.705
|
0.037
|
NA, not adopted; NS, not significantly; SCC, Squamous cell carcinoma; 95%CI, 95% confidence interval; HR, hazard ratio; Cox proportional hazards regression model. |
Higher level of circRUNX1 expression is correlated with decreased CD8+ T lymphocyte
Previous studies have been reported that circRNAs dysregulation authorize tumor immune evasion[10, 12], therefore, we wondered whether circRUNX1 overexpression might promote immunosuppressive microenvironment. Then, we explored whether forced circRUNX1 expression exhibited immune evasion signatures by qRT-PCR and IHC analysis in 30 LUAD patient tumor tissues. Interestingly, our results indicated that higher circRUNX1 expression was associated with decreased CD8+ T cells infiltration, while increased Foxp3+ cells infiltration (Fig. 2a). Scatter plot analysis demonstrated a negative correlation between circRUNX1 expression and CD8+ T cells infiltration in the LUAD tissues (Fig. 2b). Moreover, the results from scatter plot analysis also indicated a negative correlation between circRUNX1 expression and Foxp3+ T cells infiltration in the LUAD tissues (Fig. 2c). To determine whether increased circRUNX1 expression induces CD8+ T cell dysregulation, CD8+ T cells from 30 case LUAD patient tumor tissues were selected. Compared with CD8+ T cells in circRUNX1Low tissues, CD8+ T cells in circRUNX1High tissues demonstrated significant impairment of Granzyme B and perforin secretion when subsequently stimulate with a CD3 antibody (Fig. 2d and e).
Higher level of circRUNX1 expression is correlated with resistance to anti-PD-1 LUAD therapy
To determine the biological function of circRUNX1 in LUAD sensitivity to anti-PD1 therapy, we then explored retrospective data from 18 recurrent LUAD patients receiving anti-PD-1 therapy who had undergone primary tumor resection 2–24 months before the immunotherapy. Then, circRUNX1 expression in LUAD tissues were measured using qRT-PCR (Fig. 3a), and Kaplan–Meier survival analysis demonstrated that the progression-free survival for the circRUNX1-high group was much shorter than that for the circRUNX1-low group (Fig. 3b). To further determine the effects of circRUNX1 expression on the sensitivity of LUAD to anti-PD-1 immunotherapy, we analyzed circRUNX1 expression in five human LUAD cell lines using qRT-PCR (Supplementary Fig. 1a). To verify the biological functions of circRUNX1 in LUAD cells, we designed an overexpression lentivirus (circRUNX1-OE) transfected them into the NCI-H1975 and Clau-3 cell lines to upregulate circRUNX1 expression. The qRT-PCR results indicated that transfection of overexpressing circRUNX1 lentivirus vector resulted in significant forced circRUNX1 expression levels, while the expression levels of RUNX1 mRNA is not changed (Supplementary Fig. 1b and c). In addition, we also designed two shRNAs lentivirus vector targeting the back splicing site of circRUNX1 and transfected them into the PC-9 and HCC-827 cell lines to knockdown circRUNX1 (Supplementary Fig. 1d and e). Then, we explored the anti-tumor effects of the PD-1 antibody in humanized NSG mice that received LUAD cells with forced circRUNX1 expression or the respective mock cells (NCI-H1975-Mock, NCI-H1975-circRUNX1). Compared to that of the NCI-H1975-Mock cell group, the tumor growth in the NCI-H1975-circRUNX1 cell recipient xenograft mice showed a significantly phenotype of resistance to anti-PD-1 immunotherapy, and the xenograft mice had a decreased survival time (Fig. 3c-e). To further explore the molecular mechanism of circRUNX1-mediated LUAD resistance to anti-PD-1 immunotherapy, we have examined the effect of circRUNX1 on the PD-L1 expression on LUAD cells. Our results showed that the expression of PD-L1 was not changed significantly when circRUNX1 was overexpressed or knockdown in the LUAD cell lines (Fig. 3f and g). Above results indicate that circRUNX1-mediated LUAD resistance to anti-PD-1 immunotherapy in a PD-1/PD-L1-independent manner.
CircRUNX1 acts as a sponge for miR-4739 in LUAD cells
To reveal the molecular mechanisms of circRUNX1-promoting LUAD immune evasion, we predicted the potential interaction between circRUNX1 and miRNA using the Star Base v3.0. We found that circRUNX1 can bind to 9 potential miRNAs based on StarBase v3.0. To determine the critical miRNA sponging circRUNX1, we performed a pulldown assay with a biotinylated circRUNX1 probe. As shown in Fig. 4a, we selected miR-4739 was the significantly captured miRNA for circRUNX1. To determine whether circRUNX1 and miR-4739 are co-localized, the FISH assay was used to NCI-H1975 and HCC-827 cells, and the results showed that circRUNX1 and miR-4739 were co-located major in the cytoplasm (Fig. 4b). AGO2 antibody-mediated RIP assay showed that circRUNX1 and miR-4739, but not circANRIL (a circRNA verified to not bind to AGO2 protein [19]), were significantly captured in NCI-H1975 and HCC-827 cells (Fig. 4c). To further verify the predicted interaction between circRUNX1 and miR-4739, we performed RNA-pull down assays with a biotinylated miR-4739 mimics. In both NCI-H1975 and HCC-827 cells, circRUNX1 was significantly captured (Fig. 4d). In addition, we performed a luciferase assay using miR-4739 mimics or negative control (NC) mimics co-transfected with pLG3 luciferase reporter plasmids (which contained a wild-type or miR-4739-target mutant circRUNX1 sequence) into HEK-293T cells. Compared with the NC mimics, miR-4739 inhibited the luciferase reporter activity significantly in the cells with the wild-type circRUNX1 sequence but not the cells with either the wild-type- or the miR-4739-target mutant circRUNX1 sequence (Fig. 4e and f). Importantly, the levels of circRUNX1 did not show obvious changes after miR-4739 expression was either upregulated or knocked down, and the levels of miR-4739 did not show obvious changes after circRUNX1 expression was either was overexpressed or knocked down (Fig. 4g-j;Supplementary Fig. 2a and b). These results demonstrate that miR-4739 and circRUNX1 are likely not degraded by each other.
PCSK9 was a direct target of miR-4739
To further reveal the underlying biological mechanisms of miR-4739 and its down-stream critical molecular, then, Targetscan was used to predict the potential target mRNAs of miR-4739. Interestingly, PCSK9, an immune-related gene is a potential target mRNA of miR-4739 (Fig. 5a). Dual-luciferase reporter assay results demonstrated that the relative luciferase activity of PCSK9 was obviously inhibited, suggesting that the PCSK9 mRNA is a target of miR-4739 (Fig. 5b). Then, the expression of PCSK9 mRNA and protein was detected in miR-4739 overexpression LUAD cell lines. The results indicated that the expression of PCSK9 mRNA and protein was significantly decreased in miR-4739-overexpressing cell lines (Fig. 5c and d). Next, the expression of PCSK9 mRNA and protein was examined in circRUNX1 overexpression and knockdown LUAD cell lines, respectively. Our results showed that the expression of PCSK9 mRNA and protein was significantly increased in circRUNX1-overexpressing cell lines, while decreased in circRUNX1 knockdown cell lines (Fig. 5e-h). Next, a miR-4739 target sequence mutant circRUNX1 overexpression lentivirus (circRUNX1-mOE) transfected into the NCI-H1975 and Clau-3 cell lines to further determined that circRUNX1-mediated PCSK9 upregulation was in a miR-4739-depedent manner (Supplementary Fig. 3a and b). Our results showed that the expression of PCSK9 mRNA and protein was not significantly increased in mutant circRUNX1-overexpressing cell lines (Fig. 5i and j).
Knocking out PCSK9 reverses circRUNX1-induced LUAD resistance to anti-PD-1 immunotherapy
Recently, it has been reported that higher levels of PCSK9 downregulates the expression of MHC I proteins on cancer cells and induces tumor resistance to anti-PD1 immunotherapy [15]. Therefore, we conjectured that circRUNX1 promotes LUAD resistance to anti-PD-1 immunotherapy via the miR-4739/PCSK9 axis. To further determine whether circRUNX1-induced resistance to anti-PD-1 immunotherapy of LUAD through the miR-4739/PCSK9 axis, we established PCSK9-knockout NCI-H1975 cells (NCI-H1975-KOP) (Fig. 6a). The PCSK9 protein did not detected after circRUNX1 expression was upregulated in the NCI-H1975 cells (Fig. 6b). Furthermore, we analyzed the anti-tumor effects of the PD-1 antibody in humanized NSG mice that received NCI-H1975-KOP cells with forced circRUNX1 expression or the respective mock cells (NCI-H1975-KOP-Mock, NCI-H1975-KOP-circRUNX1). Compared to that of the NCI-H1975-KOP-Mock cell group, the tumor growth in the NCI-H1975-KOP-circRUNX1 cell recipient xenograft mice showed no obviously phenotype of resistance to anti-PD1 immunotherapy (Fig. 6c-e). In addition, our results showed that the levels of MHC I was significantly decreased when circRUNX1 was overexpressed in the LUAD cells (Fig. 6f). To further explore the relationship between the circRUNX1 and immune evasion, we measured the MHC I expression in tissues in the above 30 cases of LUAD tissues (Fig. 6g). The results from a scatter plot analysis showed a negative correlation between circRUNX1 expression and MHC I expression in the LUAD tissues (Fig. 6h). These results indicate that circRUNX1 may exert its immunosuppressive effects by circRUNX1/ miR-4739/PCSK9/MHC I axis.