MiR-26b-5p mediates radioresistance and immunosuppression via targeting PRKCD in non-small cell lung cancer

Abstract

Purpose: Overcoming miRNA-mediated radioresistance and enhancing its synergy with immunotherapy remained significant challenges.

Methods: A total of 23 patients with locally advanced non-small cell lung cancer (NSCLC) undergoing thoracic radiotherapy from a single center were enrolled. Pre-radiotherapy blood samples were collected and analyzed using real time qPCR array to detect miRNA expression profiles, identifying differential miRNAs between responders and non-responders. In vitro experiments further assessed the impact of radiotherapy on significant differential miRNAs. Targeted immune genes of miRNAs were predicted through bioinformatics websites and validated by cellular experiments. Using TCGA and GEO datasets, the association between immune gene of interest and survival outcomes and immune infiltration were investigated. In vivo experiment was further performed to investigate the relationship between dendritic cell (DC) expression and miR-26b-5p following radiotherapy.

Results: Using pre-radiotherapy blood samples from 23 NSCLC patients, 22 differentially expressed miRNAs were identified between responders and non-responders. Among them, miR-26b-5p exhibited significant differential expression, suggesting its role as a potential radioresistant molecule. The dual-luciferase assay confirmed miR-26b-5p targeted PRKCD, an immune-related gene. After continuous three days of 2-Gy irradiation, the expression of miR-26b-5p decreased significantly, while the expression of PRKCD increased. The effect of radiotherapy on PRCKD expression were further validated in clinical samples, which demonstrated elevated PRCKD expression after thoracic radiotherapy. Bioinformatic analysis using TCGA and GEO datasets revealed that a higher PRKCD expression was correlated with better survival outcomes, increased immune cell infiltration, and better outcomes. Further in vivo experiments showed that, after radiotherapy, the inhibition of miR-26b-5p showed a significantly higher proportion of DCs than the controls, along with increased expression of CD80, CD86, and TNFSF4.

Conclusion: miR-26b-5p and PRKCD modulates dual resistance of both radiotherapy and immunotherapy in NSCLC. These insights demonstrate that downregulating miR-26b-5p could offer a promising therapeutic strategy to enhance radiosensitivity and immune responses.

Keywords: Immunotherapy; Non-small cell lung cancer; PRKCD; Radioresistance; miR-26b-5p.

Fig. 1

The clinical information for NSCLC patients who received thoracic radiotherapy. A a flow chart for illustrating the screening of miRNAs of interest, B baseline clinical features and the best overall response of all patients, C the computed tomography images both before and after thoracic radiotherapy of three typical cases

Fig. 2

The screening of potential miRNAs affecting response to radiotherapy. A using the relative expression to screen miRNAs, B using “limma” method to screen differential miRNAs, C a Venn plot to show the intersection miRNAs, D the intersecting miRNAs identified by two methods. ^#Method 1: Calculate the ratio of miRNA in radiosensitive group to radioresistant group, and formula is y = 2-(ΔCT2-ΔCT1). miRNAs with a relative expression level ≥ 2 folds and p value < 0.05 were selected. ^##Method 2: Differentially expressed miRNAs (|logFC|> 1 & p < 0.05) between two groups identified by limma package in R software

Fig. 3

The exploration of targeted immune genes of miR-26b-5p. A a Venn plot to show the intersection genes of immune genes and predicted targeted genes of miR-26b-5p, B the regulation of miR-26b-5p on selected targeted immune genes, C binding sites of PRKCD-3′UTR targeted by miR-26b-5p, D the expression level of PRKCD decreased after transfection with miR-26b-5p mimics in wild type compared to normal control, but not in mutation type

Fig. 4

Further validation of the role of miR-26b-5p in radiotherapy. A the apoptosis rate decreased after transfection with miR-26b-5p mimics in H460 cells, but no significant change when transfected with miR-26b-5p inhibitor, B no significant change was found in A549 cells, C & D in H460 cells, the relative expression of miR-26b-5p increased after three consecutive days of 2 Gy irradiation, while the relative expression of PRKCD decreased, E & F in A549 cells, the expression of miR-26b-5p increased, while no changes in PRKCD expression, G & H in HCC 827 cells, the expression of miR-26b-5p decreased, while the expression of PRKCD increased, I In the GSE162946 cohort, a trend of elevated expression of PRCKD after radiotherapy was found in primary lung lesions

Fig. 5

PRKCD was positive associated with better survival and immune infiltration. A high PRKCD expression was significantly associated with better overall survival in TCGA dataset, B as well as in GSE11969 dataset, C PRKCD expression were positively correlated with anti-tumor immune cells in TCGA dataset, D and in GSE11969 dataset, E & F macrophage and myeloid dendritic cell were the two same anti-tumor immune cells in both datasets, G PRKCD was positively correlated with multiple immunostimulatory molecules, H especially four molecules expressed on dendritic cells, i.e. CD28, CD80, CD86, and TNFRSF4

Fig. 6

PRKCD was positive associated with better survival in an immunotherapy cohort (GSE126044). A a Sankey diagram presented the relationship between clinical features and response to immunotherapy, B patients with higher PRKCD expression had a trend of better progression-free survival, and C overall survival

Fig. 7

MiR-26b-5p inhibition promotes dendritic cell activation following radiotherapy in vivo. A Flow cytometry shows the proportion of MHC-II⁺ CD11c⁺ dendritic cells (DCs) among CD45⁺ cells in H460 tumor-bearing nude mice. The percentage of DCs was significantly increased in the miR-26b-5p antagomir group compared to the control group after radiotherapy. B–D The mean fluorescence intensity of DC activation markers CD80 (B), CD86 (C), and TNFRSF4 (D) were markedly elevated in the antagomir group compared to controls