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Dual targeting of PD-L1 and PD-L2 by PCED1B-AS1 via sponging hsa-miR-194-5p induces immunosuppression in hepatocellular carcinoma

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Abstract

Background

PD-L1 and PD-L2 are PD-1 ligands (PD-Ls). PD-Ls over-expression is associated with poor prognosis in hepatocellular carcinoma (HCC). However, little is known about how PD-Ls expression is regulated. Here, we investigated the involvement of lncRNA-microRNA network in the regulation of PD-Ls in HCC.

Methods

The expression of PD-Ls, PCED1B-AS1 and hsa-miR-194-5p was measured in 45 pairs of HCC samples. The interaction between PCED1B-AS1 and hsa-miR-194-5p was measured by microRNA pull down and in vitro binding assay. The effects of PCED1B-AS1 knockdown and over-expression on hsa-miR-194-5p and PD-Ls expression were investigated in HCC cell lines. Immunosuppression was evaluated in co-culture of HCC cell line and human T cells. Exosomes were isolated from HCC cells and their effects on receipt cells were investigated. Tumor behaviors were evaluated by in vitro and in vivo assays.

Results

PD-L1 expression was highly correlated with PD-L2 expression in HCC. PCED1B-AS1 and hsa-miR-194-5p expression was up-regulated in HCC. PCED1B-AS1 was positively correlated with PD-Ls but negatively correlated hsa-miR-194-5p in HCC. These correlations were cross-validated by TCGA-LIHC dataset. PCED1B-AS1 interacted with hsa-mir-194-5p which inhibited PD-Ls expression. PCED1B-AS1 enhanced the expression of PD-Ls via sponging hsa-mir-194-5p. PCED1B-AS1-induced PD-Ls-mediated immunosuppression in co-cultured T cells. HCC cells released PCED1B-AS1 containing exosomes and the exosomal PCED1B-AS1 enhanced PD-Ls expression in receipt HCC cells while inhibited receipt T cells and macrophages. Blood exosomal PCED1B-AS1 was correlated with HCC PD-Ls expression. Finally, PCED1B-AS1 promoted cell proliferation, colony formation and in vivo tumor formation in xenografted nude mice while inhibited apoptosis.

Conclusions

PCED1B-AS1 enhances the expression and function of PD-Ls via sponging hsa-miR-194-5p to induce immunosuppression in HCC.

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Funding

The work was supported by project of Shanghai JiaDing District Health Commission (2017-KY-02).

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Correspondence to Caifeng Liu or Bin Wu.

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Conflict of interest

Fei Fan, Keji Chen, Xiaoliang Lu, Aijun Li, Caifeng Liu, Bin Wu have no conflicts of interest to declare.

Ethical approval

The study was approved by the Review Boards of Shanghai Eastern Hepatobiliary Surgery Hospital (Shanghai, China).

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Written informed consent was obtained from each patient.

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12072_2020_10101_MOESM1_ESM.tif

Supplementary Figure 1. Expression data and correlations in TCGA LIHC dataset. (A) Expression levels (TPM) of indicated lncRNAs in TCGA LIHC dataset. (B) Scatter plots showing the positive correlation of PCED1B-AS1 with PD-L1 (CD274) or PD-L2 (PDCD1LG2) in tumor tissues from TCGA LIHC dataset. (C) Scatter plots showing the positive correlation of PCED1B-AS1 with PD-L1 (CD274) or PD-L2 (PDCD1LG2) in normal control tissues from TCGA LIHC dataset. (D) Western blots showing PD-L1 and PD-L2 protein levels in Huh-7 cells after knockdown of indicated lncRNAs (TIFF 1870 kb)

12072_2020_10101_MOESM2_ESM.tif

Supplementary Figure 2. Correlations of hsa-mir-194-5p with PCED1B-AS1 and PD-Ls in TCGA LIHC dataset. (A) Workflow of predicting PD-L1 targeting microRNAs, PD-L2 targeting microRNAs and PCED1B-AS1 targeting microRNAs using TarPmiR and RNAhybrid. Scatter plots showing the negative correlations of hsa-mir-194-5p with PD-L1 (CD274) (B), PD-L2 (PDCD1LG2) (C) or PCED1B-AS1 (D) in tumor tissues from TCGA LIHC dataset (TIFF 625 kb)

Supplementary Table 1. List of PD-Ls correlated lncRNAs in TCGA LIHC dataset (XLSX 12 kb)

Supplementary Table 2. List of microRNA prediction by TarPmiR and RNAhybrid (XLSX 26 kb)

Supplementary Table 3. Clinico-pathological characteristics of patients (DOCX 16 kb)

Supplementary Table 4. Multivarite Analysis of Factors Associated With Survival (DOCX 13 kb)

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Fan, F., Chen, K., Lu, X. et al. Dual targeting of PD-L1 and PD-L2 by PCED1B-AS1 via sponging hsa-miR-194-5p induces immunosuppression in hepatocellular carcinoma. Hepatol Int 15, 444–458 (2021). https://doi.org/10.1007/s12072-020-10101-6

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  • DOI: https://doi.org/10.1007/s12072-020-10101-6

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