miR-22 gene therapy treats HCC by promoting anti-tumor immunity and enhancing metabolism

Abstract

MicroRNA-22 (miR-22) can be induced by beneficial metabolites that have metabolic and immune effects, including retinoic acids, bile acids, vitamin D₃, and short-chain fatty acids. The tumor suppressor effects of miR-22 have been suggested, but whether miR-22 treats orthotopic hepatocellular carcinoma (HCC) is not established. The role of miR-22 in regulating tumor immunity is also poorly understood. Our data showed that miR-22 delivered by adeno-associated virus serotype 8 effectively treated HCC. Compared with FDA-approved lenvatinib, miR-22 produced better survival outcomes without noticeable toxicity. miR-22 silenced hypoxia-inducible factor 1 (HIF1α) and enhanced retinoic acid signaling in both hepatocytes and T cells. Moreover, miR-22 treatment improved metabolism and reduced inflammation. In the liver, miR-22 reduced the abundance of IL17-producing T cells and inhibited IL17 signaling by reducing the occupancy of HIF1α in the Rorc and Il17a genes. Conversely, increasing IL17 signaling ameliorated the anti-HCC effect of miR-22. Additionally, miR-22 expanded cytotoxic T cells and reduced regulatory T cells (Treg). Moreover, depleting cytotoxic T cells also abolished the anti-HCC effects of miR-22. In patients, miR-22 high HCC had upregulated metabolic pathways and reduced IL17 pro-inflammatory signaling compared with miR-22 low HCC. Together, miR-22 gene therapy can be a novel option for HCC treatment.

Keywords: HIF1α; RORγ; hepatocellular carcinoma; immunotherapy; liver cancer; retinoic acid; tumor microenvironment.

Graphical abstract

Figure 1

miR-22 treats HCC and prolongs survival in female HCC mice (A) Study design for miR-22 and lenvatinib treatment in RAS/AKT-induced HCC model, (B) representative liver morphology, (C) liver weight, L/B ratio, spleen weight, serum ALT, AST, and cholesterol levels, and (D and E) H&E-stained liver sections and Ki67 IHC staining. The cellularity of the proliferating cells is seen at high magnification (insets). The tumor score was quantitively evaluated, which is detailed in Table S3. The percentage of Ki67-positive cells was determined in five random x10 microscopic fields for each section. Scale bar, 100 μm. (F) Kaplan-Meier survival curves of overall survival of three groups (n = 15–25/group). (G) Toxicology for the studied groups. (H) Study design of miR-22 treatment in β-catenin/AKT-induced HCC model. (I) Representative liver morphology and H&E-stained liver sections (scale bar, 100 μm), (J) L/B ratio, and (K) hepatic mRNA levels of HCC markers Afp and Gpc3 for studied group. Data represent mean ± SD (n = 6–8/group for B, C, E, G, I, J, and K). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way ANOVA (C, E, F, G, J, and K).

Figure 2

miR-22 treatment restores metabolic programs and reduces inflammatory signaling accompanied by reduced HIF1α expression in the liver, hepatocytes, and T cells (A) Pathways enriched due to HCC formation or miR-22 treatment revealed by GSEA based on KEGG gene sets. miR-22-reversed pathways are underlined and highlighted in red (upregulated) or blue (downregulated). NES, normalized enrichment score. (B) Enriched IL6/JAK/STAT3 and hypoxia signaling by comparing HCC vs. healthy livers or miR-22 treated vs. untreated HCC as demonstrated by GSEA based on hallmark gene sets. (C) Human and mouse miR-22 have conserved sequences, which partially pair with the 3′ UTR of the human and mouse Hif1a gene. (D) The level of miR-22 and HIF1α in hepatocytes and T cells isolated from livers of healthy, HCC, and miR-22-treated HCC mice (n = 3). (E) The levels of indicated proteins in the HIF1α/IL6/STAT3/IL17 axis were determined by western blot (n = 3). (F) The fold changes of HIF1α-regulated metabolism-related genes are shown in the heatmap based on RNA-seq data. Data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way ANOVA (D).

Figure 3

The anti-HCC effect of miR-22 is cytotoxic T cell dependent, and miR-22 activates cytotoxic T cells to induce apoptosis of HCC cells (A) Representative flow cytometry plots and percentage of CD8⁺IFNγ⁺, CD8⁺CD107A⁺, and CD8⁺ naive and EM T cells. Hepatic lymphocytes were isolated from livers of healthy, HCC, and miR-22-treated HCC mice followed by flow cytometry. (n = 6). (B) Study design of anti-CD8 antibody blockade. (C) Representative liver morphology, (D) L/B ratio (n = 6), and (E) percentage of CD8⁺ T cells measured by flow cytometry in studied groups (n = 4). (F) Representative flow cytometry plots of Annexin V/7-AAD staining and apoptosis rates of mouse HCC Hepa1-6 cells co-cultured with hepatic T cells. Hepatic-isolated T cells from healthy livers, HCC, and miR-22-treated HCC were co-cultured with Hepa1-6 at a 1:1 ratio for 36 h. (G) The concentrations of IFNγ, granzyme B, IL17A, and IL6, and in the supernatant were quantified by ELISA. Data are representative of two independent experiments (F and G). Data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way ANOVA (A, D, E, F, and G).

Figure 4

miR-22 suppresses IL17 signaling in the T cells by reducing the recruitment of HIF1α/RORγT/STAT3 in the Il17a promoter (A) The fold changes of RA signaling and Th17/Treg-related genes in hepatic T cells were quantified by RT-PCR and are shown in the heatmap. Hepatic T cells were isolated from livers of healthy, HCC, and miR-22-treated HCC mice followed by flow cytometry (n = 3). (B) Representative flow cytometry plots and percentage of Th17 (CD4⁺IL17A⁺), Tc17 (CD8⁺IL17A⁺), Th1 (CD4⁺IFNγ⁺), and Treg (CD4⁺CD25⁺FOXP3⁺) T cells in studied groups (n = 6). (C) ChIP-qPCR using anti-HIF1α, anti-RORγt, and anti-STAT3 antibodies in hepatic T cells. Hepatic T cells that were isolated from three mice for each studied group were subjected to ChIP assay. The primers for amplifying non-binding regions were used as a negative control. The regulatory regions of the Il17a and Rorc genes are shown with the binding locations of the indicated proteins. The numbers are relative to the transcription start site. Binding enrichment was expressed relative to the IgG-negative control. CNS, conserved non-coding sequence. Data represent mean ± SD, ∗∗p < 0.01; ∗∗∗p < 0.001 by one-way ANOVA (B and C).

Figure 5

Overexpression of IL23/IL17 attenuates the anti-HCC effect of miR-22 (A) Study design for miR-22 and IL23 overexpression in male HCC mice. (B) Representative liver morphology, (C) liver weight and L/B ratio, and (D) H&E-stained liver sections in each group (n = 8); scale bar, 100 μm. (E) mRNA levels of IL17A signaling-related genes in hepatic T cells isolated from indicated groups (n = 3). (F) Hepatic mRNA levels of HCC markers in each group (n = 3). Data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way ANOVA (C, E, and F).

Figure 6

Mouse and human HCC have similar gene expression profiles based on miR-22 levels revealing human relevance of the findings (A) Fifteen common pathways were identified by comparing miR-22 Hi (high, n = 89) vs. miR-22 Lo (low, n = 92) human HCC and miR-22 treated vs. untreated mouse HCC. (B) The fold changes of RA and IL17 signaling-related genes in miR-22 Hi vs. miR-22 Lo human HCC. (C) The levels of miR-22 in human HCC vs. normal livers and different stages of HCC. The numbers in parentheses are case numbers. Data were shown with medium ±5 to 95 percentiles. (D) Kaplan-Meier survival curves of HCC patients with high and low miR-22 levels based on TCGA LIHC. p values were calculated by the log rank test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired two-tailed Student’s t test (B and C).

Figure 7

The schematic diagram summarizes miR-22 treats HCC by inducing metabolism and modulating T cell reprogramming miR-22 treatment induces the metabolism of retinol, propanoate, butanoate, fatty acid, and sugar. Therefore, the miR-22 inducer signaling is compromised in HCC and restored due to positive treatment outcomes. Meanwhile, miR-22 inhibits inflammation pathways including IL17 signaling, cytokine-receptor interaction, and ECM-receptor interaction, which are all upregulated due to HCC development. In the T cells, miR-22 inhibits IL17 signaling at multiple levels: (1) miR-22 silences HIF1α and reduces its occupancy in the Rorc promoter. (2) miR-22 reduces Rorc expressions and the recruitment of RORγT/HIF1α to the Il17a gene leading to reduced expression. (3) miR-22 deactivates STAT3 and decreases its occupancy in the Il17a promoter, which consequentially reduces Il17a gene expression. Additionally, miR-22 reduces Treg cells. The reduced inflammatory signaling as well as immunosuppressive effects permit activation of cytotoxic T cells leading to cancer cell death.