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. 2022 Jul;14(7):2493-2510.
doi: 10.21037/jtd-22-294.

The role of miR-145-5p in esophageal squamous cell carcinoma tumor-associated macrophages and selection of immunochemotherapy

Junhong Lin #  1 , Sikai Wu #  2 , Kanghao Zhu  3 , Jian Zhang  2 , Xiaoshun Shi  4 , Jianfei Shen  2 , Jianfeng Xu  5
Affiliations

The role of miR-145-5p in esophageal squamous cell carcinoma tumor-associated macrophages and selection of immunochemotherapy

Junhong Lin et al. J Thorac Dis. 2022 Jul.

Abstract

Background: The impact of miR-145-5p in immune infiltration and the potential application in esophageal squamous cell carcinoma (ESCC) immunochemotherapy remains unknown.

Methods: Transcriptomic data for ESCC tissues and normal tissues and clinical materials of patients with ESCC were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. The differences in mRNA levels in cancer tissues and noncancerous tissues were analyzed, and we subsequently investigated the association between miR-145-5p expression and the key parameters of ESCC progression and prognosis. Additionally, cytological experiments were performed to evaluate the biological functions of miR-145-5p. Pathways potentially affected by miR-145-5p were analyzed by Gene Set Enrichment Analysis (GSEA) and REACTOME. We also analyzed the function of miR-145-5p in immune infiltration through the TIMER2 (Tumor Immune Estimation Resource) database.

Results: The analysis of gene chip data from the TCGA database and GEO database (including GSE13937 and GSE43732) showed that the expression of miR-145-5p is downregulated in ESCC (P<0.05) and that patients with high miR-145-5p levels had lower survival rates (P<0.05). The expression of miR-145-5p was significantly correlated with the disease-free survival (DFS) rate (P<0.05) and M stage (P<0.05) in the TCGA database and was significantly correlated with the T stage (P<0.05) and TNM stage (P<0.05) in the GSE13937 database. Functional experiments showed that miR-145-5p attenuated proliferation (P<0.05), migration (P<0.01) and invasion (P<0.01) in the Eca109 cell line. Both GSEA gene enrichment and REACTOME gene enrichment revealed that miR-145-5p was associated with tumor signaling pathways and immune signaling pathways. Immune infiltration analysis revealed that the expression level of miR-145-5p was significantly correlated with the infiltration level of macrophages (P<0.05) and was positively correlated with the level of gene markers of M2 macrophages and tumor-associated macrophages (P<0.05).

Conclusions: MiR-145-5p acts as a tumor suppressor microRNA in ESCC and is an important noncoding RNA in the high M2-like tumor-associated macrophage infiltration of ESCC. Assessing the miR-145-5p level in ESCC samples has translational meaning, which help illustrate the immune infiltration status, predict the prognostic outcome, and select the type of immunochemotherapy.

Keywords: esophageal squamous cell carcinoma (ESCC); immunochemotherapy; miR-145-5p; prognosis.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-22-294/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The difference in miR-145-5p levels between ESCC and adjacent noncancerous ESCC tissues. (A) Based on TCGA data; (B) based on GSE43732 data; (C) based on GSE13937 data. The error bars represent the standard deviation. *, P<0.05; ****, P<0.0001. ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas.
Figure 2
Figure 2
Kaplan-Meier curve for overall survival rate in ESCC patients grouped by the expression level of miR-145-5p. (A) Based on TCGA data; (B) based on GSE43732 data; (C) based on GSE13937 data. ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas.
Figure 3
Figure 3
Kaplan-Meier curve for disease-free survival rate in ESCC patients grouped by the expression level of miR-145-5p based on TCGA data. ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas.
Figure 4
Figure 4
Kaplan-Meier curve for overall survival rate in ESCC patients with neoadjuvant chemoradiotherapy grouped by the expression level of miR-145-5p based on GSE13937 data. ESCC, esophageal squamous cell carcinoma.
Figure 5
Figure 5
MiR-145-5p inhibited the proliferation, migration and invasion of Eca109 cells. (A) The overexpression efficiency of miR-145-5p detected by qRT-PCR; (B) the absorbance values of Eca109 infected with miR-145-5p overexpressing and miR-NC lentivirus and the control group measured at 450 nm; (C,D) representative images of migration and invasion, average counts of migrated and invasive cells from five random microscopic fields (mean ± SE). Crystal violet staining. *, P<0.05; **, P<0.01. qRT-PCR, quantitative real-time polymerase chain reaction.
Figure 6
Figure 6
Network of enrichment analysis for miR-145-5p of ESCC. (A) The workflow showed that the mRNA and miR-145-5p expression of TCGA_ESCC was enriched with GSEA. The bar chart shows the top 25 enrichment pathways by size and P value. Inflammation, metastasis, and metabolism were correlated with gene enrichment. (B) The workflow showed that the targets of miR-145-5p in the miRTarBase were enriched with REACTOME. The bubble pattern shows the top 25 enrichment pathways with the entities ratio, entities found (count), and entities p-value. The bar chart demonstrates that the gene sets involved in the cancer, immune system, metabolism, and signal transduction were significantly enriched in pathways related to miR-145-5p. ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas; GSEA, Gene Set Enrichment Analysis.
Figure 7
Figure 7
Prospective target genes of mir-145-5p. (A) Network analysis of the prospective target genes of miR-145-5p. Network nodes represent proteins; colored nodes, query proteins and first shell of interactors; white nodes, second shell of interactors. Small nodes are proteins of unknown three-dimensional structure; large nodes are proteins for which some of the three-dimensional structure is known or predicted. (B) The expression of heatmaps in miR-145-5p and prospective target genes based on TCGA data. TCGA, The Cancer Genome Atlas.
Figure 8
Figure 8
Correlation between miR-145-5p and key target genes. (A) TGFBR2 (r=0.3225, P=0.0033); (B) CTGF (r=0.4137, P=0.0001); (C) CDH2 (r=0.4305, P<0.0001).
Figure 9
Figure 9
Heatmap showing the correlation between miR-145-5p and immune infiltration level. (A) Based on TIMER database; (B) based on QUANTISEQ, CIBERSORT/CIBERSORT-ABS, XCELL, EPIC, and MCPCOUNTER databases.
Figure 10
Figure 10
Correlation between miR-145-5p and macrophage infiltration level. (A) Macrophages_TIMER (r=0.2977, P=0.0070). (B) Macrophages M2_CIBERSORT-ABS (r=0.2888, P=0.0089). (C) Macrophages M2_QUANTISEQ (r=0.3205, P=0.0035). (D) Macrophages M1_CIBERSORT-ABS (r=0.2397, P=0.0311).
Figure 11
Figure 11
Correlation between miR-145-5p and macrophage gene markers. (A) CLEC10A (r=0.4534, P<0.0001). (B) CSF1R (r=0.3782, P=0.0005). (C) IL-1A (r=−0.4014, P=0.0002). (D) IL-1B (r=−0.3594, P=0.0010). (E) CCR2 (r=0.3162, P=0.0040). (F) PDGFB (r=0.2222, P=0.0461).
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