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. 2023 Mar;114(3):937-947.
doi: 10.1111/cas.15644. Epub 2022 Dec 8.

Cancer-associated fibroblasts promote tumor cell growth via miR-493-5p in intrahepatic cholangiocarcinoma

Katsuya Toshida  1 , Shinji Itoh  1 , Noboru Harada  1 , Akinari Morinaga  1 , Kyohei Yugawa  1   2 , Takahiro Tomiyama  1 , Yukiko Kosai-Fujimoto  1 , Takahiro Tomino  1 , Takeshi Kurihara  1 , Yoshihiro Nagao  1 , Kazutoyo Morita  1 , Yoshinao Oda  2 , Tomoharu Yoshizumi  1
Affiliations

Cancer-associated fibroblasts promote tumor cell growth via miR-493-5p in intrahepatic cholangiocarcinoma

Katsuya Toshida et al. Cancer Sci. 2023 Mar.

Abstract

The association between tumor microenvironment (TME) and cancer-associated fibroblasts (CAFs) in intrahepatic cholangiocarcinoma (ICC) progression is poorly understood. This study aimed to reveal whether specific microRNAs (miRNAs) in extracellular vesicles (EVs) derived from CAFs were involved in ICC progression. Conditioned medium (CM) and EVs in the CM of CAFs and normal fibroblasts (NFs) derived from ICC specimens were used to investigate the effects on tumor cell lines. miRNA microarray assay was used to examine the miRNAs of EVs derived from CAFs and NFs in ICC, and the effects of miR-493-5p on tumor cell lines were examined. Additionally, databases were used to identify miR-493-5p targets, and the relationship between prognosis of ICC patients and cocaine- and amphetamine-regulated transcript propeptide (CARTPT), one of the targets of miR-493-5p, expression in ICC tissues was retrospectively analyzed. Compared with NF-derived CM and EVs, CAF-derived CM and EVs promoted cell lines in proliferation, scratch, migration, and invasion assays. miRNA microarray analysis revealed that miR-493-5p was significantly increased in CAF-derived EVs compared to NF-derived EVs. Tumor cell lines transfected with miR-493-5p were promoted in proliferation and scratch assays. Immunohistochemical staining was performed on 76 ICC specimens; both overall and recurrence-free survival rates were significantly worse in the CARTPT-negative group. Univariate and multivariate analyses showed that low CARTPT expression was an independent poor prognostic factor for overall and recurrence-free survival. Overall, our data suggest that CAFs in the ICC TME suppress CARTPT in tumor cells and promote tumor cells via miR-493-5p in EVs.

Keywords: cancer-associated fibroblast; extracellular vesicle; intrahepatic cholangiocarcinoma; microRNA; tumor microenvironment.

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Conflict of interest statement

Y.O. is an Editorial Board Member of Cancer Science. The other authors have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Characteristics of cancer‐associated fibroblasts (CAFs) and conditioned medium (CM) derived from CAFs promote intrahepatic cholangiocarcinoma (ICC) cell progression. The expression of fibroblast markers, α‐smooth muscle actin (αSMA), fibroblast activation protein α (FAPα), Vimentin, by CAFs and normal fibroblasts (NFs) isolated from ICC specimens was confirmed by (A) fluorescent immunostaining and (B) Western blotting. Conditioned medium isolated from CAFs significantly promoted the (C) scratch assay, (D) proliferation assay, (E) transwell migration, and (F) invasion assay of HuCCT1, HuH28, and SSP25 cells compared with CM from NFs.
FIGURE 2
FIGURE 2
Extracellular vesicles (EVs) in conditioned medium (CM) secreted from cancer‐associated fibroblasts (CAFs) promote tumor cells. EVs were isolated from CM and confirmed by (A) Western blot, (B) transmission electron microscopy, and (C) Nanosight. EVs isolated from CAFs significantly promoted the (D) scratch assay, (E) proliferation assay, (F) transwell migration, and (G) invasion assay of HuCCT1, HuH28, and SSP25 cells compared with negative control (NC) and CM from normal fibroblasts (NFs).
FIGURE 3
FIGURE 3
miR‐493‐5p in extracellular vesicles (EVs) derived from cancer‐associated fibroblasts (CAFs) promote tumor cells. (A) Microarray analysis of CAF‐derived EVs with normal fibroblast (NF)‐derived EVs showed that miR‐493‐5p was significantly increased in CAFs by (B) volcano plots. miR‐493‐5p significantly promoted (C) the proliferation assay and (D) scratch assay of HuCCT1, HuH28, and SSP25 cells compared with negative control (NC).
FIGURE 4
FIGURE 4
miR‐493‐5p promotes tumor cell growth by suppressing cocaine‐ and amphetamine‐regulated transcript propeptide (CARTPT) expression. (A) The Cancer Genome Atlas data showed that low CARTPT expression was associated with significantly poorer prognosis in cholangiocarcinoma. (B) Immunohistochemical staining of CARTPT was performed on intrahepatic cholangiocarcinoma specimens at our institution (n = 76) and divided into two groups: positive (n = 20) and negative (n = 56). (C) The CARTPT‐negative group had a significantly poorer prognosis in terms of both overall survival and recurrence‐free survival.
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