The online version of this article (https://doi.org/10.1186/s12943-018-0760-x) contains supplementary material, which is available to authorized users.
Cholangiocarcinoma (CCA) has an abundance of tumor stroma which plays an important role in cancer progression via tumor-promoting signals. This study aims to explore the microRNA (miRNA) profile of CCA-associated fibroblasts (CCFs) and the roles of any identified miRNAs in CCA progression.
miRNA expression profiles of CCFs and normal skin fibroblasts were compared by microarray. Identified downregulated miRNAs and their target genes were confirmed by real-time PCR. Their binding was confirmed by a luciferase reporter assay. The effects of conditioned-media (CM) of miRNA mimic- and antagonist-transfected CCFs were tested in CCA migration in wound healing assays. Finally, the levels of miRNA and their target genes were examined by real-time PCR and immunohistochemistry in clinical CCA samples.
miR-15a was identified as a downregulated miRNA in CCFs. Moreover, PAI-2 was identified as a novel target gene of miR-15a. Recombinant PAI-2 promoted migration of CCA cells. Moreover, CM from miR-15a mimic-transfected CCFs suppressed migration of CCA cells. Lower expression of miR-15a and higher expression of PAI-2 were observed in human CCA samples compared with normal liver tissues. Importantly, PAI-2 expression correlated with poor prognosis in CCA patients.
These findings highlight the miR-15a/PAI-2 axis as a potential therapeutic target in CCA patients.
Additional file 1: Table S1. Primer sequences and product size. (DOCX 16 kb)12943_2018_760_MOESM1_ESM.docx
Additional file 2: Table S2. Primer sequences and product size of miRNA-targeted mRNA. (DOCX 17 kb)12943_2018_760_MOESM2_ESM.docx
Additional file 3: Figure S1. The expression of 11 cancer related genes and CAF markers that were previously reported was examined by real-time PCR in CCFs (B149 and C096) and SFs (SFA3 and SFA5). The expression level is normalized to an internal control, GAPDH. Bars represent mean ± SD of three measurements. (TIFF 180 kb)12943_2018_760_MOESM3_ESM.tif
Additional file 4: Figure S2. The expression levels of 15 down-regulated miRNAs in in 2 CCFs (B149 and C096) and 2 normal SFs (SFA3 and SFA5). Expression of miRNAs was examined by real-time PCR. The level is normalized to U6 snRNA. Graph shows the expression of miRNAs in 2 CCFs and the average level of 2 control SFs. (TIFF 194 kb)12943_2018_760_MOESM4_ESM.tif
Additional file 5: Table S3. A literature review of 15 miRNAs which are commonly down-regulated in CCFs. (PPTX 53 kb)12943_2018_760_MOESM5_ESM.pptx
Additional file 6: Figure S3. The up-regulated miRNAs in CCFs. (A) Vane diagram and lists of 13 up-regulated miRNAs showing the folding compared to that in SFs. (B) Expression of up-regulated miRNAs in 2 CCFs (B149 and C096) and 2 normal SFs (SFA3 and SFA5) was examined by real-time PCR. Graph shows the expression of miRNAs in 2 CCFs and the average level of 2 control SFs. (TIFF 212 kb)12943_2018_760_MOESM6_ESM.tif
Additional file 7: Figure S4. List of candidate target genes of miR-148a. (A) Four criteria of finding the candidate target genes of miR-148a. (B) The expression levels of five predicted target genes of miR-148a including WNT10B, TGFA, WNT1, TNFRSF6B and L-selectin in CCFs and SFs. Scrambled miRNAs were used as a negative control. Bars represent mean ± SD of three measurements in one experiment. (C) Expression of WNT10B in miR-148a mimic transfected C096 CCFs was examined by real-time PCR. Bars represent mean ± SD of three measurements. *P≤0.05 compared to control. (D) Expression of WNT10B in miR-148a inhibitor transfected SFA3 SFs was examined by real-time PCR. Bars represent mean ± SD of three measurements. *P≤0.05 compared to control. (TIFF 200 kb)12943_2018_760_MOESM7_ESM.tif
Additional file 8: Figure S5. VEGFA is a target gene of miR-15a. (A) Expression of VEGFA in miR-15a mimic transfected C096 CCFs was examined by real-time PCR. Scrambled miRNAs were used as negative control miRNA. Bars represent mean ± SD of three measurements. (B) Expression of VEGFA in miR-15a inhibitor transfected SFA3 SFs was examined by real-time PCR. Bars represent mean ± SD of three measurements. (C) Expression of VEGFA in 6 CCFs (B149, C095, C096, D005, D012, and D017), SFs (SFA3 and SFA5) and CCA cell lines (KKU-213 and KKU-055) was examined by real-time PCR. Bars represent means ± SD of three measurements. *P≤0.05 compared to control. (TIFF 78 kb)12943_2018_760_MOESM8_ESM.tif
Additional file 9: Figure S6. The effects of rPAI-2 on CCA tumorigenic properties. (A) Cell proliferation was examined by WST assay in KKU-213 and KKU-055 at 24, 48, and 72 h after 10 μg/ml of rPAI-2 treatment. The 2% FBS containing media are used as negative controls. (B) Cell invasion by Transwell® invasion assay in KKU-213 and KKU-055. After incubation for 18 h with 10 μg/ml of rPAI-2, invaded cells were counted. Bars represent mean ± SD of three measurements. *P < 0.05 compared to control. (TIFF 4367 kb)12943_2018_760_MOESM9_ESM.tif
Additional file 10: Supplementary information of the invasion assay method. (DOCX 12 kb)12943_2018_760_MOESM10_ESM.docx
Chuaysri C, Thuwajit P, Paupairoj A, Chau-In S, Suthiphongchai T, Thuwajit C. Alpha-smooth muscle actin-positive fibroblasts promote biliary cell proliferation and correlate with poor survival in cholangiocarcinoma. Oncol Rep. 2009;21(4):957–69. PubMed
Utispan K, Thuwajit P, Abiko Y, Charngkaew K, Paupairoj A, Chau-in S, Thuwajit C. Gene expression profiling of cholangiocarcinoma-derived fibroblast reveals alterations related to tumor progression and indicates periostin as a poor prognostic marker. Mol Cancer. 2010;9:13. CrossRefPubMedPubMedCentral
Zhang XF, Dong M, Pan YH, Chen JN, Huang XQ, Jin Y, Shao CK: Expression pattern of cancer-associated fibroblast and its clinical relevance in intrahepatic cholangiocarcinoma. Hum Pathol. 2017; pii: S0046-8177(17)30132-6. doi: https://doi.org/10.1016/j.humpath.2017.04.014.
Schoepp M, Strose AJ, Haier J: Dysregulation of miRNA expression in cancer associated fibroblasts (CAFs) and its consequences on the tumor microenvironment. Cancers (Basel). 2017; 9(6). May 24; pii: E54: doi: https://doi.org/10.3390/cancers9060054.
Shen Z, Qin X, Yan M, Li R, Chen G, Zhang J, Chen W. Cancer-associated fibroblasts promote cancer cell growth through a miR-7-RASSF2-PAR-4 axis in the tumor microenvironment. Oncotarget. 2017;8(1):1290–303. PubMed
Liu XJ, Bai XG, Teng YL, Song L, Lu N, Yang RQ. miRNA-15a-5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis. Eur Rev Med Pharmacol Sci. 2016;20(16):3319–26. PubMed
Schulte J, Weidig M, Balzer P, Richter P, Franz M, Junker K, Gajda M, Friedrich K, Wunderlich H, Ostman A, et al. Expression of the E-cadherin repressors snail, slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells. Histochem Cell Biol. 2012;138(6):847–60. CrossRefPubMed
Doldi V, Callari M, Giannoni E, D'Aiuto F, Maffezzini M, Valdagni R, Chiarugi P, Gandellini P, Zaffaroni N. Integrated gene and miRNA expression analysis of prostate cancer associated fibroblasts supports a prominent role for interleukin-6 in fibroblast activation. Oncotarget. 2015;6(31):31441–60. CrossRefPubMedPubMedCentral
- The microRNA-15a-PAI-2 axis in cholangiocarcinoma-associated fibroblasts promotes migration of cancer cells
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II