The online version of this article (doi:10.1186/s12943-017-0675-y) contains supplementary material, which is available to authorized users.
Increasing evidences demonstrate that miRNAs contribute to development and progression of hepatocellular carcinoma (HCC). Underexpression of miR-1296 is recently reported to promote growth and metastasis of human cancers. However, the expression and role of miR-1296 in HCC remain unknown.
The levels of miR-1296 in HCC tissues and cells were detected by qRT-PCR. Immunoblotting and immunofluorescence were used for detection of epithelial-to-mesenchymal transition (EMT) progression in HCC cells. Transwell assays were performed to determine migration and invasion of HCC cells. A lung metastasis mouse model was used to evaluated metastasis of HCC in vivo. The putative targets of miR-1296 were disclosed by public databases and a dual-luciferase reporter assay.
We found that the expression of miR-1296 was reduced in HCC tissues and cell lines, and it was associated with metastasis and recurrence of HCC. Notably, miR-1296 overexpression inhibited migration, invasion and EMT progress of HCCLM3 cells, while miR-1296 loss facilitated these biological behaviors of Hep3B cells in vitro and in vivo. In addition, miR-1296 inversely regulated SRPK1 abundance by directly binding to its 3′-UTR, which subsequently resulted in suppression of p-AKT. Either SRPK1 re-expression or PI3K/AKT pathway activation, at least partially, abolished the effects of miR-1296 on migration, invasion and EMT progress of HCC cells. Furthermore, miR-1296 and SRPK1 expression were markedly correlated with adverse clinical features and poor prognosis of HCC patients. We showed that hypoxia was responsible for the underexpression of miR-1296 in HCC. And the promoting effects of hypoxia on metastasis and EMT of HCC cells were reversed by miR-1296.
Underexpression of miR-1296 potentially serves as a prognostic biomarker in HCC. Hypoxia-induced miR-1296 loss promotes metastasis and EMT of HCC cells probably by targeting SRPK1/AKT pathway.
Additional file 1: Figure S1. miR-1296 suppresses EMT process of HCC cells. (A) HCCLM3 cells that were transfected with miR-1296 and miR-control, respectively, were subjected to immunoblotting for the expression of EMT-related markers including ZO-1, ZEB-1, Slug, Snail and Twist. (B) miR-1296 knockdown decreased ZO-1 expression and increased the levels of ZEB-1, Slug, Snail and Twist in Hep3B cells. *P < 0.05. (TIFF 258 kb)12943_2017_675_MOESM1_ESM.tif
Additional file 2: Figure S2. IHC staining of E-cadherin, Vimentin and SRPK1 in HCC tissues. Representative IHC results indicated that strong staining of E-cadherin and weak staining of Vimentin and SRPK1 were observed in miR-1296 high-expressing HCC tissue. Weak staining of E-cadherin and strong staining of Vimentin and SRPK1 were presented in miR-1296 low-expressing HCC tissues. (TIFF 2252 kb)12943_2017_675_MOESM2_ESM.tif
Additional file 3: Figure S3. miR-1296 does not regulate the expression of other predicted targets in HCC cells. (A) HCCLM3 cells that were transfected with miR-1296 and miR-control, respectively, were subjected to immunoblotting for the expression of ERBB2, CCND1 and MCM2. (B) miR-1296 knockdown didn’t obviously increased the levels of ERBB2, CCND1 and MCM2 protein in Hep3B cells. (TIFF 242 kb)12943_2017_675_MOESM3_ESM.tif
Additional file 4: Figure S4. SRPK1 faciliates migration, invasion and EMT progression of HCC cells. (A) HCCLM3 cells that were transfected with SRPK1 siRNA or control siRNA were detected by immunoblotting. (B) SRPK1 silencing notably restrained migration and invasion of HCCLM3 cells. (C) SRPK1 was overexpressed by plasmid transfection and confirmed by western blotting in Hep3B cells. (D) SRPK1 restoration enhanced the migratory and invasive abilities of Hep3B cells. (E) SRPK1 knockdown led to increase of E-cadherin expression and decrease of N-cadherin and Vimentin in HCCLM3 cells. (F) SRPK1 overexpression promoted the EMT progression of Hep3B cells. *P < 0.05. (TIFF 3508 kb)12943_2017_675_MOESM4_ESM.tif
Additional file 5: Figure S5. The prognostic significance of miR-1296 and SRPK1 in another cohort of HCC patients. (A) and (B) OS and DFS were compared between miR-1296 high expressing HCC patients and low expressing cases. (C) and (D) OS and DFS were compared between SRPK1 high expressing HCC patients and low expressing cases. (E) and (F) OS and DFS were compared between four subgroups of HCC patients (subgroup I: high miR-1296/low SRPK1; subgroup II: low miR-1296 /low SRPK1; subgroup III: high miR-1296/high SRPK1; subgroup IV: low miR-1296/high SRPK1). For each cohort, subgroups were divided according to the cutoff values, which were determined as the median level of miR-1296 and SRPK1 in HCC tissues. **P < 0.01. (TIFF 213 kb)12943_2017_675_MOESM5_ESM.tif
Additional file 6: Figure S6. SRPK1/AKT axis functions in hypoxia-induced metastasis and EMT process of HCC cells. (A) The levels of SRPK1 and p-AKT were increased after exposing to hypoxia condition in Hep3B cells. (B) and (C) Hypoxia induced migration and invasion of Hep3B cells. While, either SRPK1 knockdown or MK2206 treatment blocked the pro-metastatic effects of hypoxia in Hep3B cells. (D) Hypoxia induced EMT progression of Hep3B cells. Whereas, either SRPK1 knockdown or MK2206 treatment prohibited the EMT progression of Hep3B cells under hypoxia condition. *P < 0.05. (TIFF 5115 kb)12943_2017_675_MOESM6_ESM.tif
Tu K, Liu Z, Yao B, Han S, Yang W. MicroRNA-519a promotes tumor growth by targeting PTEN/PI3K/AKT signaling in hepatocellular carcinoma. Int J Oncol. 2016;48(3):965–74. PubMed
Yang W, Dou C, Wang Y, Jia Y, Li C, Zheng X, et al. MicroRNA-92a contributes to tumor growth of human hepatocellular carcinoma by targeting FBXW7. Oncol Rep. 2015;34(5):2576–84. PubMed
Bobowicz M, Skrzypski M, Czapiewski P, Marczyk M, Maciejewska A, Jankowski M, et al. Prognostic value of 5-microRNA based signature in T2-T3N0 colon cancer. Clinical & experimental metastasis. 2016;33(8):765–73. CrossRef
Liu Z, Dou C, Wang Y, Jia Y, Li Q, Zheng X, et al. Highmobility group box 1 has a prognostic role and contributes to epithelial mesenchymal transition in human hepatocellular carcinoma. Mol Med Rep. 2015;12(4):5997–6004. PubMed
Yu M, Xue H, Wang Y, Shen Q, Jiang Q, Zhang X, et al. miR-345 inhibits tumor metastasis and EMT by targeting IRF1-mediated mTOR/STAT3/AKT pathway in hepatocellular carcinoma. Int J Oncol. 2017;50(3):975–83. PubMed
- MicroRNA-1296 inhibits metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma by targeting SRPK1-mediated PI3K/AKT pathway
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II