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. 2019 Jan 8;38(1):7.
doi: 10.1186/s13046-018-1020-z.

MiR-6875-3p promotes the proliferation, invasion and metastasis of hepatocellular carcinoma via BTG2/FAK/Akt pathway

Yingjun Xie  1 , Jian Du  2 , Zefeng Liu  1 , Dan Zhang  1 , Xiaoxiao Yao  1 , Yongsheng Yang  3
Affiliations

MiR-6875-3p promotes the proliferation, invasion and metastasis of hepatocellular carcinoma via BTG2/FAK/Akt pathway

Yingjun Xie et al. J Exp Clin Cancer Res. .

Abstract

Background: Increasing evidence supports the association of microRNA with tumor occurrence and development. However, the expression of miR-6875-3p and its role in cell proliferation, invasion and metastasis in hepatocellular carcinoma (HCC) remains elusive.

Methods: The expression of miR-6875-3p and BTG2 in HCC tissues and cell lines was detected by using in situ hybridization, immunohistochemistry and qRT-PCR, respectively. A western blot assay, qRT-PCR and Luciferase reporter assay were employed to study the interaction between miR-6875-3p and BTG2. Cell proliferation invasion and metastasis were measured by MTT, transwell and matrigel analyses in vitro. In vivo, tumorigenicity and metastasis assays were performed in nude mice.

Results: We found that miR-6875-3p were elevated expressed in HCC tissues and cell lines, and negatively correlated with BTG2 expression, while positively correlated with tumor staging, size, degree of differentiation, and vascular invasion of HCC. Moreover, in vitro and in vivo assays showed that miR-6875-3p regulates EMT and improve the proliferation, metastasis and stem cell-like properties of HCC cells. BTG2 was identified as a direct and functional target of miR-6875-3p via the 3'-UTR of BTG2. We also confirmed that miR-6875-3p plays its biological functions via the BTG2/FAK/Akt pathway.

Conclusion: Our study provides evidence that high expression of miR-6875-3p can promote tumorigenesis of HCC in vitro and in vivo, so as to function as a novel oncogene in HCC. In mechanism, we found that miR-6875-3p plays its biological functions via the BTG2/FAK/Akt pathway.

Keywords: BTG2; HCC; Invasion; Metastasis; Proliferation; miR-6875-3p.

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Conflict of interest statement

Ethics approval and consent to participate

Clinical data have been approved by the Ethics Committee of Second Affiliated Hospital of Jilin University and approved by the patients. All animal experiments were approved by Animal Care and Use Committee of Second Affiliated Hospital of Jilin University.

Consent for publication

All contributing authors agree to the publication of this article.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
miR-6875-3p expression was inversely associated with BTG2 expression in HCC tissues, and high miR-6875-3p expression predicted Shorter RFS and OS. a Analysis of miR-6875-3p expression in HCC tissues and tumor-adjacent tissues using ISH. b The expression levels of miR-6875-3p were detected by qRT-PCR in 108 paired HCC and tumor-adjacent tissues. c Analysis of BTG2 expression in HCC tissues and tumor-adjacent tissues via IHC. d Expression of miR-6875-3p was inversely correlated with the mRNA level of the BTG2 (R = − 0.2638, p < 0.01, Spearman’s correlation analysis). e, f Kaplan-Meier analysis suggested that the OS and RFS rates of the low miR-6875-3p expression group were higher than those of the high miR-6875-3p expression group (p < 0.05). Scale bars, 50 mm. Data are shown as the mean ± SD of three replicates (*p < 0.05)
Fig. 2
Fig. 2
miR-6875-3p downregulated BTG2 expression via directly targeting its 3’-UTR. a Expression of miR-6875-3p were examined by qRT-PCR in seven cell lines. b, c The protein and mRNA levels of the BTG2 were analyzed after transfection with the miR-6875-3p inhibitor by Western blot and qRT-PCR. d, e The protein and mRNA level of the BTG2 were analyzed after transfection with the miR-6875-3p mimic by Western blot and qRT-PCR. f The predicted sites of miR-6875-3p binding to the 3’-UTR of the BTG2 were detected via bioinformatics prediction tools. The mutated site in the 3’-UTR of the BTG2 is shown. g The effect of miR-6875-3p on luciferase activity induced by the pMIR-BTG2-wt, pMIR-BTG2-mut-1, and pMIR-BTG2-mut-2 reporter plasmids in HL7702 cells was detected via luciferase reporter gene assays. Data are shown as the mean ± SD of three replicates (*p < 0.05)
Fig. 3
Fig. 3
The elevated expression of miR-6875-3p promoted the proliferation of HCC cells and tumorigenicity in vivo. a, b miR-6875-3p was down-regulated in Huh7 and BEL7404 cells through the transfection of a miR-6875-3p inhibitor. miR-6875-3p was up-regulated in HL7702 and HepG2 cells via the transfection of a miR-6875-3p mimic. MTT assay was used to analyze the effect of miR-6875-3p on the proliferation of these four cell lines. c, d Huh7 cells stably low-expressing miR-6875-3p or empty vector were injected into the flanks of nude mice. HL7702 cells stably overexpressing miR-6875-3p or empty vector were injected into the flanks of nude mice. Surgically removed tumor tissues from nude mice 5 weeks post-inoculation. Tumor volume at different time points and Tumor weight were measured. e, f Analysis of BTG2 expression in resected tumors tissues by IHC and Western blot. Data are shown as the mean ± SD of three replicates (*p < 0.05)
Fig. 4
Fig. 4
miR-6875-3p regulates the transition between epithelial and mesenchymal phenotypes, and promotes migratory, invasive, and metastatic of HCC cells. a, b HL7702 and HepG2 cells via the transfection of a miR-6875-3p mimic were subjected to Transwell migration (top) and Matrigel invasion assays (bottom). Quantification of migrated cells through the membrane and invaded cells through either the membrane or Matrigel for each cell line is shown as a proportion of the vector controls. c, d Huh7 and BEL7404 cells via the transfection of a miR-6875-3p inhibitor were subjected to Transwell migration (top) and Matrigel invasion assays (bottom). Quantification of migrated cells through either the membrane or Matrigel for each cell lines is shown as proportions of their vector controls. e, f Expression of epithelial (E-cadherin and a-catenin) and mesenchymal (N-cadherin and Vimentin) markers were analyzed by Western blot in transfected HL7702 and Huh7 cells. g The total number of mice with distant metastasis at 6 weeks after injection of transfected HL7702 and Huh7 cells. h, i The number of metastatic foci per section in lungs from individual mouse with injection of transfected HL7702 and Huh7 cells. Scale bars, 200 nm (a, b, c and d) and 50 mm (h and i). Data are shown as the mean ± SD of three replicates (*p < 0.05)
Fig. 5
Fig. 5
Silencing of BTG2 expression reversed proliferation, invasion and migration of HCC cells. a, b Protein and mRNA levels of the BTG2 decreased after transfection with BTG2 shRNA into miR-6875-3p-silenced Huh7 cells, as demonstrated by Western blot and qRT-PCR. c Proliferation of Huh7 cells was reversed after transfection with BTG2 shRNA into miR-6875-3p-silenced Huh7 cells, as demonstrated by MTT assay. d, e and f Transwell and Wound-healing assay demonstrated that Silencing of BTG2 expression ameliorated down-regulated miR-6875-3p-induced suppression of Huh7 cell invasion and migration. Data are shown as the mean ± SD of three replicates (*p < 0.05)
Fig. 6
Fig. 6
miR-6875-3p-BTG2 induced promotion of proliferation and migration through FAK/Akt pathway. a Supervised hierarchical clustering of the genes differentially expressed after BTG2 overexpression in Huh7 cells. b Gene-set enrichment analysis was carried out using ConceptGen. c Levels of the BTG2, phosphorylated FAK and phosphorylated Akt were detected using Western blot analysis. miR-6875-3p-silenced Huh7 cells were treated with shBTG2 RNA and Huh7 cells were treated with 10 μM of PF573228. d The proliferation-promoting effect of miR-6875-3p on Huh7 cells was blocked by PF573228, as demonstrated by an MTT assay. e, f and g The invasion and migration-promoting effect of miR-6875-3p on Huh7 cells was blocked by PF573228, as demonstrated by transwell and wound-healing assay. Data are shown as the mean ± SD of three replicates (*p < 0.05)
Fig. 7
Fig. 7
miR-6875-3p promoted emergence of stem cell-like behavior in hepatocellular carcinoma cells. a Levels of miR-6875-3p in magnetically sorted CD133+ and CD133− were measured by qRT-PCR in HL7702 and Huh7 cells (left). Levels of miR-6875-3p in magnetically sorted EPCAM+ and EPCAM− were measured by qRT-PCR in HL7702 and Huh7 cells (right). b Holoclone assays in Huh7 cells transfected with miR-6875-3p inhibitor or empty vector (left) and HL7702 cells transfected with miR-6875-3p mimic or empty vector (right) were used in three experiments (Exp. I, 100 cells/well scored on day 9; Exp. II, 100 cells/well scored on day 13; and Exp. III, 500 cells/well scored on day 7). c SP population in miR-6875-3p-overexpressing and silenced cells was determined by Hoechst 33342 efflux assays. d miR-6875-3p promotes the proliferation, invasion and metastasis of HCC cells via the BTG/FAK/Akt pathway. Data are shown as the mean ± SD of three replicates (*p < 0.05)
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