MicroRNA-155-5p promotes hepatocellular carcinoma progression by suppressing PTEN through the PI3K/Akt pathway - PubMed (original) (raw)

. 2017 Apr;108(4):620-631.

doi: 10.1111/cas.13177. Epub 2017 Apr 19.

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MicroRNA-155-5p promotes hepatocellular carcinoma progression by suppressing PTEN through the PI3K/Akt pathway

Xiao Fu et al. Cancer Sci. 2017 Apr.

Abstract

MicroRNA-155-5p (miR-155-5p) has been reported to play an oncogenic role in different human malignancies; however, its role in hepatocellular carcinoma (HCC) progression is not clearly understood. In this study, we used real-time PCR in 20 rats with chemically-induced HCC, 28 human HCC tissues, and the matched paracarcinoma tissues, and HCC cell lines to determine the expression patterns of miR-155-5p and PTEN mRNA. Algorithm-based and experimental strategies, such as dual luciferase gene reporter assays, real-time PCR and western blots were used to identify PTEN as a candidate miR-155-5p target. Gain- and loss-of-function experiments and administration of a PI3K/Akt pathway inhibitor (wortmannin) were used to identify the effects of miR-155-5p and PTEN in MTT assays, flow cytometric analysis, wound healing assays and transwell assays. The results showed that miR-155-5p was highly overexpressed; however, PTEN was underexpressed in the HCC rat models, human HCC tissues and cell lines. In addition, miR-155-5p upregulation and PTEN downregulation were significantly associated with TNM stage (P < 0.05). Through in vitro experiments, we found that miR-155-5p promoted proliferation, invasion and migration, but inhibited apoptosis in HCC by directly targeting the 3'-UTR of PTEN. Western blots showed that miR-155-5p inactivated Bax and caspase-9, but activated Bcl-2 to inhibit apoptosis, and it activated MMP to promote migration and invasion via the PI3K/Akt pathway. A xenograft tumor model was used to demonstrate that miR-155-5p targets PTEN and activates the PI3K/Akt pathway in vivo as well. Our study highlighted the importance of miR-155-5p and PTEN associated with aggressive HCC both in vitro and in vivo.

Keywords: PTEN; Hepatocellular carcinoma; PI3K/Akt pathway; hepatocellular carcinoma progression; microRNA-155-5p.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

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Figures

Figure 1

Figure 1

The expression of miR‐155‐5p in hepatocellular carcinoma (HCC) tissues and cell lines. (a) The expression of miR‐155‐5p and PTEN mRNA in chemically‐induced HCC rats, compared with normal liver tissues. *P < 0.05, **P < 0.01 and ***P < 0.001 by Student's _t_‐test. (b) The expression of miR‐155‐5p in various TNM stages, *P < 0.05, **P < 0.01 by Student's _t_‐test, and PTEN mRNA in HCC tissues according to TNM stages, and paracarcinoma liver tissues. *P < 0.05, ***P < 0.001 by Student's _t_‐test. (c) Expression of miR‐155‐5p and PTEN mRNA in HCC cell lines versus the normal hepatic cell line LO2. n = three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 by

anova

. Pca indicates matched paracarcinoma liver tissues.

Figure 2

Figure 2

PTEN is the target of miR‐155‐5p. (a) MiR‐155‐5p and its putative binding sequence in the 3′‐UTR of PTEN. Mutant miR‐155‐5p binding sites were generated in the complementary site for the seed region of miR‐155‐5p (WT, wild type; Mut, mutant type). (b) MiR‐155‐5p effects on luciferase activity in cells that carried the wild type and mutant type 3′‐UTR of PTEN. n = three repeats with similar results, *P < 0.05 by Student's _t_‐test. (c) The expression of miR‐155‐5p, and PTEN according to the dose of miR‐155‐5p inhibitor in Hep3B cells and mimics in HepG2 cells; n = three repeats with similar results; *P < 0.05, ***P < 0.001 by Student's _t_‐test. (d) The expression of PTEN and phosphorylation of Akt according to the dose of miR‐155‐5p inhibitor in Hep3B cells and mimics in HepG2 cells, the intensity of each band was quantified; the value under each lane indicates the relative expression level of the regulators; n = three repeats with similar results. p‐Akt, phosphorylated Akt.

Figure 3

Figure 3

The effects of miR‐155‐5p on hepatocellular carcinoma (HCC) cell proliferation, apoptosis, invasion and migration. (a–e) Cell viability, the percentage of apoptotic cells, wound healing and transwell assays, and the expression of Bcl‐2, Bax, caspase‐9, MMP2 and MMP9 in Hep3B and HepG2 transfected with miR‐155‐5p inhibitor and mimics, respectively; the intensity of each band was quantified; the value under each lane indicates the relative expression level of the regulators; n = three repeats with similar results; *P < 0.05, **P < 0.01, ***P < 0.001 by Student's _t_‐test versus inhibitor and mimics NC, respectively.

Figure 4

Figure 4

MiR‐155‐5p promotes hepatocellular carcinoma (HCC) malignancy through targeting PTEN. (a–e) Cell viability, the percentage of apoptotic cells, wound healing and transwell assays, and the expression of Bcl‐2, Bax, caspase‐9, MMP2 and MMP9 in Hep3B and HepG2‐transfected PTEN plasmid and PTEN siRNA with and without miR‐155‐5p mimics or inhibitor; the intensity of each band was quantified; the value under each lane indicates the relative expression level of the regulators; n = three repeats with similar results, *P < 0.05, **P < 0.01, ***P < 0.001 by Student's _t_‐test, versus inhibitor and mimics NC, respectively. #P < 0.05, ##P < 0.01, ###P < 0.001 by Student's _t_‐test, versus inhibitor + siRNA NC and mimics + PTEN vector control, respectively. PTEN, PTEN plasmid; siPTEN, PTEN siRNA.

Figure 5

Figure 5

MiR‐155‐5p promotes proliferation, invasion and migration, and inhibits apoptosis by the PI3K/Akt pathway. (a–e) Cell viability, the percentage of apoptotic cells, wound healing and transwell assay, and the expression of Akt and p‐Akt, Bcl‐2, Bax, caspase‐9, MMP2 and MMP9 in Hep3B cells treated with DMSO, wortmannin alone, wortmannin and miR mimics NC, and wortmannin and miR‐155‐5p mimics; the intensity of each band was quantified; the value under each lane indicates the relative expression level of the regulators; n = three repeats with similar results, *P < 0.05, **P < 0.01, versus Hep3B treated with DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001, versus Hep3B treated with wortmannin alone by Student's _t_‐test. WM, wortmannin.

Figure 6

Figure 6

MiR‐155‐5p promotes HCC progression in vivo. (a) Growth curves of Hep3B and HepG2 cell line xenograft tumors after injection with miR‐155‐5p antagomiR and miR‐155‐5p angomiR, respectively. Tumor volumes are showed as mean SD for each goup of six mice. (b) BALB/c nude mice resected tumors from each group are shown. (c) The top panel shows representative pictures of PTEN and p‐Akt expression in the tissues resected from mice injected Hep3B with antagomiR or antagomiR NC, or HepG2 with angomiR or angomiR NC. The bottom panel shows positive scores of PTEN and p‐Akt expression in the implanted tumors resected from mice injected Hep3B with antagomiR or antagomiR NC, or HepG2 angomiR or angomiR NC. *P < 0.05, **P < 0.01, versus antagomiR NC and angomiR NC, respectively, by Student's _t_‐test.

Figure 7

Figure 7

Schematic diagram of miR‐155‐5p in promoting HCC progression by suppressing the PTEN via the PI3K/Akt pathway.

References

    1. Dhanasekaran R, Limaye A, Cabrera R. Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis, and therapeutics. Hepat Med 2012; 4: 19–37. - PMC - PubMed
    1. El‐Serag HB. Hepatocellular carcinoma. N Engl J Med 2011; 365: 1118–27. - PubMed
    1. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3‐kinases as regulators of growth and metabolism. Nat Rev Genet 2006; 7: 606–19. - PubMed
    1. Li J, Yen C, Liaw D et al PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997; 275: 1943–7. - PubMed
    1. Salmena L, Carracedo A, Pandolfi PP. Tenets of PTEN tumor suppression. Cell 2008; 133: 403–14. - PubMed

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