MicroRNA-155-3p promotes hepatocellular carcinoma formation by suppressing FBXW7 expression - PubMed (original) (raw)
MicroRNA-155-3p promotes hepatocellular carcinoma formation by suppressing FBXW7 expression
Bo Tang et al. J Exp Clin Cancer Res. 2016.
Abstract
Background: MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignant tumors. In the present study, we analyzed the role miR-155-3p plays in Hepatocellular carcinoma (HCC), which has been reported participation in some other types of cancer.
Methods: qRT-PCR was used to measure the levels of miR-155-3p in HCC specimens and HCC cell lines. Overexpression of miR-155-3p and miR-155-3p inhibitor were transfected into HCC cell lines to investigate its role in HCC. Colony formation assay and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays were used to analyses cell proliferation in vitro. In vivo tumor formation assays were performed in BALB/c nude mice. Luciferase reporter assay was carried out to measure the translation of F-Box and WD repeat romain containing 7 (FBXW7).
Results: We found that miR-155-3p was remarkably upregulated both in HCC tissue and cell lines. Overexpression of miR-155-3p enhanced HCC cell proliferation in vitro and tumorigenesis in vivo. In addition, overexpression of miR-155-3p is correlated with decreased levels FBXW7 mainly through inhibiting the expression of FBXW7.
Conclusions: Our studies suggest that miR-155-3p plays an important role in the pathogenesis of HCC and implicates its potential applications in the treatment of HCC cancer.
Keywords: FBXW7; Hepatocellular carcinoma; miR-155-3p.
Figures
Fig. 1
The expression of miR-155-3p is elevated in Hepatocellular carcinoma tissues. a miR-155-3p expression in Hepatocellular carcinoma in comparison to normal tissues was measured by qRT-PCR. b miR-155-3p expression levels are significantly elevated in Hepatocellular carcinoma in comparison to normal tissues. c miR-155-3p expression levels are significantly increased in patients of stage III-IV in comparison to the stage I-II. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 2
Elevated expression of miR-155-3p is associated with poor patient survival of Hepatocellular carcinoma
Fig. 3
The overexpression of miR-155-3p enhances tumorigenesis in vitro. a Expression profile of miR-155-3p in Hepatocellular carcinoma cell lines. miR-155-3p RNA levels relative to normal liver cell line THLE-3 were determined by qRT-PCR. Gene expression was normalized to U6. Data are presented as means ± Standard deviation. b Establishment of BEL-7405-expressing miR-155-3p cells. The results were analyzed by qRT-PCR. c Proliferation of BEL-7405-miR-155-3p cells is significantly accelerated compared to normal BEL-7405 control cells measured by MTT assay. d Representative stained colonies are displayed. e The number of colonies was counted. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 4
The overexpression of miR-155-3p enhances tumorigenesis in vivo. a Subcutaneous tumors in the five nude mice are displayed. b The weights of tumors are shown. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 5
Downregulation of miR-155-3p reduce tumorigenesis in vitro and in vivo. a Establishment of downregulation of miR-155-3p in HepG2 cells. The results were analyzed by qRT-PCR. b Proliferation of HepG2-miR-155-3p cells is significantly accelerated compared to normal HepG2 control cells measured by MTT assay. c Representative stained colonies are displayed. d The number of colonies was counted. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 6
Downregulation of miR-155-3p reduce tumorigenesis in vivo. a Subcutaneous tumors in the five nude mice are displayed. b The weights of tumors are shown in the right graph. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 7
miR-155-3p regulates the expression of FBXW7. a Supervised hierarchical clustering of the genes differentially expressed after miR-155-3p overexpression in BEL-7405 cells. b Gene set enrichment analysis was carried out using ConceptGen
Fig. 8
FBXW7 is the potential target of miR-155-3p. a The expression of FBXW7 mRNA was analyzed by qRT-PCR in BEL-7405-miR-155-3p and its control cells. b Western blot analysis was performed to detect the expression of FBXW7 and internal control β-actin in BEL-7405-miR-155-3p and its control cells. c The expression of FBXW7 protein was measured by immunohistochemical analysis in BEL-7405-miR-155-3p and its control cells in vivo tumor samples. Representative results are shown in micrographs. d The expression of FBXW7 mRNA was analyzed by qRT-PCR in HepG2-anti-miR-155-3p and its control cells. e Western blot analysis was performed to detect the expression of FBXW7 and internal control β-actin in in HepG2-anti-miR-155-3p and its control cells. f The expression of FBXW7 protein was measured by immunohistochemical analysis in in HepG2-anti-miR-155-3p and its control cells in vivo tumor samples. Representative results are shown in micrographs. All experiments were performed in triplicate. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 9
FBXW7 is a direct target of miR-155-3p. a The potential binding site for miR-155-3p in 3’UTR of FBXW7 mRNA. b FBXW7 3’-UTR possessing a mutation in the putative miR-155-3p binding site. c The luciferase activity after transfection in BEL-7405-miR-155-3p and its control cells of the indicated 3’-UTR-driven reporter constructs. Reporter plasmids containing no oligonucleotides as a Control, the wild-type 3’UTR region of FBXW7 as a Wild type and the mutant 3’UTR region as a Mutant. d The luciferase activity after transfection in HepG2-anti-miR-155-3p and its control cells of the indicated 3’-UTR-driven reporter constructs. Reporter plasmids containing no oligonucleotides as a Control, the wild-type 3’UTR region of FBXW7 as a Wild type and the mutant 3’UTR region as a Mutant. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 10
FBXW7 expression suppresses the colony formation and proliferation phenotype of BEL-7405 cells. a BEL-7405-miR-155-3p were transfected FBXW7, the results of immunoblotting for FBXW7 and β-actin are shown. b BEL-7405-miR-155-3p transfected with FBXW7 show a significant reduction in proliferation in comparison to control cells. c BEL-7405-miR-155-3p transfected with FBXW7 reduces colony formation. **P < 0.01, unpaired two-tailed Student’s t-test
Fig. 11
Reduced FBXW7 expression enhances the colony formation and proliferation phenotype of HepG2-Anti-miR-155-3p cells. a Knockdown of FBXW7 in HepG2-Anti-miR155-3p cells, the results of immunoblotting for FBXW7 and β-actin are shown. b Knockdown of FBXW7 in HepG2-Anti-miR-155-3p cells facilitates the proliferation ability of HepG2-Anti-miR-155-3p cells. c Knockdown of FBXW7 in HepG2-Anti-miR-155-3p cells increases colony formation. **P < 0.01, unpaired two-tailed Student’s t-test
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