MiR-122 Targets SerpinB3 and Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma - PubMed (original) (raw)

MiR-122 Targets SerpinB3 and Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma

Cristian Turato et al. J Clin Med. 2019.

Abstract

The only first-line treatment approved for advanced hepatocellular carcinoma (HCC) is sorafenib. Since many patients experience drug resistance, the discovery of more effective therapeutic strategies represents an unmet clinical need. MicroRNA (MiR)-122 is downregulated in most HCCs, while oncogenic SerpinB3 is upregulated. Here, we assessed the relationship between miR-122 and SerpinB3 and their influence on cell phenotype and sorafenib resistance in HCC. A bioinformatics analysis identified SerpinB3 among hypothetical miR-122 targets. In SerpinB3-overexpressing HepG2 cells, miR-122 transfection decreased SerpinB3 mRNA and protein levels, whereas miR-122 inhibition increased SerpinB3 expression. Luciferase assay demonstrated the interaction between miR-122 and SerpinB3 mRNA. In an HCC rat model, high miR-122 levels were associated with negative SerpinB3 expression, while low miR-122 levels correlated with SerpinB3 positivity. A negative correlation between miR-122 and SerpinB3 or stem cell markers was found in HCC patients. Anti-miR-122 transfection increased cell viability in sorafenib-treated Huh-7 cells, while miR-122 overexpression increased sorafenib sensitivity in treated cells, but not in those overexpressing SerpinB3. In conclusion, we demonstrated that miR-122 targets SerpinB3, and its low levels are associated with SerpinB3 positivity and a stem-like phenotype in HCC. MiR-122 replacement therapy in combination with sorafenib deserves attention as a possible therapeutic strategy in SerpinB3-negative HCCs.

Keywords: hepatocellular carcinoma; micro RNA; molecular targets.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

(A) miR-122 binding site in the SerpinB3 3’untranslated region (3′UTR) as reported by the TargetScan algorithm. (B) Dual-luciferase assay in HepG2 cells. The SerpinB3 3’UTR-containing vector was co-transfected with miR-122 or negative control (NC). MiR-122 overexpression determined a decrease of the reporter gene activity in pGL3-SerpinB3 co-transfected HepG2 cells. (C) qPCR analysis of miR-122 levels following miR-122 inhibitor (AM-122) or miR-122 mimic transfection in HepG2 cells stably overexpressing SerpinB3 (HepG2/SerpinB3) with respect to controls. U6RNA was used as housekeeping gene. Y-axis reports 2−ΔΔCt levels expressed in logarithmic form. (D,E) qPCR and Western blot analysis of SerpinB3 in transfected HepG2/SerpinB3 cells. β-Actin was used as housekeeping gene. Y-axis reports 2−ΔΔCt levels. NCi: miRNA inhibitor negative control; NC: miRNA precursor negative control. (* p ˂ 0.05). Data shown are representative results of at least three independent experiments.

Figure 2

Figure 2

(A) SerpinB3 and (B) miR-122 expression in hepatocellular carcinoma (HCC) nodules and matched surrounding liver (SL) tissues from diethyl nitrosamine (DEN)-induced HCC rats. Y-axis reports 2−ΔΔCt levels from qPCR analysis. The values are represents as box-and-whiskers graph with the minimum, maximum, and median data. (* p ˂ 0.05 Mann–Whitney test). (C) MicroRNA (MiR)-122 expression in tumor tissues from HCC rats in relation to SerpinB3 expression. HCCs were grouped on the basis of SerpinB3 expression as detected by qPCR analysis (* p ˂ 0.05 Mann–Whitney test). Specifically, the high SerpinB3 group included HCC specimens with a cycle threshold (Ct) value lower than 35, whereas the low SerpinB3 group included HCC specimens with a Ct value higher than 35. (D) Box plot graph representing tumor size in HCC rats in relation to SerpinB3 expression (* p ˂ 0.05 Mann–Whitney test). Tumor size was represented by the value of major diameter (cm).

Figure 3

Figure 3

(A) Correlation graphs in human HCC tissues between miR-122 and SerpinB3 mRNA and (B) protein levels, as detected by qPCR and tissue microarray, respectively. Regarding the qPCR analysis, we considered only HCC samples with a Ct value lower than 35, which resulted in 19 out of 35 tested HCC samples. β-Actin was used as housekeeping gene for SerpinB3 normalization; U6RNA was used as housekeeping gene for miR-122 analysis. (C) Correlation graphs in human HCC tissues between SerpinB3 and caspase-3 mRNA levels, as detected by qPCR analysis. (D) Representative images of tissue microarray (TMA) in HCC samples from three patients (Pt2, Pt21 and Pt30) with different expression of miR-122 and SerpinB3 in sequential tissue slides. Scale bar 100 µM.

Figure 4

Figure 4

(A) Correlation graphs between tissue miR-122 and PROM1/CD133 or (B) SerpinB3. (C) Correlation graph between EpCAM/CD326 and SerpinB3 or (D) miR-122 levels in the same HCC patient cohort. The X- and Y-axes report 2−∆∆Ct values from qPCR analysis converted in a log2 form. U6RNA was used as housekeeping gene for miR-122 normalization, whereas β-Actin was used for gene normalization. (E) PROM1/CD133 and EpCAM protein detected by WB analysis in SerpinB3-overexpressing and empty vector-bearing (CTR) HCC cell lines. β-Actin was used as housekeeping gene. (F) PROM1/CD133 and (G) EpCAM mRNA levels in miR-122-overexpressing (miR) or silenced (AM) HCC cell lines. The Y-axis reports relative values with respect to negative controls. (H) Immunophenotype analysis of PROM1/CD133 and EpCAM levels in miR-122 (miR) or anti-miR-122 (AM)-transfected Huh-7 cells. The percentage of positive cells is reported on the top left of the histogram graph. NC: negative control precursor miRNA. NCi: negative control inhibitor miRNA.

Figure 5

Figure 5

(A–C) In each panel, from the top to the bottom: cell viability and caspase 3/7 activity assays and western blot analysis of cleaved caspase-3 in HepG2 and Hep3B cells overexpressing miR-122 (A,B) and in Huh-7 cells transfected with miR-122 inhibitor (AM-122) (C) following vehicle (NT) or sorafenib administration. β-Actin was used as housekeeping gene in western blot analysis and numbers represent fold-change values. NC: miRNA precursor negative control; NCi: miRNA inhibitor’s negative control. (D) Cell viability assay in SerpinB3-overexpressing HepG2 cells following sorafenib treatment (10 and 50 µM for 48 h). (* p ˂ 0.05 Mann–Whitney test). (E) MiR-122 quantification in SerpinB3-overexpressing HepG2 cells following sorafenib treatment. (* p ˂ 0.05 Mann–Whitney test). (F) Western blot analysis of cleaved Poly(ADP-ribose) polymerase 1 (PARP) and cleaved caspase-3 in HepG2 cells overexpressing SerpinB3 (HepG2/SerpinB3) transfected with miR-122 in untreated or sorafenib (10 µM)-treated cells (48 h). β-Actin was used as housekeeping gene in western blot analysis and numbers represent fold-change values. NC: miRNA precursor negative control.

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