An artificial lncRNA targeting multiple miRNAs overcomes sorafenib resistance in hepatocellular carcinoma cells - PubMed (original) (raw)

An artificial lncRNA targeting multiple miRNAs overcomes sorafenib resistance in hepatocellular carcinoma cells

Shuyao Tang et al. Oncotarget. 2016.

Abstract

Sorafenib resistance remains a major obstacle for the effective treatment of hepatocellular carcinoma (HCC), and a number of miRNAs contribute to this resistance. However, the regulatory networks of miRNAs are very complex, thus inhibiting a single miRNA may sequentially activate other compensatory pathways. In the present study, we generated an artificial long non-coding RNA (AlncRNA), which simultaneously targets multiple miRNAs including miR-21, miR-153, miR-216a, miR-217, miR-494 and miR-10a-5p. These miRNAs have been shown to be upregulated in sorafenib-resistant cells and participate in the mechanisms underlying sorafenib resistance. The AlncRNA contains tandem sequences of 6 copies of the complementary binding sequences to the target miRNAs and is expressed by an adenoviral vector (Ad5-AlncRNA). Infection of Ad5-AlncRNA into sorafenib-resistant HCC cells blocked the function of miRNAs, and sequentially inhibited the downregulation of PTEN and activation of AKT. Ad5-AlncRNA significantly inhibited proliferation and induced apoptosis of sorafenib-resistant cells and enhanced the effects of sorafenib in vitro and in animal models. Inhibition of autophagy decreased the sensitivity of sorafenib-resistant cells to Ad5-AlncRNA, while its induction had the opposite effect. These results indicate that targeting multiple miRNAs by the artificial lncRNA could be a potential promising strategy for overcoming sorafenib resistance in the treatment of HCC.

Keywords: drug resistance; hepatocellular carcinoma; miRNA; sorafenib; lncRNA.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

Figures

Figure 1

Figure 1. Exposure to sorafenib induces upregulation of multiple miRNAs

(A) HepG2, HepG2-SR, Huh7 and Huh7-SR cells were incubated with sorafenib (2.5 μM) for 96 h, and then the expression of miRNAs was measured by real-time RT-PCR. The level of miRNA from untreated parental cells was defined as 1. “*” (P < 0.05) and “**” (P < 0.001) indicate a significant difference. (B) Schematic diagram of Ad5-AlncRNA. The expression cassette of whole-length encoding sequence of AlncRNA was inserted into the _EcoR_I/_BamH_I sites of adenovirus E1 region, to generate the recombinant adenovirus Ad5-AlncRNA. ITR, inverted terminal repeats; ψ, adenovirus 5 packaging signal; mCMV, mouse cytomegalovirus promoter.

Figure 2

Figure 2. Expression of AlncRNA in HCC cells after Ad5-AlncRNA infection

(A) HepG2 and Huh7 cells were infected with Ad5-EGFP at MOIs of 10, 20, 100 and 200 pfu/cell for 3 h, and then observed under fluorescent microscopy. (B, C) HepG2, HepG2-SR, Huh7 and Huh7-SR cells were infected with Ad5-AlncRNA at an MOI of 100 pfu/cell for 3 h. Cells were harvested and subjected to conventional RT-PCR and PCR products were electrophoresed (B), or real-time PCR to quantify the expression of AlncRNA (C). “**” (P < 0.001) indicates a significant difference.

Figure 3

Figure 3. AlncRNA increases PTEN expression and inhibits AKT phosphorylation by targeting miR-21 and miR-153

(A) Predicted paring of miR-21 (or miR-153) to the 3′-UTR of the human PTEN gene, and the diagram of a pMIR-REPORT luciferase reporter vector containing the 3′-UTR of PTEN with miR-21 (or miR-153) seed site (pMIR-luc-PTEN3′UTR-miR21 or pMIR-luc-PTEN3′UTR-miR153). (B) Luciferase activities in HepG2, HepG2-SR, Huh7 and Huh7-SR cells transfected with pMIR-luc-PTEN3′UTR-miR21, pMIR-luc-PTEN3′UTR-miR153 or a control vector. Cells were mock co-transfected or co-transfected with negative control (NC), anti-miR-21 or anti-miR153 oligonucleotides, or infected with AlncRNA. Relative luciferase activity was calculated as the percentage of luciferase activity in luciferase reporter vectors -transfected cells over those with the control vector. (C, D) Huh7 and Huh7-SR cells were mock transfected or transfected with NC, anti-miR-21, anti-miR153 or AlncRNA. Twenty-four hours later, cells were subjected to real-time RT-PCR to measure PTEN mRNA expression (C), or immunoblotted for detecting protein expression (D). The density of each band was measured and normalized to respective β-actin. “**” (P < 0.001) indicates a significant difference. “†” (P < 0.05) and “‡” (P < 0.001) indicate a significant increase; while “#” (P < 0.05) and “##” (P < 0.001), a significant reduction, versus mock transfected cells. “f” (P < 0.05) indicates a significant difference from anti-miR-21-transfected cells, while “Ñ” (P < 0.05), from anti-miR-153-transfected cells.

Figure 4

Figure 4. AlncRNA enhances the effects of sorafenib-induced growth inhibition and apoptosis in sorafenib-resistant HCC cells

(A) HepG2-SR or Huh7-SR cells transfected with miRNA negative control, anti-miR-21 or anti-miR-153 oligonucleotides, or infected with Ad5-AlncRNA or Ad5-EGFP, were incubated with serial concentrations of sorafenib for 48 h. Cell viability (%) was measured and normalized with the corresponding untreated cells. (B) The above cells incubated with sorafenib (0, 2.5, 5 or 10 μM) for 48 h were analyzed to measure apoptosis rates (%). (C) Representative dot plots were from the above cytometrically analyzed Huh7-SR cells incubated with sorafenib (5 μM). (D) Huh7-SR cells untreated or incubated with sorafenib (5 μM), or infected with Ad5-AlncRNA, or the combination for 48 h. Cells were then subjected to immunoblotting. “*” (P < 0.05) and “**” (P < 0.001) indicate a significant difference. “†” (P < 0.05)and “‡” (P < 0.001) indicate a significant increase from miR control-treated cells. “#” (P < 0.05) and “##” (P < 0.001) indicate a significant increase from Ad5-EGFP-infected cells. “f” (P < 0.05) indicates a significant difference from anti-miR-21-transfected cells, while “Ñ” (P < 0.05), from anti-miR-153-transfected cells.

Figure 5

Figure 5. AlncRNA enhances sorafenib-induced autophagy in sorafenib-resistant cells

Untreated or Ad5-AlncRNA-infected Huh7-SR cells were incubated in the presence or absence of sorafenib (5 μM) for 48 h. (A) Representative images were taken from acridine orange-stained cells. (B) The above cells were subjected to flow cytometry, and the fold change of acridine orange fluorescence intensity (FL3) versus untreated cells was calculated. (C) The above cells were immunoblotted. The density of each band was measured and normalized to respective β-actin. (D) Untreated or Ad5-AlncRNA-infected cells were incubated for 48 h in the presence or absence of 3-MA (3-methyladenine) (5 mM), RAP (rapamycin) (10 nM), or a mixture of E-64d (10 μg/ml) and pepstatin A (10 μg/ml). Cell viability (%) was compared to untreated cells. (E, F) Huh7-SR cells infected with Ad5-AlncRNA were incubated with sorafenib (5 μM) for 48 h in the presence or absence of 3-MA (5 mM). Cells was subjected to apoptosis assays (E) and immunoblotting (F). Untreated cells served as controls. “*” (P < 0.05) and “**” (P < 0.001) indicate a significant difference. “#” (P < 0.05) and “##” (P < 0.001) indicate a significant increase, while “†” (P < 0.05) and “‡” (P < 0.001), a significant reduction, from untreated control cells. “f” (P < 0.05) indicates a significant difference, while “Ñ” (P < 0.05), a significant reduction, from Ad5-AlncRNA-infected cells.

Figure 6

Figure 6. AlncRNA enhances the efficacy of sorafenib to suppress sorafenib-resistant tumors in vivo

Subcutaneous tumors were established in mice, which received different treatments for 21 days as described in MATERIALS AND METHODS. (A) Illustrated are representative fluorescence microscopy-viewed tumor sections from vehicle and Ad5-EGFP-treated mice prepared 10 days after commencement of treatments. (B) The sizes of tumors were recorded. (C) Tumors were excised and weighed at the end of experiments. (D) Sections of tumors were stained with an anti-Ki67 Ab (upper panel, magnification ×100) or TUNEL (middle panel, magnification ×200). (E, F) Proliferation index (E) and apoptosis index (F) were quantified. “*” (P < 0.05) and “**” (P < 0.001) indicate a significant difference.

Figure 7

Figure 7. Proposed mechanisms by which an artificial lncRNA targeting multiple miRNAs to overcome the molecular mechanism contributing sorafenib resistance by regulating the PTEN/AKT pathway

“→” indicates positive regulation or activation; “⊥”, negative regulation or blockade; “+ P”, regulation by phosphorylation. A dotted line indicates the mechanisms not investigated in the present study. Abbreviations: 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; GSK-3β, glycogen synthase kinase 3β; LC3, microtubule-associated protein 1 light chain 3; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; S6K, ribosomal protein S6 kinase.

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