MicroRNA-92b augments sorafenib resistance in hepatocellular carcinoma via targeting PTEN to activate PI3K/AKT/mTOR signaling

Sorafenib (SOR) resistance is still a significant challenge for the effective treatment of hepatocellular carcinoma (HCC). The mechanism of sorafenib resistance remains unclear. Several microRNAs (miRNAs) have been identified as playing a role in impairing the sensitivity of tumor cells to treatment. We examined the mechanism behind the role of miR-92b in mediating sorafenib resistance in HCC cells. We detected that miR-92b expression was significantly upregulated in SOR-resistant HepG2/SOR cells compared to parental HepG2/WT cells. After transfection with miR-92b inhibitor, the proliferation of HepG2/SOR cells was remarkably weakened and rates of apoptosis significantly increased. PTEN was considered to be a functional target of miR-92b according to a luciferase reporter assay. Knockdown of PTEN significantly impaired the ability of miR-92b inhibitor on increasing sorafenib sensitivity of HepG2/SOR cells. Furthermore, we confirmed by western blotting and immunofluorescence that miR-92b can mediate sorafenib resistance by activating the PI3K/AKT/mTOR pathway in HCC cells by directly targeting PTEN. These findings further validate the mechanism of miR-92b in SOR resistance in HCC treatment.

MicroRNA-92b; Sorafenib; Resistance; PTEN; Hepatocellular carcinoma

Authorship

Zhouyang Cheng

Department of General Surgery, The First Affiliated Hospital of Soochow University, Soochow, ChinaThe First Affiliated Hospital of Soochow UniversityChinaSoochow, ChinaDepartment of General Surgery, The First Affiliated Hospital of Soochow University, Soochow, China

http://orcid.org/0000-0001-6715-2787

Qingfeng Ni

Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, ChinaAffiliated Hospital of Nantong UniversityChinaNantong, ChinaDepartment of General Surgery, Affiliated Hospital of Nantong University, Nantong, China

http://orcid.org/0000-0001-8833-4344

Lei Qin

http://orcid.org/0000-0002-5743-1368

Yang Shi Correspondence: Yang Shi: <shiyang999@126.com>

http://orcid.org/0000-0002-1330-5335

Correspondence: Yang Shi: <shiyang999@126.com>

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (6)

Thumbnail

Figure 1
miR-92b is overexpressed in sorafenib-resistant HepG2 cells. A, Rates of inhibition and half maximal inhibitory concentration (IC₅₀) values of sorafenib (SOR) in HepG2/SOR or HepG2/WT (wild type) cells. B, Flow cytometry was conducted to measure the apoptosis of HepG2/SOR and HepG2/WT cells treated with SOR (0.5 μM). C, qRT-PCR analysis of miR-92b expression in HepG2/WT or HepG2/SOR cells. Data are reported as means±SD, ***P<0.001 (Student's t-test).

Thumbnail

Figure 2
Down-regulation of miR-92b increases sorafenib (SOR) sensitivity in HepG2/SOR cells. A, qRT-PCR analysis of miR-92b expression at different transfections. B and C, Rates of inhibition and half maximal inhibitory concentration (IC₅₀) values of SOR in HepG2/SOR cells. D, Flow cytometry measured apoptosis rates of HepG2/SOR cells at different transfections and incubation with SOR (0.5 μM). Data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001 (ANOVA). NC: negative control.

Thumbnail

Figure 3
PTEN is a target of miR-92b. A, Binding site of miR-92b is located on PTEN mRNA 3′UTR. B, Relative luciferase activities in HepG2/SOR cells co-transfected with miR-92b mimics and luciferase reporters containing PTEN-WT (wild type) or PTEN-MUT (mutant) transcript. C, The mRNA expression and (D) PTEN protein expression was measured using qRT-PCR and western blot in HepG2/SOR cells at different transfections. Data are reported as means±SD. **P<0.01, ***P<0.001 (ANOVA and Student's t-test). NC: negative control.

Thumbnail

Figure 4
Knockdown of PTEN partially reversed the effect of miR-92b inhibitor in HepG2/SOR cells. A, mRNA expression and (B) PTEN protein expression were measured using qRT-PCR and western blot in HepG2/SOR cells at different transfections. C, Rates of inhibition and half maximal inhibitory concentration (IC₅₀) values of sorafenib in HepG2/SOR cells at different transfections. D, Flow cytometry was performed to confirm the effect of PTEN on apoptosis of miR-92b-downregulated HepG2/SOR cells under sorafenib treatment. Data are reported as means±SD. **P<0.01, ***P<0.001 (ANOVA). NC: negative control.

Thumbnail

Figure 5
Knockdown of miR-92b suppresses PI3K/AKT/mTOR signaling pathways in HepG2/SOR cells and down-regulation of PTEN reverses the effect. A, Western blot analysis of p-mTOR, mTOR, p-AKT, and AKT. B, Representative immunofluorescence photomicrograph of p-AKT and p-mTOR (red). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data are reported as means±SD. ***P<0.001 (ANOVA). NC: negative control.

Thumbnail

Figure 6
Inhibition of p-mTOR attenuated the sorafenib resistance of HepG2/SOR cells induced by miR-92b. A, Rates of inhibition and (B) half maximal inhibitory concentration (IC₅₀) values of sorafenib in HepG2/SOR cells transfected with mimics negative control (NC), miR-92b mimics, or miR-92b mimics+rapamycin. C, Flow cytometry was conducted to measure apoptosis of HepG2/SOR cells transfected with mimics NC, miR-92b mimics, or miR-92b mimics+rapamycin. Data are reported as means±SD. **P<0.01, ***P<0.001 (ANOVA).

Figure 1 miR-92b is overexpressed in sorafenib-resistant HepG2 cells. A, Rates of inhibition and half maximal inhibitory concentration (IC₅₀) values of sorafenib (SOR) in HepG2/SOR or HepG2/WT (wild type) cells. B, Flow cytometry was conducted to measure the apoptosis of HepG2/SOR and HepG2/WT cells treated with SOR (0.5 μM). C, qRT-PCR analysis of miR-92b expression in HepG2/WT or HepG2/SOR cells. Data are reported as means±SD, ***P<0.001 (Student's t-test).

Figure 2 Down-regulation of miR-92b increases sorafenib (SOR) sensitivity in HepG2/SOR cells. A, qRT-PCR analysis of miR-92b expression at different transfections. B and C, Rates of inhibition and half maximal inhibitory concentration (IC₅₀) values of SOR in HepG2/SOR cells. D, Flow cytometry measured apoptosis rates of HepG2/SOR cells at different transfections and incubation with SOR (0.5 μM). Data are reported as means±SD. P<0.05, P<0.01, P<0.001 (ANOVA). NC: negative control.

Figure 3 PTEN is a target of miR-92b. A, Binding site of miR-92b is located on PTEN mRNA 3′UTR. B, Relative luciferase activities in HepG2/SOR cells co-transfected with miR-92b mimics and luciferase reporters containing PTEN-WT (wild type) or PTEN-MUT (mutant) transcript. C, The mRNA expression and (D) PTEN protein expression was measured using qRT-PCR and western blot in HepG2/SOR cells at different transfections. Data are reported as means±SD. P<0.01, *P<0.001 (ANOVA and Student's t-test). NC: negative control.

Figure 4 Knockdown of PTEN partially reversed the effect of miR-92b inhibitor in HepG2/SOR cells. A, mRNA expression and (B) PTEN protein expression were measured using qRT-PCR and western blot in HepG2/SOR cells at different transfections. C, Rates of inhibition and half maximal inhibitory concentration (IC₅₀) values of sorafenib in HepG2/SOR cells at different transfections. D, Flow cytometry was performed to confirm the effect of PTEN on apoptosis of miR-92b-downregulated HepG2/SOR cells under sorafenib treatment. Data are reported as means±SD. P<0.01, *P<0.001 (ANOVA). NC: negative control.

Figure 5 Knockdown of miR-92b suppresses PI3K/AKT/mTOR signaling pathways in HepG2/SOR cells and down-regulation of PTEN reverses the effect. A, Western blot analysis of p-mTOR, mTOR, p-AKT, and AKT. B, Representative immunofluorescence photomicrograph of p-AKT and p-mTOR (red). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data are reported as means±SD. ***P<0.001 (ANOVA). NC: negative control.

Figure 6 Inhibition of p-mTOR attenuated the sorafenib resistance of HepG2/SOR cells induced by miR-92b. A, Rates of inhibition and (B) half maximal inhibitory concentration (IC₅₀) values of sorafenib in HepG2/SOR cells transfected with mimics negative control (NC), miR-92b mimics, or miR-92b mimics+rapamycin. C, Flow cytometry was conducted to measure apoptosis of HepG2/SOR cells transfected with mimics NC, miR-92b mimics, or miR-92b mimics+rapamycin. Data are reported as means±SD. P<0.01, *P<0.001 (ANOVA).

How to cite

copy

Associação Brasileira de Divulgação Científica Av. Bandeirantes, 3900, 14049-900 Ribeirão Preto SP Brazil, Tel. / Fax: +55 16 3315-9120 - Ribeirão Preto - SP - Brazil
E-mail: bjournal@terra.com.br

Acompanhe os números deste periódico no seu leitor de RSS

Versão para download de PDF

PDF

Inglês

Versões e tradução automática

Versão original do texto

English

Tradução automática

Google Translator
Microsoft Translator

Como citar

RIS

BIBTEX

Outros formatos de citação e exportação:

SciELO - Scientific Electronic Library Online
Rua Dr. Diogo de Faria, 1087 – 9º andar – Vila Clementino 04037-003 São Paulo/SP - Brasil
E-mail: scielo@scielo.org

Leia a Declaração de Acesso Aberto

Métricas

MicroRNA-92b augments sorafenib resistance in hepatocellular carcinoma via targeting PTEN to activate PI3K/AKT/mTOR signaling

PlumX

Mensagem

[1] Correspondence: Yang Shi: <shiyang999@126.com>