International Journal of
Radiation Oncology biology
physics
www.redjournal.org
Biology Contribution
MiR-20a Induces Cell Radioresistance by Activating the PTEN/PI3K/Akt Signaling Pathway in Hepatocellular Carcinoma Yuqin Zhang, MD, PhD,* Lin Zheng, MD, PhD,y,z Yi Ding, MD, PhD,* Qi Li, MD, PhD,x Rong Wang, MS,* Tongxin Liu, MD, PhD,k Quanquan Sun, MD, PhD,k Hua Yang, MD,{ Shunli Peng, MS,* Wei Wang, MD, PhD,* and Longhua Chen, MD, PhD* Departments of *Radiation Oncology, zPathology, and xGastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China; yDepartment of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong Province, China; kDepartment of Radiation Oncology, Cancer Hospital, Hangzhou, Zhejiang Province, China; and {Department of Radiation Oncology, Nanhai Hospital, Southern Medical University, Guangzhou, Guangdong Province, China Received Oct 21, 2014, and in revised form Mar 11, 2015. Accepted for publication Apr 5, 2015.
Summary miR-20a is overexpressed in HCC tissues and cell lines, and PTEN is inversely correlated with it. miR-20a could induce HCC cell radioresistance. PTEN is a functional target of miR-20a for the induction of radioresistance. PTEN could rescue the effect of miR-20a on cell radioresistance. PTEN could rescue the effect of miR-20a on cell
Purpose: To investigate the role of miR-20a in hepatocellular carcinoma (HCC) cell radioresistance, which may reveal potential strategies to improve treatment. Methods and Materials: The expression of miR-20a and PTEN were detected in HCC cell lines and paired primary tissues by quantitative real-time polymerase chain reaction. Cell radiation combined with colony formation assays was administrated to discover the effect of miR-20a on radiosensitivity. Bioinformatics prediction and luciferase assay were used to identify the target of miR-20a. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit phosphorylation of Akt, to verify whether miR-20a affects HCC cell radioresistance through activating the PTEN/PI3K/Akt pathway. Results: MiR-20a levels were increased in HCC cell lines and tissues, whereas PTEN was inversely correlated with it. Overexpression of miR-20a in Bel-7402 and SMMC7721 cells enhances their resistance to the effect of ionizing radiation, and the inhibition of miR-20a in HCCLM3 and QGY-7701 cells sensitizes them to it. PTEN was identified as a direct functional target of miR-20a for the induction of radioresistance.
Reprint requests to: Longhua Chen, MD, PhD, Department of Radiation Oncology, Southern Medical University, Nanfang Hospital, Guangzhou, Guangdong Province, China. Tel: (þ86) (20) 6164-2136; E-mail: [email protected] or Wei Wang, MD, PhD, Department of Radiation Oncology, Southern Medical University, Nanfang Hospital, Guangzhou, Guangdong Province, China. Tel: (+86) (20) 6164-2133; E-mail: [email protected]
Int J Radiation Oncol Biol Phys, Vol. -, No. -, pp. 1e9, 2015 0360-3016/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2015.04.007
This work was supported by the National Natural Scientific Foundation of China (grant 81272507), the Natural Science Foundation of Guangdong Province (grant S2013010014185), and the Pearl River Nova Program of Guangzhou, Guangdong Province (grant 2014J2200015). Y.Z., L.Z., and Y.D. contributed equally to this work. Conflict of interest: none. Supplementary material for this article can be found at www.redjournal.org.
2
International Journal of Radiation Oncology Biology Physics
Zhang et al.
radioresistance. miR-20a induces cell radioresistance by activating the PTEN/PI3K/ Akt signaling pathway in HCC.
Overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway. Additionally, the kinase inhibitor LY294002 could reverse the effect of miR-20aeinduced radioresistance. Conclusion: MiR-20a induces HCC cell radioresistance by activating the PTEN/PI3K/ Akt pathway, which suggests that miR-20a/PTEN/PI3K/Akt might represent a target of investigation for developing effective therapeutic strategies against HCC. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies in the world. Surgical resection provides the best chance for a cure in select patients (1). Unfortunately, surgery is often followed by a high rate of tumor recurrence. The 5-year rate of HCC recurrence after resection has been reported as high as 70% (2). It has been proposed that the best way to reduce the rate of tumor recurrence in HCC is through improvements in adjuvant therapies (3). Radiation therapy (RT) is used in approximately one-third of all patients with cancer. The development of a 3-dimensional conformal technique and intensitymodulated radiation therapy techniques allows the delivery of higher doses of radiation to the target tissue, thereby sparing the majority of the liver from radiation damage. In a series of retrospective and prospective studies, it was determined that RT is a safe and effective local therapy with potential benefits for patients who are unable to undergo surgery (4-6). There are certain limitations to the use of RT in patients with HCC, however. A crucial limitation is that it is one of several forms of therapy to which HCC has demonstrated resistance, the others being chemotherapy, targeted therapy, and various forms of adjuvant treatments. If cellular sensitivity to radiation can be enhanced, we may be able to improve the benefit of RT in HCC and reduce the toxicity of this approach. It was recently determined that microRNAs (miRNAs) may play a role in the cellular response to RT (7, 8). MiR20a, a member of the miR-17-92 cluster, has been shown to function as an oncomir in many human cancers, including lung cancer (9), HCC (10), and gastric cancer (11). Research has shown that irradiation can up-regulate miR20a activity (12). We found that miR-20a levels are increased in primary HCC tissues and HCC cell lines. miR-20a enhanced the resistance of Bel-7402 and SMMC-7721 cells to radiation, and inhibition of miR-20a in HCCLM3 and QGY-7701 cell lines sensitized these cells to radiation treatment. Previous studies confirmed the relationship between radioresistance and the expression of genes that induce the DNA damage checkpoint response and increase the capacity for DNA repair (13-15). The tumor suppressor gene PTEN plays a vital role in the inhibition of progression through various intracellular regulatory
survival pathways, including PI3K/Akt and mitogenactivated protein kinase pathways (16). Downregulation of PTEN is important in the development of multi-drug resistance and radioresistance (17, 18). We identified PTEN as a direct target of miR-20a during the development of radioresistance. Specifically, overexpression of PTEN in cells in which miR-20a is continuously overexpressed inhibited the effects of it. Moreover, overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway, and miR-20ae induced radioresistance was inhibited using LY294002 to increase Akt activity. These findings suggest that miR20a induces cell radioresistance by activating the PTEN/ PI3K/Akt pathway, indicating an appropriate target for the development of a new therapeutic approach to HCC.
Methods and Materials Patient specimens and cell culture Hepatocellular carcinoma tissue samples were collected from 28 patients at Nanfang Hospital, Southern Medical University Hospital. Written informed consent was obtained from every study participant. Human liver cancer cell lines Bel-7402, Bel-7404, SMMC-7721, QGY-7701, and HCCLM3 and normal human cell line LO2 were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, USA) in a humid wet atmosphere containing 5% CO2 at 37 C.
Cell reagents Full-length PTEN complementary DNA (cDNA) lacking the 30 untranslated region (UTR) was purchased from GeneCopeia (Rockville, MD) and subcloned into the eukaryotic expression vector pcDNA3.1(þ) (Invitrogen). The pre-miR-20a sequence was amplified and cloned into pCDH-CMV-MCS-EF1-coGFP constructs (System Biosciences, Mountain View, CA). miR-20a and PTEN knockdown virus were purchased from GENECHEM (Shanghai, China). MiR-20a mimics, a nonspecific miRcontrol, anti-miR-20a, and a nonspecific anti-miR control were purchased from Thermo Scientific Dharmacon
Volume - Number - 2015
miR-20a induces HCC cell radioresistance
(Pittsburgh, USA). The PI3K tyrosine kinase inhibitor LY294002 was purchased from Calbiochem (Darmstadt, Germany), and an Annexin V-fluorescein isothiocyanate/ 7-aminoactinomcin D kit was purchased from Beckman Coulter (Jersey City, NJ).
analysis of miR-20a activity were performed using a qSYBR-greenecontaining PCR kit (GenePharma, Shanghai, China), with U6 used as an endogenous control. The precursor form of miR-20a was amplified. To detect PTEN mRNA, cDNA was synthesized from 1 mg of total RNA using the reverse transcription reaction kit according to the manufacturer’s instructions (Promega, Madison, WI). Human glyceraldehyde-3phosphate dehydrogenase was amplified in parallel as an internal control. The primers are listed in Table S1 (available online at www.redjournal.org). All samples
Quantitative real-time polymerase chain reaction Total RNA was extracted from the cells using a TRIzol reagent (Invitrogen). Reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR)
A
C
Relative miR-20a/U6 expression
10
3
N1 0.89
Normal Tumor
C1 0.08
N2 0.32
C2 0.11
C5
N6 C6 1.66 0.53
N3 0.91
C3 N4 C4 0.41 0.78 0.17
PTEN
8
GAPDH N5 1.32
6
0.65
N7 C7 1.59 0.71
N8 1.12
C8 0.81
PTEN
4
GAPDH
2 N9 1.52
0
C10 N11 0.59 0.95
C11 N12 C12 0.46 1.08 0.41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
PTEN GAPDH
2.0
Normal Tumor
N13 C13 N14 C14 N15 C15 N16 0.82 0.63 1.59 0.41 1.67 0.58 0.43
Relative PTEN/GAPDH expression
B
C9 N10 0.76 1.63
C16 0.78
PTEN
1.5
GAPDH
1.0
N17 C17 0.86 0.21
N18 0.23
C18 0.01
N19 C19 N20 C20 1.21 0.32 0.45 0.008
N21 0.34
C21 0.59
N22 C22 N23 C23 0.69 0.21 0.59 0.28
N25 0.45
C25 N26 C26 N27 C27 0.51 0.43 0.11 0.53 0.09
PTEN
0.5
GAPDH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
0.0
40 2
70 1
M3
GAPDH
HC
QG
Be
l-7
CL
40
Y7
4
72 1 Be
1.01
l-7
MC SM
2 LO
1.87
1.65
0.48
0.62
1.21
PTEN
C28 0.38
PTEN
F 1.5
*
8
*
6 4
*
*
*
2
Relative PTEN mRNA expression
10
1.0
*
*
0.5
* *
*
40 4
CL M3 QG Y77 01 Be l-7 40 2
HC
l-7
-7 72 1
Be
MC SM
40 2
Be
l-7
70 1
QG
Y7
M3
4
CL HC
l-7 40
72 1
Be
-7
LO
MC SM
LO 2
0.0
0 2
Relative miR-20a expression
N28 0.39
GAPDH
GAPDH
E
C24 0.19
PTEN
-7
D
N24 0.67
Fig. 1. Expression of miR-20a and PTEN in primary hepatocellular carcinoma (HCC) tissues and cell lines. (A) miR-20a levels in HCC tissues compared with matched normal tissues. U6 was used as an internal control. (B, C) PTEN levels in HCC tissues compared with matched normal tissues, detected by quantitative real-time polymerase chain reaction and Western blot, respectively. (D) PTEN levels in HCC cell lines, detected by Western blot. (E) miR-20a levels in HCC cell lines compared with a normal HCC cell line. U6 was used as an internal control. *P
Radiation Oncology biology
physics
www.redjournal.org
Biology Contribution
MiR-20a Induces Cell Radioresistance by Activating the PTEN/PI3K/Akt Signaling Pathway in Hepatocellular Carcinoma Yuqin Zhang, MD, PhD,* Lin Zheng, MD, PhD,y,z Yi Ding, MD, PhD,* Qi Li, MD, PhD,x Rong Wang, MS,* Tongxin Liu, MD, PhD,k Quanquan Sun, MD, PhD,k Hua Yang, MD,{ Shunli Peng, MS,* Wei Wang, MD, PhD,* and Longhua Chen, MD, PhD* Departments of *Radiation Oncology, zPathology, and xGastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China; yDepartment of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong Province, China; kDepartment of Radiation Oncology, Cancer Hospital, Hangzhou, Zhejiang Province, China; and {Department of Radiation Oncology, Nanhai Hospital, Southern Medical University, Guangzhou, Guangdong Province, China Received Oct 21, 2014, and in revised form Mar 11, 2015. Accepted for publication Apr 5, 2015.
Summary miR-20a is overexpressed in HCC tissues and cell lines, and PTEN is inversely correlated with it. miR-20a could induce HCC cell radioresistance. PTEN is a functional target of miR-20a for the induction of radioresistance. PTEN could rescue the effect of miR-20a on cell radioresistance. PTEN could rescue the effect of miR-20a on cell
Purpose: To investigate the role of miR-20a in hepatocellular carcinoma (HCC) cell radioresistance, which may reveal potential strategies to improve treatment. Methods and Materials: The expression of miR-20a and PTEN were detected in HCC cell lines and paired primary tissues by quantitative real-time polymerase chain reaction. Cell radiation combined with colony formation assays was administrated to discover the effect of miR-20a on radiosensitivity. Bioinformatics prediction and luciferase assay were used to identify the target of miR-20a. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit phosphorylation of Akt, to verify whether miR-20a affects HCC cell radioresistance through activating the PTEN/PI3K/Akt pathway. Results: MiR-20a levels were increased in HCC cell lines and tissues, whereas PTEN was inversely correlated with it. Overexpression of miR-20a in Bel-7402 and SMMC7721 cells enhances their resistance to the effect of ionizing radiation, and the inhibition of miR-20a in HCCLM3 and QGY-7701 cells sensitizes them to it. PTEN was identified as a direct functional target of miR-20a for the induction of radioresistance.
Reprint requests to: Longhua Chen, MD, PhD, Department of Radiation Oncology, Southern Medical University, Nanfang Hospital, Guangzhou, Guangdong Province, China. Tel: (þ86) (20) 6164-2136; E-mail: [email protected] or Wei Wang, MD, PhD, Department of Radiation Oncology, Southern Medical University, Nanfang Hospital, Guangzhou, Guangdong Province, China. Tel: (+86) (20) 6164-2133; E-mail: [email protected]
Int J Radiation Oncol Biol Phys, Vol. -, No. -, pp. 1e9, 2015 0360-3016/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2015.04.007
This work was supported by the National Natural Scientific Foundation of China (grant 81272507), the Natural Science Foundation of Guangdong Province (grant S2013010014185), and the Pearl River Nova Program of Guangzhou, Guangdong Province (grant 2014J2200015). Y.Z., L.Z., and Y.D. contributed equally to this work. Conflict of interest: none. Supplementary material for this article can be found at www.redjournal.org.
2
International Journal of Radiation Oncology Biology Physics
Zhang et al.
radioresistance. miR-20a induces cell radioresistance by activating the PTEN/PI3K/ Akt signaling pathway in HCC.
Overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway. Additionally, the kinase inhibitor LY294002 could reverse the effect of miR-20aeinduced radioresistance. Conclusion: MiR-20a induces HCC cell radioresistance by activating the PTEN/PI3K/ Akt pathway, which suggests that miR-20a/PTEN/PI3K/Akt might represent a target of investigation for developing effective therapeutic strategies against HCC. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies in the world. Surgical resection provides the best chance for a cure in select patients (1). Unfortunately, surgery is often followed by a high rate of tumor recurrence. The 5-year rate of HCC recurrence after resection has been reported as high as 70% (2). It has been proposed that the best way to reduce the rate of tumor recurrence in HCC is through improvements in adjuvant therapies (3). Radiation therapy (RT) is used in approximately one-third of all patients with cancer. The development of a 3-dimensional conformal technique and intensitymodulated radiation therapy techniques allows the delivery of higher doses of radiation to the target tissue, thereby sparing the majority of the liver from radiation damage. In a series of retrospective and prospective studies, it was determined that RT is a safe and effective local therapy with potential benefits for patients who are unable to undergo surgery (4-6). There are certain limitations to the use of RT in patients with HCC, however. A crucial limitation is that it is one of several forms of therapy to which HCC has demonstrated resistance, the others being chemotherapy, targeted therapy, and various forms of adjuvant treatments. If cellular sensitivity to radiation can be enhanced, we may be able to improve the benefit of RT in HCC and reduce the toxicity of this approach. It was recently determined that microRNAs (miRNAs) may play a role in the cellular response to RT (7, 8). MiR20a, a member of the miR-17-92 cluster, has been shown to function as an oncomir in many human cancers, including lung cancer (9), HCC (10), and gastric cancer (11). Research has shown that irradiation can up-regulate miR20a activity (12). We found that miR-20a levels are increased in primary HCC tissues and HCC cell lines. miR-20a enhanced the resistance of Bel-7402 and SMMC-7721 cells to radiation, and inhibition of miR-20a in HCCLM3 and QGY-7701 cell lines sensitized these cells to radiation treatment. Previous studies confirmed the relationship between radioresistance and the expression of genes that induce the DNA damage checkpoint response and increase the capacity for DNA repair (13-15). The tumor suppressor gene PTEN plays a vital role in the inhibition of progression through various intracellular regulatory
survival pathways, including PI3K/Akt and mitogenactivated protein kinase pathways (16). Downregulation of PTEN is important in the development of multi-drug resistance and radioresistance (17, 18). We identified PTEN as a direct target of miR-20a during the development of radioresistance. Specifically, overexpression of PTEN in cells in which miR-20a is continuously overexpressed inhibited the effects of it. Moreover, overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway, and miR-20ae induced radioresistance was inhibited using LY294002 to increase Akt activity. These findings suggest that miR20a induces cell radioresistance by activating the PTEN/ PI3K/Akt pathway, indicating an appropriate target for the development of a new therapeutic approach to HCC.
Methods and Materials Patient specimens and cell culture Hepatocellular carcinoma tissue samples were collected from 28 patients at Nanfang Hospital, Southern Medical University Hospital. Written informed consent was obtained from every study participant. Human liver cancer cell lines Bel-7402, Bel-7404, SMMC-7721, QGY-7701, and HCCLM3 and normal human cell line LO2 were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, USA) in a humid wet atmosphere containing 5% CO2 at 37 C.
Cell reagents Full-length PTEN complementary DNA (cDNA) lacking the 30 untranslated region (UTR) was purchased from GeneCopeia (Rockville, MD) and subcloned into the eukaryotic expression vector pcDNA3.1(þ) (Invitrogen). The pre-miR-20a sequence was amplified and cloned into pCDH-CMV-MCS-EF1-coGFP constructs (System Biosciences, Mountain View, CA). miR-20a and PTEN knockdown virus were purchased from GENECHEM (Shanghai, China). MiR-20a mimics, a nonspecific miRcontrol, anti-miR-20a, and a nonspecific anti-miR control were purchased from Thermo Scientific Dharmacon
Volume - Number - 2015
miR-20a induces HCC cell radioresistance
(Pittsburgh, USA). The PI3K tyrosine kinase inhibitor LY294002 was purchased from Calbiochem (Darmstadt, Germany), and an Annexin V-fluorescein isothiocyanate/ 7-aminoactinomcin D kit was purchased from Beckman Coulter (Jersey City, NJ).
analysis of miR-20a activity were performed using a qSYBR-greenecontaining PCR kit (GenePharma, Shanghai, China), with U6 used as an endogenous control. The precursor form of miR-20a was amplified. To detect PTEN mRNA, cDNA was synthesized from 1 mg of total RNA using the reverse transcription reaction kit according to the manufacturer’s instructions (Promega, Madison, WI). Human glyceraldehyde-3phosphate dehydrogenase was amplified in parallel as an internal control. The primers are listed in Table S1 (available online at www.redjournal.org). All samples
Quantitative real-time polymerase chain reaction Total RNA was extracted from the cells using a TRIzol reagent (Invitrogen). Reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR)
A
C
Relative miR-20a/U6 expression
10
3
N1 0.89
Normal Tumor
C1 0.08
N2 0.32
C2 0.11
C5
N6 C6 1.66 0.53
N3 0.91
C3 N4 C4 0.41 0.78 0.17
PTEN
8
GAPDH N5 1.32
6
0.65
N7 C7 1.59 0.71
N8 1.12
C8 0.81
PTEN
4
GAPDH
2 N9 1.52
0
C10 N11 0.59 0.95
C11 N12 C12 0.46 1.08 0.41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
PTEN GAPDH
2.0
Normal Tumor
N13 C13 N14 C14 N15 C15 N16 0.82 0.63 1.59 0.41 1.67 0.58 0.43
Relative PTEN/GAPDH expression
B
C9 N10 0.76 1.63
C16 0.78
PTEN
1.5
GAPDH
1.0
N17 C17 0.86 0.21
N18 0.23
C18 0.01
N19 C19 N20 C20 1.21 0.32 0.45 0.008
N21 0.34
C21 0.59
N22 C22 N23 C23 0.69 0.21 0.59 0.28
N25 0.45
C25 N26 C26 N27 C27 0.51 0.43 0.11 0.53 0.09
PTEN
0.5
GAPDH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
0.0
40 2
70 1
M3
GAPDH
HC
QG
Be
l-7
CL
40
Y7
4
72 1 Be
1.01
l-7
MC SM
2 LO
1.87
1.65
0.48
0.62
1.21
PTEN
C28 0.38
PTEN
F 1.5
*
8
*
6 4
*
*
*
2
Relative PTEN mRNA expression
10
1.0
*
*
0.5
* *
*
40 4
CL M3 QG Y77 01 Be l-7 40 2
HC
l-7
-7 72 1
Be
MC SM
40 2
Be
l-7
70 1
QG
Y7
M3
4
CL HC
l-7 40
72 1
Be
-7
LO
MC SM
LO 2
0.0
0 2
Relative miR-20a expression
N28 0.39
GAPDH
GAPDH
E
C24 0.19
PTEN
-7
D
N24 0.67
Fig. 1. Expression of miR-20a and PTEN in primary hepatocellular carcinoma (HCC) tissues and cell lines. (A) miR-20a levels in HCC tissues compared with matched normal tissues. U6 was used as an internal control. (B, C) PTEN levels in HCC tissues compared with matched normal tissues, detected by quantitative real-time polymerase chain reaction and Western blot, respectively. (D) PTEN levels in HCC cell lines, detected by Western blot. (E) miR-20a levels in HCC cell lines compared with a normal HCC cell line. U6 was used as an internal control. *P
