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Radiation-induced miR-208a increases the proliferation and radioresistance by targeting p21 in human lung cancer cells

Tang, Yiting, Cui, Yayun, Li, Zengpeng, Jiao, Zhuqing, Zhang, Yong, He, Yan, Chen, Guangxia, Zhou, Qunyan, Wang, Wenjie, Zhou, Xifa, Luo, Judong, Zhang, Shuyu·2016·Journal of Experimental & Clinical Cancer Research
DOI 10.1186/s13046-016-0285-3PMC PMC4710038
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Fig. 1 miR-208a in pairs of serum samples from 30 lung cancer patients before and after radiotherapy and in lung cancer cells was examined by qRT-PCR. a The average serum miR-208a expression in 30 paired serum samples from lung cancer patients before and after radiotherapy. b qRT-PCR analysis of the expression of miR-208a in human lung cancer cell lines (A549, H1299, H1975 and H460) and non-cancerous bronchial epithelial cell lines (BEAS-2B and HBE). The relative expression levels of miR-208a in (c) A549, (d) H1299 and (e) HBE cells after exposure to 0, 2, 4 or 8 Gy X-ray irradiation. The relative serum miR-208a expression level normalized to miR-16 and cellular miR-208a expression normalized to U6 were calculated using the equation described in the Materials and Methods. *P < 0.05 and **P < 0.01 compared with the negative control
Fig. 1 miR-208a in pairs of serum samples from 30 lung cancer patients before and after radiotherapy and in lung cancer cells was examined by qRT-PCR. a The average serum miR-208a expression in 30 paired serum samples from lung cancer patients before and after radiotherapy. b qRT-PCR analysis of the expression of miR-208a in human lung cancer cell lines (A549, H1299, H1975 and H460) and non-cancerous bronchial epithelial cell lines (BEAS-2B and HBE). The relative expression levels of miR-208a in (c) A549, (d) H1299 and (e) HBE cells after exposure to 0, 2, 4 or 8 Gy X-ray irradiation. The relative serum miR-208a expression level normalized to miR-16 and cellular miR-208a expression normalized to U6 were calculated using the equation described in the Materials and Methods. *P < 0.05 and **P < 0.01 compared with the negative controlCC BY · PubMed Central
Fig. 2 miR-208a affected the levels of the p21/AKT/mTOR pathway in A549 cells. A549 cells were transfected with the miR-208a mimics, negative control miRNA (miRNA-NC), miR-208a inhibitor or inhibitor-NC for 24 h before lysis. a Western blot analysis the expression levels of p21. b Putative miR-208a binding sequences in the 3′-UTR of p21 mRNA. c Full-length 3′-UTR of p21 and the mutant sequences were cloned into the downstream of the luciferase reporter gene named as pGL3-UTR-WT and pGL3-UTR-MUT, respectively. Each of the plasmids and pRL-TK, were cotransfected into A549 cells, either with miR-208a mimics or inhibitor as indicated. Luciferase activity was assayed 48 h after transfection. All experiments were repeated three times independently. The results are presented as the means ± SEM (n = 3). *P < 0.05. d The membranes were incubated with antibodies to AKT, p-AKT, mTOR, or p-mTOR. The samples were derived from the same experiment and analyzed under the same experimental conditions
Fig. 2 miR-208a affected the levels of the p21/AKT/mTOR pathway in A549 cells. A549 cells were transfected with the miR-208a mimics, negative control miRNA (miRNA-NC), miR-208a inhibitor or inhibitor-NC for 24 h before lysis. a Western blot analysis the expression levels of p21. b Putative miR-208a binding sequences in the 3′-UTR of p21 mRNA. c Full-length 3′-UTR of p21 and the mutant sequences were cloned into the downstream of the luciferase reporter gene named as pGL3-UTR-WT and pGL3-UTR-MUT, respectively. Each of the plasmids and pRL-TK, were cotransfected into A549 cells, either with miR-208a mimics or inhibitor as indicated. Luciferase activity was assayed 48 h after transfection. All experiments were repeated three times independently. The results are presented as the means ± SEM (n = 3). *P < 0.05. d The membranes were incubated with antibodies to AKT, p-AKT, mTOR, or p-mTOR. The samples were derived from the same experiment and analyzed under the same experimental conditionsCC BY · PubMed Central
Fig. 3 Over-expression of miR-208a promoted cellular proliferation. a The effect of miR-208a on viability of the A549 cells was analyzed using the MTT assay, and the miRNA-NC-transfected cells were normalized as 100 %. b The proliferating A549 cells were labeled with EdU. The click-it reaction revealed the EdU staining (red). The cell nuclei were stained with Hoechst 33342 (blue). The images are representative of the results obtained. c Colony formation assays of A549 cells transfected with miR-208a mimics or miR-208a inhibitor. One thousand cells were seeded onto each plate. After 10 days, the cells were stained with crystal violet. The colonies consisting of more than 50 cells were counted. The data are presented as the means ± SEM (n = 4). *P < 0.05 and **P < 0.01 compared with the negative control
Fig. 3 Over-expression of miR-208a promoted cellular proliferation. a The effect of miR-208a on viability of the A549 cells was analyzed using the MTT assay, and the miRNA-NC-transfected cells were normalized as 100 %. b The proliferating A549 cells were labeled with EdU. The click-it reaction revealed the EdU staining (red). The cell nuclei were stained with Hoechst 33342 (blue). The images are representative of the results obtained. c Colony formation assays of A549 cells transfected with miR-208a mimics or miR-208a inhibitor. One thousand cells were seeded onto each plate. After 10 days, the cells were stained with crystal violet. The colonies consisting of more than 50 cells were counted. The data are presented as the means ± SEM (n = 4). *P < 0.05 and **P < 0.01 compared with the negative controlCC BY · PubMed Central
Fig. 4 miR-208a increased the cell apoptosis and disturbed the cell cycle of lung cancer cells. a A549 and (b) H1975 cells were transfected with the indicated vectors. Two days after transfection, the percentage of apoptotic cells was evaluated using flow cytometry. Western blot analyzed the apoptotic molecular component including PARP1, Bcl2 and Bax in (c) A549 and (d) H1975 cells. The cell cycle distributions of (e) A549 and (f) H1975 cells were measured using flow cytometry. *P < 0.05 and **P < 0.01 compared with the negative control
Fig. 4 miR-208a increased the cell apoptosis and disturbed the cell cycle of lung cancer cells. a A549 and (b) H1975 cells were transfected with the indicated vectors. Two days after transfection, the percentage of apoptotic cells was evaluated using flow cytometry. Western blot analyzed the apoptotic molecular component including PARP1, Bcl2 and Bax in (c) A549 and (d) H1975 cells. The cell cycle distributions of (e) A549 and (f) H1975 cells were measured using flow cytometry. *P < 0.05 and **P < 0.01 compared with the negative controlCC BY · PubMed Central
Fig. 5 miR-208a enhanced the radioresistance of A549 cells. a Colony formation assays and (b) clonogenic cell survival curves were generated. c D0, Dq and calculated SER values of miRNA-NC- and miR-208a mimic-transfected groups are shown; (d) Colony formation assays and (e) clonogenic cell survival curves were generated. f D0, Dq and calculated SER values of inhibitor-NC- and miR-208a inhibitor-transfected groups are shown. The SER was calculated according to the multi-target single hit model
Fig. 5 miR-208a enhanced the radioresistance of A549 cells. a Colony formation assays and (b) clonogenic cell survival curves were generated. c D0, Dq and calculated SER values of miRNA-NC- and miR-208a mimic-transfected groups are shown; (d) Colony formation assays and (e) clonogenic cell survival curves were generated. f D0, Dq and calculated SER values of inhibitor-NC- and miR-208a inhibitor-transfected groups are shown. The SER was calculated according to the multi-target single hit modelCC BY · PubMed Central
Fig. 6 Identification and characterization of serum exosome miR-208a translocation into A549 cells. Exosomes were isolated using centrifugation, filtration and ultracentrifugation as described in the Materials and Methods. a Confirmation of the exosome marker CD63 by Western blot. b qRT-PCR analysis of miR-208a and miR-16 contained in the exosomes. c qRT-PCR analysis of miR-208a in culture medium of A549 cells. The conditional medium incubated with A549 cells collected 24 h after exposure to 0 or 4 Gy X-ray irradiation. d Microscopic analysis of the translocation of the pre-labeled exosomes. The DiI-labeled exosomes displayed red color. The cell nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. *P < 0.05 compared with BR or the group treated with 0 Gy. BR: before radiotherapy; AR: after receiving 60 Gy radiotherapy
Fig. 6 Identification and characterization of serum exosome miR-208a translocation into A549 cells. Exosomes were isolated using centrifugation, filtration and ultracentrifugation as described in the Materials and Methods. a Confirmation of the exosome marker CD63 by Western blot. b qRT-PCR analysis of miR-208a and miR-16 contained in the exosomes. c qRT-PCR analysis of miR-208a in culture medium of A549 cells. The conditional medium incubated with A549 cells collected 24 h after exposure to 0 or 4 Gy X-ray irradiation. d Microscopic analysis of the translocation of the pre-labeled exosomes. The DiI-labeled exosomes displayed red color. The cell nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. *P < 0.05 compared with BR or the group treated with 0 Gy. BR: before radiotherapy; AR: after receiving 60 Gy radiotherapyCC BY · PubMed Central

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Metadata

Authors
Tang, Y., Cui, Y., Li, Z., Jiao, Z., Zhang, Y., He, Y., Chen, G., Zhou, Q., Wang, W., Zhou, X., Luo, J., & Zhang, S.
Venue
Journal of Experimental & Clinical Cancer Research
Publisher
Springer Science and Business Media LLC
Published
2016-01-12
Volume
35
Issue
1
ISSN
1756-9966
DOI
10.1186/s13046-016-0285-3
PMC
PMC4710038
Cited by
152
References
4
Language
en
Update notices
correction (2016-01-28)
Funders
  • National Natural Science Foundation of China (81402518, 51307010)
  • National Natural Science Foundation of China (CN) (81472920, 81502038)
  • Jiangsu Provincial Special Program of Medical Science (BE2015631; BK20151174)
  • Changzhou Scientific Program (2014260; CJ20140050; CJ20159038; CE20155046; QN201503)

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