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MiR-106b induces cell radioresistance via the PTEN/PI3K/AKT pathways and p21 in colorectal cancer.

Zheng L, Zhang Y, Liu Y, Zhou M, Lu Y, Yuan L, Zhang C, Hong M, Wang S, Li X - J Transl Med (2015)

Bottom Line: Correspondingly, knocking down miR-106b in SW480 yielded the opposite effect.Restoring the expression of PTEN or p21 in stably miR-106b-overexpressed cells could rescue the effect of miR-106b on cell radioresistance.These observations illustrated that miR-106b could induce cell radioresistance by directly targeting PTEN and p21, this process was accompanied by tumour-initiating cell capacity enhancement, which is universally confirmed to be associated with radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, Guangdong Province, China. 147938636@qq.com.

ABSTRACT

Background: Radioresistance is a challenge in the treatment of patients with colorectal cancer (CRC). Individuals display different therapeutic responses to preoperative radiotherapy, and the need of targeted therapies is urgent. MicroRNAs (miRNAs) are involved in essential biological activities, including chemoresistance and radioresistance. Several research studies have indicated that miRNA played an important role in sensitizing cells to ionizing radiation (IR). MiR-106b, a member of the miR-106b-25 cluster, is frequently dysregulated in many human cancers, including CRC. However, the function of miR-106b in radioresistance is currently poorly understood.

Methods: A series of in vitro and in vivo studies were performed to investigate the roles of miR-106b on cell radioresistance in CRC.

Results: We found overexpression of miR-106b could induce resistance to IR in vitro and in vivo in SW620 cells. Correspondingly, knocking down miR-106b in SW480 yielded the opposite effect. In addition, overexpression of miR-106b could enhance the tumour-initiating cell capacity without or with IR condition, such as the colony sphere formation capacity and the upregulation of stemness-related genes (CD133, Sox2). We further identified PTEN and p21 as novel direct targets of miR-106b by using target prediction algorithms and a luciferase assay. Overexpression of miR-106b reduced the expression of PTEN and p21 and increased the expression of p-AKT, which is a downstream of PTEN. Restoring the expression of PTEN or p21 in stably miR-106b-overexpressed cells could rescue the effect of miR-106b on cell radioresistance. Together, the acquisition of tumour-initiating cell capacity endowed CRC cells with the potential of resistance to irradiation.

Conclusions: These observations illustrated that miR-106b could induce cell radioresistance by directly targeting PTEN and p21, this process was accompanied by tumour-initiating cell capacity enhancement, which is universally confirmed to be associated with radioresistance. Our data suggested that miR-106b at least partly induces cell radioresistance in CRC.

No MeSH data available.


Related in: MedlinePlus

MiR-106b enhances the cell radioresistance. a The detection of cell sensitivity to irradiation by MTT after miR-106b overexpression or downregulation. Data shown are the mean and SE from three independent experiments.*p<0.05 **p<0.01. b Impact of miR-106b on cell survival foci formation when exposed to irradiation (2, 4, 6, 8 Gy). The cell survival curve of the clonogenic assay was obtained using the L-Q Linearity Quadri-model.*p<0.05. c Effect of miR-106b on DNA damage detected by immunofluorescence when exposed to radiation (4 Gy, 6 h). The γ-H2AX staining is shown in the left panels and the numbers of γ-H2AX foci are shown in the right panels.*p<0.05. d γ-H2AX and caspase-3 were examined by western blot when cells were exposed to radiation (4 Gy, 48 h). e Effects of miR-106b on the xenograft radiosensitivity. Tumour sizes were measured at different time points until the mice were sacrificed. The tumour inhibition ratio was calculated every 4 days after exposure to radiation (8 Gy). **p<0.01.
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Fig1: MiR-106b enhances the cell radioresistance. a The detection of cell sensitivity to irradiation by MTT after miR-106b overexpression or downregulation. Data shown are the mean and SE from three independent experiments.*p<0.05 **p<0.01. b Impact of miR-106b on cell survival foci formation when exposed to irradiation (2, 4, 6, 8 Gy). The cell survival curve of the clonogenic assay was obtained using the L-Q Linearity Quadri-model.*p<0.05. c Effect of miR-106b on DNA damage detected by immunofluorescence when exposed to radiation (4 Gy, 6 h). The γ-H2AX staining is shown in the left panels and the numbers of γ-H2AX foci are shown in the right panels.*p<0.05. d γ-H2AX and caspase-3 were examined by western blot when cells were exposed to radiation (4 Gy, 48 h). e Effects of miR-106b on the xenograft radiosensitivity. Tumour sizes were measured at different time points until the mice were sacrificed. The tumour inhibition ratio was calculated every 4 days after exposure to radiation (8 Gy). **p<0.01.

Mentions: First, we analysed the expression pattern of miR-106b in CRC cell lines with different degrees of differentiation, including LOVO (undifferentiated), HT-29 (highly differentiated), SW620 (poorly differentiated), and SW480 (highly differentiated) cells. We found that the expression of miR-106b was higher in the highly differentiated cells lines HT-29 and SW480 (Additional file 5: Figure S1A). Cell lines that stably overexpressed miR-106b or expressed reduced amounts of miR-106b were established via lentiviral transduction, and this procedure is shown in Additional file 5: Figure S1B (*p<0.05). MTT assays were carried out to evaluate the radiosensitivity when cells were exposed to X-rays at a dose of 4 Gy. The data indicated that miR-106b could enhance the cell radioresistance (*p<0.05 **p<0.01; Fig. 1a). The colony survival assay is considered a canonical standard to determine radiosensitivity. Thus, we sought to explore the effect of miR-106b on the colony survival of CRC cells in the presence of ionizing radiation. The cells were irradiated with various doses of X-rays (0, 2, 4, 6 and 8 Gy), and colony formation assays were performed to evaluate the survival fraction. The clonogenic assay results confirmed that cells that overexpressed miR-160b were more resistant to IR than their counterparts, while miR-106b knockdown could enhance cell radiosensitivity (*p<0.05; Fig. 1b).Fig. 1


MiR-106b induces cell radioresistance via the PTEN/PI3K/AKT pathways and p21 in colorectal cancer.

Zheng L, Zhang Y, Liu Y, Zhou M, Lu Y, Yuan L, Zhang C, Hong M, Wang S, Li X - J Transl Med (2015)

MiR-106b enhances the cell radioresistance. a The detection of cell sensitivity to irradiation by MTT after miR-106b overexpression or downregulation. Data shown are the mean and SE from three independent experiments.*p<0.05 **p<0.01. b Impact of miR-106b on cell survival foci formation when exposed to irradiation (2, 4, 6, 8 Gy). The cell survival curve of the clonogenic assay was obtained using the L-Q Linearity Quadri-model.*p<0.05. c Effect of miR-106b on DNA damage detected by immunofluorescence when exposed to radiation (4 Gy, 6 h). The γ-H2AX staining is shown in the left panels and the numbers of γ-H2AX foci are shown in the right panels.*p<0.05. d γ-H2AX and caspase-3 were examined by western blot when cells were exposed to radiation (4 Gy, 48 h). e Effects of miR-106b on the xenograft radiosensitivity. Tumour sizes were measured at different time points until the mice were sacrificed. The tumour inhibition ratio was calculated every 4 days after exposure to radiation (8 Gy). **p<0.01.
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Fig1: MiR-106b enhances the cell radioresistance. a The detection of cell sensitivity to irradiation by MTT after miR-106b overexpression or downregulation. Data shown are the mean and SE from three independent experiments.*p<0.05 **p<0.01. b Impact of miR-106b on cell survival foci formation when exposed to irradiation (2, 4, 6, 8 Gy). The cell survival curve of the clonogenic assay was obtained using the L-Q Linearity Quadri-model.*p<0.05. c Effect of miR-106b on DNA damage detected by immunofluorescence when exposed to radiation (4 Gy, 6 h). The γ-H2AX staining is shown in the left panels and the numbers of γ-H2AX foci are shown in the right panels.*p<0.05. d γ-H2AX and caspase-3 were examined by western blot when cells were exposed to radiation (4 Gy, 48 h). e Effects of miR-106b on the xenograft radiosensitivity. Tumour sizes were measured at different time points until the mice were sacrificed. The tumour inhibition ratio was calculated every 4 days after exposure to radiation (8 Gy). **p<0.01.
Mentions: First, we analysed the expression pattern of miR-106b in CRC cell lines with different degrees of differentiation, including LOVO (undifferentiated), HT-29 (highly differentiated), SW620 (poorly differentiated), and SW480 (highly differentiated) cells. We found that the expression of miR-106b was higher in the highly differentiated cells lines HT-29 and SW480 (Additional file 5: Figure S1A). Cell lines that stably overexpressed miR-106b or expressed reduced amounts of miR-106b were established via lentiviral transduction, and this procedure is shown in Additional file 5: Figure S1B (*p<0.05). MTT assays were carried out to evaluate the radiosensitivity when cells were exposed to X-rays at a dose of 4 Gy. The data indicated that miR-106b could enhance the cell radioresistance (*p<0.05 **p<0.01; Fig. 1a). The colony survival assay is considered a canonical standard to determine radiosensitivity. Thus, we sought to explore the effect of miR-106b on the colony survival of CRC cells in the presence of ionizing radiation. The cells were irradiated with various doses of X-rays (0, 2, 4, 6 and 8 Gy), and colony formation assays were performed to evaluate the survival fraction. The clonogenic assay results confirmed that cells that overexpressed miR-160b were more resistant to IR than their counterparts, while miR-106b knockdown could enhance cell radiosensitivity (*p<0.05; Fig. 1b).Fig. 1

Bottom Line: Correspondingly, knocking down miR-106b in SW480 yielded the opposite effect.Restoring the expression of PTEN or p21 in stably miR-106b-overexpressed cells could rescue the effect of miR-106b on cell radioresistance.These observations illustrated that miR-106b could induce cell radioresistance by directly targeting PTEN and p21, this process was accompanied by tumour-initiating cell capacity enhancement, which is universally confirmed to be associated with radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, Guangdong Province, China. 147938636@qq.com.

ABSTRACT

Background: Radioresistance is a challenge in the treatment of patients with colorectal cancer (CRC). Individuals display different therapeutic responses to preoperative radiotherapy, and the need of targeted therapies is urgent. MicroRNAs (miRNAs) are involved in essential biological activities, including chemoresistance and radioresistance. Several research studies have indicated that miRNA played an important role in sensitizing cells to ionizing radiation (IR). MiR-106b, a member of the miR-106b-25 cluster, is frequently dysregulated in many human cancers, including CRC. However, the function of miR-106b in radioresistance is currently poorly understood.

Methods: A series of in vitro and in vivo studies were performed to investigate the roles of miR-106b on cell radioresistance in CRC.

Results: We found overexpression of miR-106b could induce resistance to IR in vitro and in vivo in SW620 cells. Correspondingly, knocking down miR-106b in SW480 yielded the opposite effect. In addition, overexpression of miR-106b could enhance the tumour-initiating cell capacity without or with IR condition, such as the colony sphere formation capacity and the upregulation of stemness-related genes (CD133, Sox2). We further identified PTEN and p21 as novel direct targets of miR-106b by using target prediction algorithms and a luciferase assay. Overexpression of miR-106b reduced the expression of PTEN and p21 and increased the expression of p-AKT, which is a downstream of PTEN. Restoring the expression of PTEN or p21 in stably miR-106b-overexpressed cells could rescue the effect of miR-106b on cell radioresistance. Together, the acquisition of tumour-initiating cell capacity endowed CRC cells with the potential of resistance to irradiation.

Conclusions: These observations illustrated that miR-106b could induce cell radioresistance by directly targeting PTEN and p21, this process was accompanied by tumour-initiating cell capacity enhancement, which is universally confirmed to be associated with radioresistance. Our data suggested that miR-106b at least partly induces cell radioresistance in CRC.

No MeSH data available.


Related in: MedlinePlus