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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

PTEN and p21 are targets of miR-106b. a PTEN and p21 3′UTRs contain predicted miR-106b binding sites. In the figure the alignment of the seed regions of miR-106b with PTEN and p21 3′UTRs is shown. b The expression levels of PTEN and p21 after the inhibition of miR-106b via lentiviral transduction in SW480 cells or the overexpression of the same miRNA by oligonucleotide transfection or lentiviral transduction in SW620 cells were detected using western blot. c The mRNA expression levels of PTEN after the inhibition of miR-106b in SW480 cells or the overexpression of the same miRNA in SW620 cells was detected using qRT-PCR. **p<0.01. d PTEN 3′UTRs are targets of miR-106b. pluc3-PTEN that contained a wild-type or mutated PTEN 3′UTRs (indicated as WT or mut on the X-axis) was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results. **p<0.01. e p21 3′UTRs are targets of miR-106b. pluc3-p21 that contained a wild-type p21 3′UTRs was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results.**p<0.01.
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Fig3: PTEN and p21 are targets of miR-106b. a PTEN and p21 3′UTRs contain predicted miR-106b binding sites. In the figure the alignment of the seed regions of miR-106b with PTEN and p21 3′UTRs is shown. b The expression levels of PTEN and p21 after the inhibition of miR-106b via lentiviral transduction in SW480 cells or the overexpression of the same miRNA by oligonucleotide transfection or lentiviral transduction in SW620 cells were detected using western blot. c The mRNA expression levels of PTEN after the inhibition of miR-106b in SW480 cells or the overexpression of the same miRNA in SW620 cells was detected using qRT-PCR. **p<0.01. d PTEN 3′UTRs are targets of miR-106b. pluc3-PTEN that contained a wild-type or mutated PTEN 3′UTRs (indicated as WT or mut on the X-axis) was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results. **p<0.01. e p21 3′UTRs are targets of miR-106b. pluc3-p21 that contained a wild-type p21 3′UTRs was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results.**p<0.01.

Mentions: We focused on the targets of miR-106b and found via a bioinformatics search in Targetscan (http://www.targetscan.org) that the 3′-UTRs of human PTEN and p21 contained regions that matched the seed sequences of miR-106b (Fig. 3a). PTEN is an important negative regulator of PI3K-AKT signalling that is involved in the complex response to IR via the induction of cell cycle arrest in the G2/M phase and apoptosis [21, 22]. CDKN1A (p21), a key inhibitor of the cell cycle, is also frequently dysfunctional in human cancer [23]. Increasing the endogenous miR-106b levels by either oligonucleotide transfection (*p<0.05; Additional file 7: Figure S3A) or lentiviral transduction could significantly decrease PTEN expression both at the RNA and protein levels, but the expression of P21 was only decreased at the protein level. The inhibition of miR-106b yielded the same effect (Fig. 3b, c).Fig. 3


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)

PTEN and p21 are targets of miR-106b. a PTEN and p21 3′UTRs contain predicted miR-106b binding sites. In the figure the alignment of the seed regions of miR-106b with PTEN and p21 3′UTRs is shown. b The expression levels of PTEN and p21 after the inhibition of miR-106b via lentiviral transduction in SW480 cells or the overexpression of the same miRNA by oligonucleotide transfection or lentiviral transduction in SW620 cells were detected using western blot. c The mRNA expression levels of PTEN after the inhibition of miR-106b in SW480 cells or the overexpression of the same miRNA in SW620 cells was detected using qRT-PCR. **p<0.01. d PTEN 3′UTRs are targets of miR-106b. pluc3-PTEN that contained a wild-type or mutated PTEN 3′UTRs (indicated as WT or mut on the X-axis) was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results. **p<0.01. e p21 3′UTRs are targets of miR-106b. pluc3-p21 that contained a wild-type p21 3′UTRs was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results.**p<0.01.
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Fig3: PTEN and p21 are targets of miR-106b. a PTEN and p21 3′UTRs contain predicted miR-106b binding sites. In the figure the alignment of the seed regions of miR-106b with PTEN and p21 3′UTRs is shown. b The expression levels of PTEN and p21 after the inhibition of miR-106b via lentiviral transduction in SW480 cells or the overexpression of the same miRNA by oligonucleotide transfection or lentiviral transduction in SW620 cells were detected using western blot. c The mRNA expression levels of PTEN after the inhibition of miR-106b in SW480 cells or the overexpression of the same miRNA in SW620 cells was detected using qRT-PCR. **p<0.01. d PTEN 3′UTRs are targets of miR-106b. pluc3-PTEN that contained a wild-type or mutated PTEN 3′UTRs (indicated as WT or mut on the X-axis) was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results. **p<0.01. e p21 3′UTRs are targets of miR-106b. pluc3-p21 that contained a wild-type p21 3′UTRs was transfected into SW620 or SW480 cells. The relative repression of firefly luciferase was standardized to a transfection control. The reporter assays were performed three times with essentially identical results.**p<0.01.
Mentions: We focused on the targets of miR-106b and found via a bioinformatics search in Targetscan (http://www.targetscan.org) that the 3′-UTRs of human PTEN and p21 contained regions that matched the seed sequences of miR-106b (Fig. 3a). PTEN is an important negative regulator of PI3K-AKT signalling that is involved in the complex response to IR via the induction of cell cycle arrest in the G2/M phase and apoptosis [21, 22]. CDKN1A (p21), a key inhibitor of the cell cycle, is also frequently dysfunctional in human cancer [23]. Increasing the endogenous miR-106b levels by either oligonucleotide transfection (*p<0.05; Additional file 7: Figure S3A) or lentiviral transduction could significantly decrease PTEN expression both at the RNA and protein levels, but the expression of P21 was only decreased at the protein level. The inhibition of miR-106b yielded the same effect (Fig. 3b, c).Fig. 3

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