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Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells.

Tao YJ, Li YJ, Zheng W, Zhao JJ, Guo MM, Zhou Y, Qin NL, Zheng J, Xu L - Cancer Cell Int. (2015)

Bottom Line: Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells.Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells.Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Zunyi Medical College, Zunyi, 563003 Guizhou China.

ABSTRACT

Background: Colon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated.

Methods: In present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot.

Results: We found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered.

Conclusions: Therefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.

No MeSH data available.


Related in: MedlinePlus

MiRNA-21 ASO reduced the proliferation of human colon carcinoma cells. a The schematic of a eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO). b Human colon carcinoma cell line HCT116 cells were cultured in 96 well plate and transiently transfected with p-miR-21-ASO or p-Cont (5 μg). After 48 h, the expression of GFP protein was observed by fluorescent microscopy. c The expression level of miR-21 in HCT116 cells was also determined by Real-time PCR assay and calculated. d At indicated time point, the proliferation of HCT116 cells in p-miR-21-ASO transfected group and p-Cont group also were detected by CCK-8 assay. One representative of three experiments was shown. *p < 0.05.
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Fig1: MiRNA-21 ASO reduced the proliferation of human colon carcinoma cells. a The schematic of a eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO). b Human colon carcinoma cell line HCT116 cells were cultured in 96 well plate and transiently transfected with p-miR-21-ASO or p-Cont (5 μg). After 48 h, the expression of GFP protein was observed by fluorescent microscopy. c The expression level of miR-21 in HCT116 cells was also determined by Real-time PCR assay and calculated. d At indicated time point, the proliferation of HCT116 cells in p-miR-21-ASO transfected group and p-Cont group also were detected by CCK-8 assay. One representative of three experiments was shown. *p < 0.05.

Mentions: To investigate the possible effect of miR-21-based therapeutic strategy against human colon carcinoma cells, we firstly identified the relative expression of miRNA-21 in a set of three colon carcinoma cell lines and a normal colonic cell line using Real-time PCR assay. As shown in Additional file 1: Figure S1, miRNA-21 levels were up-regulated in all carcinoma cell lines compared with normal control (p < 0.05), which was consistent with previous report [7]. Then we constructed an eukaryotic expression vector encoding antisense oligonucleotides (ASOs) against miR-21 (termed as p-miR-21-ASO), and then transiently transfected into colon carcinoma cell line HCT116 cells (Fig. 1a). The transfection efficiency was verified by detecting the expression of GFP by fluorescence microscopy. As shown in Fig. 1b, the mean proportion of GFP positive cells was about 70% in each group at 48 h after transfection. As expected, real-time PCR analysis showed that the expression of miRNA-21 in p-miR-21-ASO transfection group was significantly decreased compared with that in p-Cont transfection group (Fig. 1c, p < 0.05), suggesting that miR-21 ASO could significantly reduce the expression of intrinsic miR-21 in colon carcinoma cells.Fig. 1


Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells.

Tao YJ, Li YJ, Zheng W, Zhao JJ, Guo MM, Zhou Y, Qin NL, Zheng J, Xu L - Cancer Cell Int. (2015)

MiRNA-21 ASO reduced the proliferation of human colon carcinoma cells. a The schematic of a eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO). b Human colon carcinoma cell line HCT116 cells were cultured in 96 well plate and transiently transfected with p-miR-21-ASO or p-Cont (5 μg). After 48 h, the expression of GFP protein was observed by fluorescent microscopy. c The expression level of miR-21 in HCT116 cells was also determined by Real-time PCR assay and calculated. d At indicated time point, the proliferation of HCT116 cells in p-miR-21-ASO transfected group and p-Cont group also were detected by CCK-8 assay. One representative of three experiments was shown. *p < 0.05.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522075&req=5

Fig1: MiRNA-21 ASO reduced the proliferation of human colon carcinoma cells. a The schematic of a eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO). b Human colon carcinoma cell line HCT116 cells were cultured in 96 well plate and transiently transfected with p-miR-21-ASO or p-Cont (5 μg). After 48 h, the expression of GFP protein was observed by fluorescent microscopy. c The expression level of miR-21 in HCT116 cells was also determined by Real-time PCR assay and calculated. d At indicated time point, the proliferation of HCT116 cells in p-miR-21-ASO transfected group and p-Cont group also were detected by CCK-8 assay. One representative of three experiments was shown. *p < 0.05.
Mentions: To investigate the possible effect of miR-21-based therapeutic strategy against human colon carcinoma cells, we firstly identified the relative expression of miRNA-21 in a set of three colon carcinoma cell lines and a normal colonic cell line using Real-time PCR assay. As shown in Additional file 1: Figure S1, miRNA-21 levels were up-regulated in all carcinoma cell lines compared with normal control (p < 0.05), which was consistent with previous report [7]. Then we constructed an eukaryotic expression vector encoding antisense oligonucleotides (ASOs) against miR-21 (termed as p-miR-21-ASO), and then transiently transfected into colon carcinoma cell line HCT116 cells (Fig. 1a). The transfection efficiency was verified by detecting the expression of GFP by fluorescence microscopy. As shown in Fig. 1b, the mean proportion of GFP positive cells was about 70% in each group at 48 h after transfection. As expected, real-time PCR analysis showed that the expression of miRNA-21 in p-miR-21-ASO transfection group was significantly decreased compared with that in p-Cont transfection group (Fig. 1c, p < 0.05), suggesting that miR-21 ASO could significantly reduce the expression of intrinsic miR-21 in colon carcinoma cells.Fig. 1

Bottom Line: Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells.Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells.Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Zunyi Medical College, Zunyi, 563003 Guizhou China.

ABSTRACT

Background: Colon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated.

Methods: In present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot.

Results: We found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered.

Conclusions: Therefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.

No MeSH data available.


Related in: MedlinePlus