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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 impaired the invasion and migration ability of human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). Then, the ability of invasion of cells was analyzed by Transwell assay (a) and calculated (b). c The ability of migration of cells also was determined by Wound-healing assay and calculated (d). e HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of VEGF was analyzed by western blotting and calculated (f). One representative of three experiments was shown. *p < 0.05.
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Fig3: MiRNA-21 ASO impaired the invasion and migration ability of human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). Then, the ability of invasion of cells was analyzed by Transwell assay (a) and calculated (b). c The ability of migration of cells also was determined by Wound-healing assay and calculated (d). e HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of VEGF was analyzed by western blotting and calculated (f). One representative of three experiments was shown. *p < 0.05.

Mentions: Then, we further investigated whether down-regulation of miR-21 by miR-21 ASO could affect the invasion ability of colon cancer cells in vitro. As shown in Fig. 2a, b, transwell assay showed that the invasion cell number in p-miR-21-ASO transfected group was obviously decreased compared with that in p-Cont transfected group (Fig. 3a, b, p < 0.5). To analysis the possible effect of down-regulation of miR-21 on the migration capacity of colon carcinoma cells, scratch wound assay was also performed. Data showed that the migration capacity of HCT116 cells was also impaired in p-miR-21-ASO transfected group (Fig. 3c, d, p < 0.5).Fig. 3


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 impaired the invasion and migration ability of human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). Then, the ability of invasion of cells was analyzed by Transwell assay (a) and calculated (b). c The ability of migration of cells also was determined by Wound-healing assay and calculated (d). e HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of VEGF was analyzed by western blotting and calculated (f). 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

Fig3: MiRNA-21 ASO impaired the invasion and migration ability of human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). Then, the ability of invasion of cells was analyzed by Transwell assay (a) and calculated (b). c The ability of migration of cells also was determined by Wound-healing assay and calculated (d). e HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of VEGF was analyzed by western blotting and calculated (f). One representative of three experiments was shown. *p < 0.05.
Mentions: Then, we further investigated whether down-regulation of miR-21 by miR-21 ASO could affect the invasion ability of colon cancer cells in vitro. As shown in Fig. 2a, b, transwell assay showed that the invasion cell number in p-miR-21-ASO transfected group was obviously decreased compared with that in p-Cont transfected group (Fig. 3a, b, p < 0.5). To analysis the possible effect of down-regulation of miR-21 on the migration capacity of colon carcinoma cells, scratch wound assay was also performed. Data showed that the migration capacity of HCT116 cells was also impaired in p-miR-21-ASO transfected group (Fig. 3c, d, p < 0.5).Fig. 3

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