Limits...
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 reversed PTEN expression in human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of PTEN was analyzed by Western blotting (a) and calculated (b). c The expression of PTEN also analyzed by FACS and then the mean fluorescence intensity (MFI) was calculated (d). Gray line p-Cont transfected group, black line p-miR-21-ASO transfected group. One representative of three experiments was shown. *p < 0.05.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: MiRNA-21 ASO reversed PTEN expression in human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of PTEN was analyzed by Western blotting (a) and calculated (b). c The expression of PTEN also analyzed by FACS and then the mean fluorescence intensity (MFI) was calculated (d). Gray line p-Cont transfected group, black line p-miR-21-ASO transfected group. One representative of three experiments was shown. *p < 0.05.

Mentions: Recent studies have shown that PTEN, a critical tumor suppressor in the occurrence and progression of various tumors, was one of target molecules of miR-21 [15–17]. Moreover, PTEN also could inhibit angiogenesis that associated with decreased expression of VEGF [18]. To investigate the possible mechanism of down-regulation of miRNA-21 by miR-21 ASO in the proliferation and migration of colon carcinoma, we further detected the expression of PTEN in human colon carcinoma HCT116 cells. As shown Fig. 4a, b, the expression of PTEN protein was significantly elevated in p-miR-21-ASO transfected group compared with that in p-Cont transfected group (p < 0.5). To further verify the expression of PTEN, we also analyzed the expression of PTEN protein in HCT116 cells using FACS analysis and similar result was obtained (Fig. 4c, d, p < 0.5).Fig. 4


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 reversed PTEN expression in human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of PTEN was analyzed by Western blotting (a) and calculated (b). c The expression of PTEN also analyzed by FACS and then the mean fluorescence intensity (MFI) was calculated (d). Gray line p-Cont transfected group, black line p-miR-21-ASO transfected group. 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

Fig4: MiRNA-21 ASO reversed PTEN expression in human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of PTEN was analyzed by Western blotting (a) and calculated (b). c The expression of PTEN also analyzed by FACS and then the mean fluorescence intensity (MFI) was calculated (d). Gray line p-Cont transfected group, black line p-miR-21-ASO transfected group. One representative of three experiments was shown. *p < 0.05.
Mentions: Recent studies have shown that PTEN, a critical tumor suppressor in the occurrence and progression of various tumors, was one of target molecules of miR-21 [15–17]. Moreover, PTEN also could inhibit angiogenesis that associated with decreased expression of VEGF [18]. To investigate the possible mechanism of down-regulation of miRNA-21 by miR-21 ASO in the proliferation and migration of colon carcinoma, we further detected the expression of PTEN in human colon carcinoma HCT116 cells. As shown Fig. 4a, b, the expression of PTEN protein was significantly elevated in p-miR-21-ASO transfected group compared with that in p-Cont transfected group (p < 0.5). To further verify the expression of PTEN, we also analyzed the expression of PTEN protein in HCT116 cells using FACS analysis and similar result was obtained (Fig. 4c, d, p < 0.5).Fig. 4

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