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miR-191 promotes tumorigenesis of human colorectal cancer through targeting C/EBPβ.

Zhang XF, Li KK, Gao L, Li SZ, Chen K, Zhang JB, Wang D, Tu RF, Zhang JX, Tao KX, Wang G, Zhang XD - Oncotarget (2015)

Bottom Line: In this study, we show that miR-191 is highly expressed in colon tumor tissues, and that inhibition of miR-191 leads to decreased cell growth, proliferation and tumorigenicity in a xenograft model.Overexpression of miR-191 in colorectal cancer cell lines alters cell cycle progression and cell resistance to 5-Fu induced cell apoptosis.Mechanistic studies demonstrated that miR-191 directly binds to the 3'UTR of the C/EBPβ mRNA and mediates a decrease in the mRNA and protein expression of C/EBPβ.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Wuhan University, Wuhan 430072, PR China.

ABSTRACT
MicroRNA-191 (miR-191), a small non-coding RNA, is involved in disease development and cancer diagnosis and prognosis. However, how miR-191 functions in colorectal cancer remains largely unclear. In this study, we show that miR-191 is highly expressed in colon tumor tissues, and that inhibition of miR-191 leads to decreased cell growth, proliferation and tumorigenicity in a xenograft model. Overexpression of miR-191 in colorectal cancer cell lines alters cell cycle progression and cell resistance to 5-Fu induced cell apoptosis. Mechanistic studies demonstrated that miR-191 directly binds to the 3'UTR of the C/EBPβ mRNA and mediates a decrease in the mRNA and protein expression of C/EBPβ. We further showed that C/EBPβ induces growth arrest in a colorectal cancer cell line and that its expression is negatively correlated with the miR-191 level in patient samples. Our findings suggest that miR-191 may be a potential gene therapy target for the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

The involvement of miR-191 in the 5-Fu-induced cell apoptotic pathway in HCT116 cellsHCT116 cells were treated with various commonly-used chemotherapeutic drugs, including 5-fluorouracil (5-Fu, 10 μg/ml), indomethacin (Indo, 10 μg/ml), cisplatin (Cis, 10 μg/ml) and etoposide (Eto, 2 μM). (A) Cell viability was assessed by CCK8 assays. (B) RT-PCR analysis of the relative expression of miR-191. (C) 5-Fu down-regulated miR-191 in a dose-dependent manner. HCT116 cells were treated with the indicated concentration of 5-Fu for 48 hours, total mRNA was isolated, and the miR-191 level was analyzed by RT-PCR (*P < 0.05 versus DMSO treated cells). HCT116 cells were transfected with the indicated oligos for 24 hours and then treated with 5-Fu (25 μg/ml) for another 24 hours. (D) Cell viability was assessed by CCK8 assays (*P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (E) The mRNA levels of Bax and Bcl-2 was determined by RT-PCR (*P < 0.05 versus DMSO treated mimic-ctrl/inhibitor-ctrl cells, #P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (F) The protein levels of Bax, Bcl-2, caspase-3 and cleaved-caspase-3 were detected by western blotting analysis in HCT116 cells transfected with mimic-ctrl/miR-191-mimic (left panel) and inhibitor-ctrl/miR-191-inhibitor (right panel) after DMSO or 5-Fu treatment. (mc, mimic ctrl; m, miR-191-mimic; ic, inhibitor ctrl; i, miR-191-inhibitor) (G) Cell apoptosis analysis of transfected cells after treatment. GAPDH served as a loading control. The data represents the means ± SDs.
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Figure 4: The involvement of miR-191 in the 5-Fu-induced cell apoptotic pathway in HCT116 cellsHCT116 cells were treated with various commonly-used chemotherapeutic drugs, including 5-fluorouracil (5-Fu, 10 μg/ml), indomethacin (Indo, 10 μg/ml), cisplatin (Cis, 10 μg/ml) and etoposide (Eto, 2 μM). (A) Cell viability was assessed by CCK8 assays. (B) RT-PCR analysis of the relative expression of miR-191. (C) 5-Fu down-regulated miR-191 in a dose-dependent manner. HCT116 cells were treated with the indicated concentration of 5-Fu for 48 hours, total mRNA was isolated, and the miR-191 level was analyzed by RT-PCR (*P < 0.05 versus DMSO treated cells). HCT116 cells were transfected with the indicated oligos for 24 hours and then treated with 5-Fu (25 μg/ml) for another 24 hours. (D) Cell viability was assessed by CCK8 assays (*P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (E) The mRNA levels of Bax and Bcl-2 was determined by RT-PCR (*P < 0.05 versus DMSO treated mimic-ctrl/inhibitor-ctrl cells, #P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (F) The protein levels of Bax, Bcl-2, caspase-3 and cleaved-caspase-3 were detected by western blotting analysis in HCT116 cells transfected with mimic-ctrl/miR-191-mimic (left panel) and inhibitor-ctrl/miR-191-inhibitor (right panel) after DMSO or 5-Fu treatment. (mc, mimic ctrl; m, miR-191-mimic; ic, inhibitor ctrl; i, miR-191-inhibitor) (G) Cell apoptosis analysis of transfected cells after treatment. GAPDH served as a loading control. The data represents the means ± SDs.

Mentions: 5-Fu is a widely used anticancer drug, but due to its high-dose regimen in the clinic, the drug's side effects were observed. In our drug screening assay, we found that endogenous miR-191 was modulated by various drugs. Interestingly, although the changes of cell viability were similar when HCT116 cells were treated with different drugs, the alteration of the level of miR-191 was the most apparent in cells treated with 5-Fu (Figure 4A, 4B). We further demonstrated that 5-Fu decreased the endogenous miR-191 level in a dose-dependent manner (Figure 4C). The induction of the pro-apoptotic pathway by 5-Fu is crucial for its anticancer role, so we hypothesized that miR-191 might be involved in the 5-Fu induced cell apoptotic pathway. HCT116 cells were transfected with miR-191 mimic or inhibitor for 24 hours and then treated with 25 μg/ml 5-Fu for another 24 hours, and the cell viability was measured by CCK8. We observed an almost 50% decrease in cell viability when cells were treated with 5-Fu. Notably, when exposed to 5-Fu, cells transfected with the miR-191 mimic exhibited higher cell viability compared to cells transfected with the mimic control. The introduction of miR-191 inhibitor led to a significant decrease in cell viability when compared with the inhibitor control (Figure 4D).


miR-191 promotes tumorigenesis of human colorectal cancer through targeting C/EBPβ.

Zhang XF, Li KK, Gao L, Li SZ, Chen K, Zhang JB, Wang D, Tu RF, Zhang JX, Tao KX, Wang G, Zhang XD - Oncotarget (2015)

The involvement of miR-191 in the 5-Fu-induced cell apoptotic pathway in HCT116 cellsHCT116 cells were treated with various commonly-used chemotherapeutic drugs, including 5-fluorouracil (5-Fu, 10 μg/ml), indomethacin (Indo, 10 μg/ml), cisplatin (Cis, 10 μg/ml) and etoposide (Eto, 2 μM). (A) Cell viability was assessed by CCK8 assays. (B) RT-PCR analysis of the relative expression of miR-191. (C) 5-Fu down-regulated miR-191 in a dose-dependent manner. HCT116 cells were treated with the indicated concentration of 5-Fu for 48 hours, total mRNA was isolated, and the miR-191 level was analyzed by RT-PCR (*P < 0.05 versus DMSO treated cells). HCT116 cells were transfected with the indicated oligos for 24 hours and then treated with 5-Fu (25 μg/ml) for another 24 hours. (D) Cell viability was assessed by CCK8 assays (*P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (E) The mRNA levels of Bax and Bcl-2 was determined by RT-PCR (*P < 0.05 versus DMSO treated mimic-ctrl/inhibitor-ctrl cells, #P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (F) The protein levels of Bax, Bcl-2, caspase-3 and cleaved-caspase-3 were detected by western blotting analysis in HCT116 cells transfected with mimic-ctrl/miR-191-mimic (left panel) and inhibitor-ctrl/miR-191-inhibitor (right panel) after DMSO or 5-Fu treatment. (mc, mimic ctrl; m, miR-191-mimic; ic, inhibitor ctrl; i, miR-191-inhibitor) (G) Cell apoptosis analysis of transfected cells after treatment. GAPDH served as a loading control. The data represents the means ± SDs.
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Figure 4: The involvement of miR-191 in the 5-Fu-induced cell apoptotic pathway in HCT116 cellsHCT116 cells were treated with various commonly-used chemotherapeutic drugs, including 5-fluorouracil (5-Fu, 10 μg/ml), indomethacin (Indo, 10 μg/ml), cisplatin (Cis, 10 μg/ml) and etoposide (Eto, 2 μM). (A) Cell viability was assessed by CCK8 assays. (B) RT-PCR analysis of the relative expression of miR-191. (C) 5-Fu down-regulated miR-191 in a dose-dependent manner. HCT116 cells were treated with the indicated concentration of 5-Fu for 48 hours, total mRNA was isolated, and the miR-191 level was analyzed by RT-PCR (*P < 0.05 versus DMSO treated cells). HCT116 cells were transfected with the indicated oligos for 24 hours and then treated with 5-Fu (25 μg/ml) for another 24 hours. (D) Cell viability was assessed by CCK8 assays (*P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (E) The mRNA levels of Bax and Bcl-2 was determined by RT-PCR (*P < 0.05 versus DMSO treated mimic-ctrl/inhibitor-ctrl cells, #P < 0.05 versus 5-Fu treated mimic-ctrl/inhibitor-ctrl cells). (F) The protein levels of Bax, Bcl-2, caspase-3 and cleaved-caspase-3 were detected by western blotting analysis in HCT116 cells transfected with mimic-ctrl/miR-191-mimic (left panel) and inhibitor-ctrl/miR-191-inhibitor (right panel) after DMSO or 5-Fu treatment. (mc, mimic ctrl; m, miR-191-mimic; ic, inhibitor ctrl; i, miR-191-inhibitor) (G) Cell apoptosis analysis of transfected cells after treatment. GAPDH served as a loading control. The data represents the means ± SDs.
Mentions: 5-Fu is a widely used anticancer drug, but due to its high-dose regimen in the clinic, the drug's side effects were observed. In our drug screening assay, we found that endogenous miR-191 was modulated by various drugs. Interestingly, although the changes of cell viability were similar when HCT116 cells were treated with different drugs, the alteration of the level of miR-191 was the most apparent in cells treated with 5-Fu (Figure 4A, 4B). We further demonstrated that 5-Fu decreased the endogenous miR-191 level in a dose-dependent manner (Figure 4C). The induction of the pro-apoptotic pathway by 5-Fu is crucial for its anticancer role, so we hypothesized that miR-191 might be involved in the 5-Fu induced cell apoptotic pathway. HCT116 cells were transfected with miR-191 mimic or inhibitor for 24 hours and then treated with 25 μg/ml 5-Fu for another 24 hours, and the cell viability was measured by CCK8. We observed an almost 50% decrease in cell viability when cells were treated with 5-Fu. Notably, when exposed to 5-Fu, cells transfected with the miR-191 mimic exhibited higher cell viability compared to cells transfected with the mimic control. The introduction of miR-191 inhibitor led to a significant decrease in cell viability when compared with the inhibitor control (Figure 4D).

Bottom Line: In this study, we show that miR-191 is highly expressed in colon tumor tissues, and that inhibition of miR-191 leads to decreased cell growth, proliferation and tumorigenicity in a xenograft model.Overexpression of miR-191 in colorectal cancer cell lines alters cell cycle progression and cell resistance to 5-Fu induced cell apoptosis.Mechanistic studies demonstrated that miR-191 directly binds to the 3'UTR of the C/EBPβ mRNA and mediates a decrease in the mRNA and protein expression of C/EBPβ.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Wuhan University, Wuhan 430072, PR China.

ABSTRACT
MicroRNA-191 (miR-191), a small non-coding RNA, is involved in disease development and cancer diagnosis and prognosis. However, how miR-191 functions in colorectal cancer remains largely unclear. In this study, we show that miR-191 is highly expressed in colon tumor tissues, and that inhibition of miR-191 leads to decreased cell growth, proliferation and tumorigenicity in a xenograft model. Overexpression of miR-191 in colorectal cancer cell lines alters cell cycle progression and cell resistance to 5-Fu induced cell apoptosis. Mechanistic studies demonstrated that miR-191 directly binds to the 3'UTR of the C/EBPβ mRNA and mediates a decrease in the mRNA and protein expression of C/EBPβ. We further showed that C/EBPβ induces growth arrest in a colorectal cancer cell line and that its expression is negatively correlated with the miR-191 level in patient samples. Our findings suggest that miR-191 may be a potential gene therapy target for the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus