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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

miR-191 down-regulates the expression of C/EBPβ(A) The predicted binding sites of miR-191 in the 3′ UTRs of C/EBPβ of different species. (B) Schematic description of wild type (WT) and mutated 3′ UTRs of the C/EBPβ mRNA. The WT and mutated 3′ UTR sequences (~400 bp) were cloned into the psiCHECK2 vector. (C) Luciferase analysis was used to detect the reporter activity. HCT116 cells in 24 well plates were co-transfected with 200 ng plemiR/plemiR-191 or psiCHECK2-WT 3′ UTR/psiCHECK2-mutated 3′ UTR (100 ng) for 48 hours and then subjected to luciferase assays according to the Materials and Methods. (D-E) miR-191 inhibits the mRNA and protein levels of C/EBPβ in HCT116 cells. HCT116 cells were transfected with the miR-191 mimic/mimic control (D) or miR-191 inhibitor/inhibitor control (E) for 48 hours and total mRNA and protein were extracted and analyzed by RT-PCR and western blotting analysis, respectively. GAPDH served as a loading control. (F) RT-PCR analysis of the relative expression of C/EBPβ in 16 colon cancer tissues. (G) Western blotting analysis of C/EBPβ in 16 colon cancer tissues. GAPDH served as a loading control. (H) Inverse correlation between miR-191 and C/EBPβ in colon cancer tissues. miR-191 level was normalized to the U6 level, and the C/EBPβ level was normalized to the GAPDH level. Statistical analysis was performed using Person's correlation coefficient analysis. The data represents the means ± SDs.
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Figure 5: miR-191 down-regulates the expression of C/EBPβ(A) The predicted binding sites of miR-191 in the 3′ UTRs of C/EBPβ of different species. (B) Schematic description of wild type (WT) and mutated 3′ UTRs of the C/EBPβ mRNA. The WT and mutated 3′ UTR sequences (~400 bp) were cloned into the psiCHECK2 vector. (C) Luciferase analysis was used to detect the reporter activity. HCT116 cells in 24 well plates were co-transfected with 200 ng plemiR/plemiR-191 or psiCHECK2-WT 3′ UTR/psiCHECK2-mutated 3′ UTR (100 ng) for 48 hours and then subjected to luciferase assays according to the Materials and Methods. (D-E) miR-191 inhibits the mRNA and protein levels of C/EBPβ in HCT116 cells. HCT116 cells were transfected with the miR-191 mimic/mimic control (D) or miR-191 inhibitor/inhibitor control (E) for 48 hours and total mRNA and protein were extracted and analyzed by RT-PCR and western blotting analysis, respectively. GAPDH served as a loading control. (F) RT-PCR analysis of the relative expression of C/EBPβ in 16 colon cancer tissues. (G) Western blotting analysis of C/EBPβ in 16 colon cancer tissues. GAPDH served as a loading control. (H) Inverse correlation between miR-191 and C/EBPβ in colon cancer tissues. miR-191 level was normalized to the U6 level, and the C/EBPβ level was normalized to the GAPDH level. Statistical analysis was performed using Person's correlation coefficient analysis. The data represents the means ± SDs.

Mentions: To explore the molecular mechanism responsible for the function of miR-191 in CRC, we used three publicly available databases (TargetScan, picTar and miRanda) to search for predicted direct target genes of miR-191. The predicted targets were arranged according to the binding probability score. The targets with high score and shared by the three databases were chosen. Seven candidate targets of miR-191 (unpublished and we are doing further research) were screened. C/EBPβ was chosen for further analysis, because previous reports have shown that C/EBPβ is an important regulator of cell growth [26, 27], cell apoptosis [28] and tumorigenicity [29]. miR-191 has conserved binding sites in the 3′UTRs of C/EBPβ of different species (Figure 5A). The wild-type (WT) or mutant 3′ UTR, in which the seed region was mutated to abolish miR-191 binding, was cloned into the psiCheck-2 plasmid (Figure 5B), and a dual-luciferase reporter system was employed to verify whether C/EBPBβ is a direct target of miR-191. The luciferase activity of the reporter containing the WT 3′ UTR of C/EBPβ was decreased in cells transfected with plemiR-191, whereas the activity of the mutant reporter was not significantly altered following plemiR-191 transfection (Figure 5C).


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)

miR-191 down-regulates the expression of C/EBPβ(A) The predicted binding sites of miR-191 in the 3′ UTRs of C/EBPβ of different species. (B) Schematic description of wild type (WT) and mutated 3′ UTRs of the C/EBPβ mRNA. The WT and mutated 3′ UTR sequences (~400 bp) were cloned into the psiCHECK2 vector. (C) Luciferase analysis was used to detect the reporter activity. HCT116 cells in 24 well plates were co-transfected with 200 ng plemiR/plemiR-191 or psiCHECK2-WT 3′ UTR/psiCHECK2-mutated 3′ UTR (100 ng) for 48 hours and then subjected to luciferase assays according to the Materials and Methods. (D-E) miR-191 inhibits the mRNA and protein levels of C/EBPβ in HCT116 cells. HCT116 cells were transfected with the miR-191 mimic/mimic control (D) or miR-191 inhibitor/inhibitor control (E) for 48 hours and total mRNA and protein were extracted and analyzed by RT-PCR and western blotting analysis, respectively. GAPDH served as a loading control. (F) RT-PCR analysis of the relative expression of C/EBPβ in 16 colon cancer tissues. (G) Western blotting analysis of C/EBPβ in 16 colon cancer tissues. GAPDH served as a loading control. (H) Inverse correlation between miR-191 and C/EBPβ in colon cancer tissues. miR-191 level was normalized to the U6 level, and the C/EBPβ level was normalized to the GAPDH level. Statistical analysis was performed using Person's correlation coefficient analysis. The data represents the means ± SDs.
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Figure 5: miR-191 down-regulates the expression of C/EBPβ(A) The predicted binding sites of miR-191 in the 3′ UTRs of C/EBPβ of different species. (B) Schematic description of wild type (WT) and mutated 3′ UTRs of the C/EBPβ mRNA. The WT and mutated 3′ UTR sequences (~400 bp) were cloned into the psiCHECK2 vector. (C) Luciferase analysis was used to detect the reporter activity. HCT116 cells in 24 well plates were co-transfected with 200 ng plemiR/plemiR-191 or psiCHECK2-WT 3′ UTR/psiCHECK2-mutated 3′ UTR (100 ng) for 48 hours and then subjected to luciferase assays according to the Materials and Methods. (D-E) miR-191 inhibits the mRNA and protein levels of C/EBPβ in HCT116 cells. HCT116 cells were transfected with the miR-191 mimic/mimic control (D) or miR-191 inhibitor/inhibitor control (E) for 48 hours and total mRNA and protein were extracted and analyzed by RT-PCR and western blotting analysis, respectively. GAPDH served as a loading control. (F) RT-PCR analysis of the relative expression of C/EBPβ in 16 colon cancer tissues. (G) Western blotting analysis of C/EBPβ in 16 colon cancer tissues. GAPDH served as a loading control. (H) Inverse correlation between miR-191 and C/EBPβ in colon cancer tissues. miR-191 level was normalized to the U6 level, and the C/EBPβ level was normalized to the GAPDH level. Statistical analysis was performed using Person's correlation coefficient analysis. The data represents the means ± SDs.
Mentions: To explore the molecular mechanism responsible for the function of miR-191 in CRC, we used three publicly available databases (TargetScan, picTar and miRanda) to search for predicted direct target genes of miR-191. The predicted targets were arranged according to the binding probability score. The targets with high score and shared by the three databases were chosen. Seven candidate targets of miR-191 (unpublished and we are doing further research) were screened. C/EBPβ was chosen for further analysis, because previous reports have shown that C/EBPβ is an important regulator of cell growth [26, 27], cell apoptosis [28] and tumorigenicity [29]. miR-191 has conserved binding sites in the 3′UTRs of C/EBPβ of different species (Figure 5A). The wild-type (WT) or mutant 3′ UTR, in which the seed region was mutated to abolish miR-191 binding, was cloned into the psiCheck-2 plasmid (Figure 5B), and a dual-luciferase reporter system was employed to verify whether C/EBPBβ is a direct target of miR-191. The luciferase activity of the reporter containing the WT 3′ UTR of C/EBPβ was decreased in cells transfected with plemiR-191, whereas the activity of the mutant reporter was not significantly altered following plemiR-191 transfection (Figure 5C).

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