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microRNA-34a inhibits epithelial mesenchymal transition in human cholangiocarcinoma by targeting Smad4 through transforming growth factor-beta/Smad pathway.

Qiao P, Li G, Bi W, Yang L, Yao L, Wu D - BMC Cancer (2015)

Bottom Line: Cell morphology, invasion and migration assays were further applied to confirm the anti-carcinogenic effects of miR-34a through the downstream target. miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues and normal bile duct tissues. miR-34a was found correlated with the migration and invasion in EHCC patients.Moreover, activation of miR-34a suppressed invasion and migration through TGF-beta/Smad4 signaling pathway by epithelial-mesenchymal transition (EMT) in vitro.Taken together, our results suggest that miR-34a inhibits invasion and migration by targeting Smad4 to suppress EMT through TGF- beta/Smad signaling pathway in human EHCC.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Peoples Republic of China. lunwenqpf@126.com.

ABSTRACT

Background: Extrahepatic Cholangiocarcinoma (EHCC) is one of the uncommon malignancies in the digestive system which is characterized by a poor prognosis. Aberrations of miRNAs have been shown involved in the progression of this disease. In this study, we evaluated the expression and effects of miR-34a on EHCC.

Methods: miR-34a expression levels were detected in EHCC tissues, adjacent non-tumor tissues, normal bile duct (NBD) specimens of patients and cholangiocarcinoma (CC) cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Relationships between miR-34a with clinical characteristics of EHCC patients were further analyzed. Computational search, functional luciferase assay and western blot were further used to demonstrate the downstream target of miR-34a in CC cells. Immunohistochemistry was carried on to identify the downstream target gene of miR-34a in EHCC patients. Cell morphology, invasion and migration assays were further applied to confirm the anti-carcinogenic effects of miR-34a through the downstream target.

Results: miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues and normal bile duct tissues. miR-34a was found correlated with the migration and invasion in EHCC patients. Smad4 was over-expressed in most of the EHCC patients and was further demonstrated as one of the downstream targets of miR-34a, which was involved in the progression of EHCC. Moreover, activation of miR-34a suppressed invasion and migration through TGF-beta/Smad4 signaling pathway by epithelial-mesenchymal transition (EMT) in vitro.

Conclusions: Taken together, our results suggest that miR-34a inhibits invasion and migration by targeting Smad4 to suppress EMT through TGF- beta/Smad signaling pathway in human EHCC.

No MeSH data available.


Related in: MedlinePlus

miR-34a suppresses EHCC cells invasion and migration through TGF-β/Smad4 signaling pathway. a The migration and invasive properties of EHCC cells treated with the empty vector, miR-34a mimic or miR-34a inhibitor were analyzed using a cell invasion assay in transwell chambers. Representative images of cells that had migrated into the lower chamber are shown (left panel, original magnification: ×100), and quantitative data are also presented (right panel, **P < 0.05 and ***P < 0.01). The average numbers of cells per field of view from three different experiments are plotted. b Both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without 5 ng/ml of TGF-β for 24 hrs to evaluate cell invasion and migration activities. Cells transfected with miR-34a scramble oligos were used as the controls. Representative images of cells in the lower section of a transwell chamber are shown to demonstrate the migration and invasive properties of QBC939 and HuCCT1 cells when transfected with the empty vector, miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β (left panel). Quantitative analyses of the migrated and invasion cells are also shown (right panel, **P < 0.05 and ***P < 0.01 indicates miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β vs. NC. $$$P < 0.01 means a mixed miR-34a and TGF-β vs. TGF-β). Data are plotted as the average number of cells per field of view from three different experiments (original magnification: ×100)
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Fig5: miR-34a suppresses EHCC cells invasion and migration through TGF-β/Smad4 signaling pathway. a The migration and invasive properties of EHCC cells treated with the empty vector, miR-34a mimic or miR-34a inhibitor were analyzed using a cell invasion assay in transwell chambers. Representative images of cells that had migrated into the lower chamber are shown (left panel, original magnification: ×100), and quantitative data are also presented (right panel, **P < 0.05 and ***P < 0.01). The average numbers of cells per field of view from three different experiments are plotted. b Both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without 5 ng/ml of TGF-β for 24 hrs to evaluate cell invasion and migration activities. Cells transfected with miR-34a scramble oligos were used as the controls. Representative images of cells in the lower section of a transwell chamber are shown to demonstrate the migration and invasive properties of QBC939 and HuCCT1 cells when transfected with the empty vector, miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β (left panel). Quantitative analyses of the migrated and invasion cells are also shown (right panel, **P < 0.05 and ***P < 0.01 indicates miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β vs. NC. $$$P < 0.01 means a mixed miR-34a and TGF-β vs. TGF-β). Data are plotted as the average number of cells per field of view from three different experiments (original magnification: ×100)

Mentions: EMT is often linked to a gain in the migratory and invasive properties of cells [19]. To further investigate whether miR-34a suppresses cell invasion and migration through TGF-β/Smad4 signaling pathway in EHCC, both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without TGF-β. Transwell migration and invasion assays were then performed after transfection. It was found that transfection with miR-34a significantly suppressed cell migration in both QBC939 and HuCCT1 cells. Similarly, invasion capacity was also significantly down-regulated in both of the cell lines (Fig. 5a). The cell migration and in invasion capacities were all induced in both QBC939 and HuCCT1 cell lines after treated with TGF-β (Fig. 5b). However, these inductions were inhibited by the treatment with miR-34a mimics in both QBC939 and HuCCT1 cells (Fig. 5b). Moreover, Snail protein expression level was decreased by transfection with miR-34a mimics but increased by transfection with miR-34a inhibitor in both QBC939 and HuCCT1 cells (Fig. 3d). Snail is one of the specific downstream targets of TGF-β/Smad4 in regulation of EMT [25]. These results indicate that miR-34a participates in the regulation of cell migration and invasion in EHCC cells through down-regulation of TGF-β/Smad4 signaling pathway.Fig. 5


microRNA-34a inhibits epithelial mesenchymal transition in human cholangiocarcinoma by targeting Smad4 through transforming growth factor-beta/Smad pathway.

Qiao P, Li G, Bi W, Yang L, Yao L, Wu D - BMC Cancer (2015)

miR-34a suppresses EHCC cells invasion and migration through TGF-β/Smad4 signaling pathway. a The migration and invasive properties of EHCC cells treated with the empty vector, miR-34a mimic or miR-34a inhibitor were analyzed using a cell invasion assay in transwell chambers. Representative images of cells that had migrated into the lower chamber are shown (left panel, original magnification: ×100), and quantitative data are also presented (right panel, **P < 0.05 and ***P < 0.01). The average numbers of cells per field of view from three different experiments are plotted. b Both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without 5 ng/ml of TGF-β for 24 hrs to evaluate cell invasion and migration activities. Cells transfected with miR-34a scramble oligos were used as the controls. Representative images of cells in the lower section of a transwell chamber are shown to demonstrate the migration and invasive properties of QBC939 and HuCCT1 cells when transfected with the empty vector, miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β (left panel). Quantitative analyses of the migrated and invasion cells are also shown (right panel, **P < 0.05 and ***P < 0.01 indicates miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β vs. NC. $$$P < 0.01 means a mixed miR-34a and TGF-β vs. TGF-β). Data are plotted as the average number of cells per field of view from three different experiments (original magnification: ×100)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: miR-34a suppresses EHCC cells invasion and migration through TGF-β/Smad4 signaling pathway. a The migration and invasive properties of EHCC cells treated with the empty vector, miR-34a mimic or miR-34a inhibitor were analyzed using a cell invasion assay in transwell chambers. Representative images of cells that had migrated into the lower chamber are shown (left panel, original magnification: ×100), and quantitative data are also presented (right panel, **P < 0.05 and ***P < 0.01). The average numbers of cells per field of view from three different experiments are plotted. b Both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without 5 ng/ml of TGF-β for 24 hrs to evaluate cell invasion and migration activities. Cells transfected with miR-34a scramble oligos were used as the controls. Representative images of cells in the lower section of a transwell chamber are shown to demonstrate the migration and invasive properties of QBC939 and HuCCT1 cells when transfected with the empty vector, miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β (left panel). Quantitative analyses of the migrated and invasion cells are also shown (right panel, **P < 0.05 and ***P < 0.01 indicates miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β vs. NC. $$$P < 0.01 means a mixed miR-34a and TGF-β vs. TGF-β). Data are plotted as the average number of cells per field of view from three different experiments (original magnification: ×100)
Mentions: EMT is often linked to a gain in the migratory and invasive properties of cells [19]. To further investigate whether miR-34a suppresses cell invasion and migration through TGF-β/Smad4 signaling pathway in EHCC, both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without TGF-β. Transwell migration and invasion assays were then performed after transfection. It was found that transfection with miR-34a significantly suppressed cell migration in both QBC939 and HuCCT1 cells. Similarly, invasion capacity was also significantly down-regulated in both of the cell lines (Fig. 5a). The cell migration and in invasion capacities were all induced in both QBC939 and HuCCT1 cell lines after treated with TGF-β (Fig. 5b). However, these inductions were inhibited by the treatment with miR-34a mimics in both QBC939 and HuCCT1 cells (Fig. 5b). Moreover, Snail protein expression level was decreased by transfection with miR-34a mimics but increased by transfection with miR-34a inhibitor in both QBC939 and HuCCT1 cells (Fig. 3d). Snail is one of the specific downstream targets of TGF-β/Smad4 in regulation of EMT [25]. These results indicate that miR-34a participates in the regulation of cell migration and invasion in EHCC cells through down-regulation of TGF-β/Smad4 signaling pathway.Fig. 5

Bottom Line: Cell morphology, invasion and migration assays were further applied to confirm the anti-carcinogenic effects of miR-34a through the downstream target. miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues and normal bile duct tissues. miR-34a was found correlated with the migration and invasion in EHCC patients.Moreover, activation of miR-34a suppressed invasion and migration through TGF-beta/Smad4 signaling pathway by epithelial-mesenchymal transition (EMT) in vitro.Taken together, our results suggest that miR-34a inhibits invasion and migration by targeting Smad4 to suppress EMT through TGF- beta/Smad signaling pathway in human EHCC.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Peoples Republic of China. lunwenqpf@126.com.

ABSTRACT

Background: Extrahepatic Cholangiocarcinoma (EHCC) is one of the uncommon malignancies in the digestive system which is characterized by a poor prognosis. Aberrations of miRNAs have been shown involved in the progression of this disease. In this study, we evaluated the expression and effects of miR-34a on EHCC.

Methods: miR-34a expression levels were detected in EHCC tissues, adjacent non-tumor tissues, normal bile duct (NBD) specimens of patients and cholangiocarcinoma (CC) cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Relationships between miR-34a with clinical characteristics of EHCC patients were further analyzed. Computational search, functional luciferase assay and western blot were further used to demonstrate the downstream target of miR-34a in CC cells. Immunohistochemistry was carried on to identify the downstream target gene of miR-34a in EHCC patients. Cell morphology, invasion and migration assays were further applied to confirm the anti-carcinogenic effects of miR-34a through the downstream target.

Results: miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues and normal bile duct tissues. miR-34a was found correlated with the migration and invasion in EHCC patients. Smad4 was over-expressed in most of the EHCC patients and was further demonstrated as one of the downstream targets of miR-34a, which was involved in the progression of EHCC. Moreover, activation of miR-34a suppressed invasion and migration through TGF-beta/Smad4 signaling pathway by epithelial-mesenchymal transition (EMT) in vitro.

Conclusions: Taken together, our results suggest that miR-34a inhibits invasion and migration by targeting Smad4 to suppress EMT through TGF- beta/Smad signaling pathway in human EHCC.

No MeSH data available.


Related in: MedlinePlus