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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

The expression levels of Smad4 in the primary EHCC and NBD specimens. a The mRNA expression level of Smad4 in the primary EHCC, the adjacent non-tumor tissues and NBD specimens determined by qRT-PCR. U6 was used as the internal control. b Immunohistochemical staining of Smad4 protein expression in primary EHCC and NBD samples. The arrows indicated nuclear staining of Smad4 in both NBD and EHCC samples. (I) Low Smad4 protein expression in a NBD. (II) High Smad4 protein expression in a NBD. (III) Reduced Smad4 protein expression in a primary EHCC. (IV) High Smad4 protein expression in a primary EHCC. Original magnification, 100× and 400× respectively for each slide
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Fig2: The expression levels of Smad4 in the primary EHCC and NBD specimens. a The mRNA expression level of Smad4 in the primary EHCC, the adjacent non-tumor tissues and NBD specimens determined by qRT-PCR. U6 was used as the internal control. b Immunohistochemical staining of Smad4 protein expression in primary EHCC and NBD samples. The arrows indicated nuclear staining of Smad4 in both NBD and EHCC samples. (I) Low Smad4 protein expression in a NBD. (II) High Smad4 protein expression in a NBD. (III) Reduced Smad4 protein expression in a primary EHCC. (IV) High Smad4 protein expression in a primary EHCC. Original magnification, 100× and 400× respectively for each slide

Mentions: In order to determine the clinical significance of miR-34a target genes in EHCC, Sanger miRNA database (http://www.mirbase.org/) and Targetscan (http://www.targetscan.org/) were used to predict the candidates of miR-34a. Moreover, both miR-34a and TGF-β/Smad4 pathway have been shown to involve in mediating metastasis and invasion in various types of cancers including cholangiocarcinoma [23, 24]. Thus, we examined Smad4 expression in the human clinical specimens. It was found that the mRNA level of Smad4 was not pronouncedly inhibited in human EHCC tissues compared with NBD and adjacent non-tumor tissues (Fig. 2a). The expression of Smad4 protein levels were further detected in 27 EHCC and 7 NBD specimens by IHC. As shown in Fig. 2b, positive staining of Smad4 was mainly identified in the cell cytoplasm of cancer cells in 18 of 27 tumor tissues and negative staining for Smad4 protein in 5 of 7 NBD tissues. Even a down-regulation of miR-34a with negative staining for Smad4 proteins were found in 7 patients, low expression of miR-34a is associated with positive staining for Smad4 protein in 18 EHCC samples. Logistic regression analysis was performed to determine a negative correlation between Smad4 protein expression level and miR-34a level (B = −3.035, P = 0.01) among the total 27 EHCC tissues. These data suggest that miR-34a expression is inversely correlated with Smad4 in EHCC patients, and miR-34a might play a critical role on Smad4 regulation in over a half but not all of the EHCC patients.Fig. 2


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)

The expression levels of Smad4 in the primary EHCC and NBD specimens. a The mRNA expression level of Smad4 in the primary EHCC, the adjacent non-tumor tissues and NBD specimens determined by qRT-PCR. U6 was used as the internal control. b Immunohistochemical staining of Smad4 protein expression in primary EHCC and NBD samples. The arrows indicated nuclear staining of Smad4 in both NBD and EHCC samples. (I) Low Smad4 protein expression in a NBD. (II) High Smad4 protein expression in a NBD. (III) Reduced Smad4 protein expression in a primary EHCC. (IV) High Smad4 protein expression in a primary EHCC. Original magnification, 100× and 400× respectively for each slide
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477414&req=5

Fig2: The expression levels of Smad4 in the primary EHCC and NBD specimens. a The mRNA expression level of Smad4 in the primary EHCC, the adjacent non-tumor tissues and NBD specimens determined by qRT-PCR. U6 was used as the internal control. b Immunohistochemical staining of Smad4 protein expression in primary EHCC and NBD samples. The arrows indicated nuclear staining of Smad4 in both NBD and EHCC samples. (I) Low Smad4 protein expression in a NBD. (II) High Smad4 protein expression in a NBD. (III) Reduced Smad4 protein expression in a primary EHCC. (IV) High Smad4 protein expression in a primary EHCC. Original magnification, 100× and 400× respectively for each slide
Mentions: In order to determine the clinical significance of miR-34a target genes in EHCC, Sanger miRNA database (http://www.mirbase.org/) and Targetscan (http://www.targetscan.org/) were used to predict the candidates of miR-34a. Moreover, both miR-34a and TGF-β/Smad4 pathway have been shown to involve in mediating metastasis and invasion in various types of cancers including cholangiocarcinoma [23, 24]. Thus, we examined Smad4 expression in the human clinical specimens. It was found that the mRNA level of Smad4 was not pronouncedly inhibited in human EHCC tissues compared with NBD and adjacent non-tumor tissues (Fig. 2a). The expression of Smad4 protein levels were further detected in 27 EHCC and 7 NBD specimens by IHC. As shown in Fig. 2b, positive staining of Smad4 was mainly identified in the cell cytoplasm of cancer cells in 18 of 27 tumor tissues and negative staining for Smad4 protein in 5 of 7 NBD tissues. Even a down-regulation of miR-34a with negative staining for Smad4 proteins were found in 7 patients, low expression of miR-34a is associated with positive staining for Smad4 protein in 18 EHCC samples. Logistic regression analysis was performed to determine a negative correlation between Smad4 protein expression level and miR-34a level (B = −3.035, P = 0.01) among the total 27 EHCC tissues. These data suggest that miR-34a expression is inversely correlated with Smad4 in EHCC patients, and miR-34a might play a critical role on Smad4 regulation in over a half but not all of the EHCC patients.Fig. 2

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