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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 expression in human EHCC tissues and CC cell lines, and the relationship between miR-34a expression and disease-free survival in EHCC patients. a The mRNA expression profile of miR-34a in 27 primary EHCC tissues compared to the adjacent non-tumor tissues and 7 normal bile duct tissues (NBD) determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. b The mRNA expression level of miR-34a in 4 CC cell lines (QBC939, HuCCT1, RBE and HCCC9810) compared to HiBECs determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. c miR-34a predicts disease-free survival in EHCC patients. The Kaplan-Meier curve of disease-free survival in patients with high miR-34a expression (n = 13) and low miR-34a expression (n = 14) (***P < 0.01). The median disease-free survival time was 13.07 months and 23.54 months in low- and high- miR-34a group, respectively (*P < 0.05)
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Fig1: miR-34a expression in human EHCC tissues and CC cell lines, and the relationship between miR-34a expression and disease-free survival in EHCC patients. a The mRNA expression profile of miR-34a in 27 primary EHCC tissues compared to the adjacent non-tumor tissues and 7 normal bile duct tissues (NBD) determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. b The mRNA expression level of miR-34a in 4 CC cell lines (QBC939, HuCCT1, RBE and HCCC9810) compared to HiBECs determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. c miR-34a predicts disease-free survival in EHCC patients. The Kaplan-Meier curve of disease-free survival in patients with high miR-34a expression (n = 13) and low miR-34a expression (n = 14) (***P < 0.01). The median disease-free survival time was 13.07 months and 23.54 months in low- and high- miR-34a group, respectively (*P < 0.05)

Mentions: The relative expression level of miR-34a in EHCC tissues was significantly lower than the NBD and the adjacent non-tumor tissues (P < 0.01, Fig. 1a). No significant difference was found between the NBD tissues and the adjacent non-tumor tissues. For further characterization of miR-34a expression in CC cell lines, HiBECs, EHCC cell line QBC939, HuCCT1 and the IHCC cell lines RBE and HCCC9810 were examined. qRT-PCR analysis revealed that the expression level of miR-34a was markedly decreased in all of the CC cell lines in comparison with the expression levels in HiBECs (P < 0.01, Fig. 1b). To further evaluate the clinical value of miR-34a in EHCC patients, we divided the patients into two groups according to the median value (5.113) of the expression level of miR-34a. The correlation between miR-34a and clinicopathological characteristics was then analyzed (Table 1). miR-34a showed lower expression levels in specimens with lymphatic metastasis (P = 0.004, Table 1) and in the advanced clinical stages (stage III, IV vs I, II) (P < 0.001, Table 1). The results of multivariate logistic regression analysis also showed that miR-34a expression related with clinical stages (P = 0.0013, Table 2). However, no association of miR-34a was observed with age, gender, tumor size, different pathological types, cell differentiation and Bismuth classification (P > 0.05, Table 1). Kaplan-Meier analysis showed that down-regulation of miR-34a was correlated with decreased disease-free survival (Fig. 1c, P = 0.004). Furthermore, Cox regression analysis was performed to analyze all the factors in Table 1 by backward variable selection. The results indicated that only miR-34a was selected into the model (P = 0.002). Thus, miR-34a was an independent prognostic indicator in EHCC.Fig. 1


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 expression in human EHCC tissues and CC cell lines, and the relationship between miR-34a expression and disease-free survival in EHCC patients. a The mRNA expression profile of miR-34a in 27 primary EHCC tissues compared to the adjacent non-tumor tissues and 7 normal bile duct tissues (NBD) determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. b The mRNA expression level of miR-34a in 4 CC cell lines (QBC939, HuCCT1, RBE and HCCC9810) compared to HiBECs determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. c miR-34a predicts disease-free survival in EHCC patients. The Kaplan-Meier curve of disease-free survival in patients with high miR-34a expression (n = 13) and low miR-34a expression (n = 14) (***P < 0.01). The median disease-free survival time was 13.07 months and 23.54 months in low- and high- miR-34a group, respectively (*P < 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4477414&req=5

Fig1: miR-34a expression in human EHCC tissues and CC cell lines, and the relationship between miR-34a expression and disease-free survival in EHCC patients. a The mRNA expression profile of miR-34a in 27 primary EHCC tissues compared to the adjacent non-tumor tissues and 7 normal bile duct tissues (NBD) determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. b The mRNA expression level of miR-34a in 4 CC cell lines (QBC939, HuCCT1, RBE and HCCC9810) compared to HiBECs determined by qRT-PCR (***P < 0.01). U6 was used as the internal control. c miR-34a predicts disease-free survival in EHCC patients. The Kaplan-Meier curve of disease-free survival in patients with high miR-34a expression (n = 13) and low miR-34a expression (n = 14) (***P < 0.01). The median disease-free survival time was 13.07 months and 23.54 months in low- and high- miR-34a group, respectively (*P < 0.05)
Mentions: The relative expression level of miR-34a in EHCC tissues was significantly lower than the NBD and the adjacent non-tumor tissues (P < 0.01, Fig. 1a). No significant difference was found between the NBD tissues and the adjacent non-tumor tissues. For further characterization of miR-34a expression in CC cell lines, HiBECs, EHCC cell line QBC939, HuCCT1 and the IHCC cell lines RBE and HCCC9810 were examined. qRT-PCR analysis revealed that the expression level of miR-34a was markedly decreased in all of the CC cell lines in comparison with the expression levels in HiBECs (P < 0.01, Fig. 1b). To further evaluate the clinical value of miR-34a in EHCC patients, we divided the patients into two groups according to the median value (5.113) of the expression level of miR-34a. The correlation between miR-34a and clinicopathological characteristics was then analyzed (Table 1). miR-34a showed lower expression levels in specimens with lymphatic metastasis (P = 0.004, Table 1) and in the advanced clinical stages (stage III, IV vs I, II) (P < 0.001, Table 1). The results of multivariate logistic regression analysis also showed that miR-34a expression related with clinical stages (P = 0.0013, Table 2). However, no association of miR-34a was observed with age, gender, tumor size, different pathological types, cell differentiation and Bismuth classification (P > 0.05, Table 1). Kaplan-Meier analysis showed that down-regulation of miR-34a was correlated with decreased disease-free survival (Fig. 1c, P = 0.004). Furthermore, Cox regression analysis was performed to analyze all the factors in Table 1 by backward variable selection. The results indicated that only miR-34a was selected into the model (P = 0.002). Thus, miR-34a was an independent prognostic indicator in EHCC.Fig. 1

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