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Upregulated lncRNA-UCA1 contributes to progression of hepatocellular carcinoma through inhibition of miR-216b and activation of FGFR1/ERK signaling pathway.

Wang F, Ying HQ, He BS, Pan YQ, Deng QW, Sun HL, Chen J, Liu X, Wang SK - Oncotarget (2015)

Bottom Line: The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been recently shown to be dysregulated, which plays an important role in the progression of several cancers.Taken together, our data highlights the pivotal role of UCA1 in the tumorigenesis of HCC.Moreover, the present study elucidates a novel lncRNA-miRNA-mRNA regulatory network that is UCA1-miR-216b-FGFR1-ERK signaling pathway in HCC, which may help to lead a better understanding the pathogenesis of HCC and probe the feasibility of lncRNA-directed diagnosis and therapy for this deadly disease.

View Article: PubMed Central - PubMed

Affiliation: Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China.

ABSTRACT
The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been recently shown to be dysregulated, which plays an important role in the progression of several cancers. However, the biological role and clinical significance of UCA1 in the carcinogenesis of hepatocellular carcinoma (HCC) remain unclear. Herein, we found that UCA1 was aberrantly upregulated in HCC tissues and associated with TNM stage, metastasis and postoperative survival. UCA1 depletion inhibited the growth and metastasis of HCC cell lines in vitro and in vivo. Furthermore, UCA1 could act as an endogenous sponge by directly binding to miR-216b and downregulation miR-216b expression. In addition, UCA1 could reverse the inhibitory effect of miR-216b on the growth and metastasis of HCC cells, which might be involved in the derepression of fibroblast growth factor receptor 1 (FGFR1) expression, a target gene of miR-216b, and the activation of ERK signaling pathway. Taken together, our data highlights the pivotal role of UCA1 in the tumorigenesis of HCC. Moreover, the present study elucidates a novel lncRNA-miRNA-mRNA regulatory network that is UCA1-miR-216b-FGFR1-ERK signaling pathway in HCC, which may help to lead a better understanding the pathogenesis of HCC and probe the feasibility of lncRNA-directed diagnosis and therapy for this deadly disease.

No MeSH data available.


Related in: MedlinePlus

Experimental verification the relationship between UCA1 and the miR-216b mRNA target, FGFR1(A) Representative immunohistochemical staining for FGFR1 from HCC tissues and corresponding nontumourous tissue. Upper: H&E staining; Lower: immunostaining (×200). (B) The statistical graph showed that immunohistochemistry (IHC) scores of FGFR1 in high-UCA1 HCC tissues were significantly higher than that of low-UCA1 HCC tissues. (C) The correlation analyses were performed between IHC scores of FGFR1 and the levels of UCA1 expression (left panel) or miR-216b expression (right panel) in HCC tissues. (D) Putative miR-216b-binding 3′UTR sequence of FGFR1 mRNA. Mutation was generated on the FGFR1 mRNA 3′UTR sequence in the complementary site for the seed region of miR-216b. The wild type or mutant miR-216b-binding FGFR1 mRNA 3′UTR sequence was cloned into pMIR luciferase reporter. (E) The wild type (FGFR1/3′-UTR-WT) and mutant (FGFR1/3′-UTR-Mut) pMIR luciferase reporter were co-transfected into SMMC-7721 cells with miR-NC, miR-216b, miR-216b and pcDNA-NC or miR-216b and pcDNA/UCA1. The normalized luciferase activity in the control group was set as relative luciferase activity. qRT-PCR (F) and western blotting analyses (G) of the levels of FGFR1 mRNA and protein expression following treatment of SMMC-7721 cells with miR-NC, miR-216b, miR-216b + pcDNA-NC or miR-216b + pcDNA/UCA1, and HepG2 cells with siRNA-NC, siUCA1, pcDNA-NC or pcDNA/UCA1, GAPDH and β-actin were used as controls, respectively. The results were reproducible in three independent experiments, mean ± SD, **P < 0.01.
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Figure 6: Experimental verification the relationship between UCA1 and the miR-216b mRNA target, FGFR1(A) Representative immunohistochemical staining for FGFR1 from HCC tissues and corresponding nontumourous tissue. Upper: H&E staining; Lower: immunostaining (×200). (B) The statistical graph showed that immunohistochemistry (IHC) scores of FGFR1 in high-UCA1 HCC tissues were significantly higher than that of low-UCA1 HCC tissues. (C) The correlation analyses were performed between IHC scores of FGFR1 and the levels of UCA1 expression (left panel) or miR-216b expression (right panel) in HCC tissues. (D) Putative miR-216b-binding 3′UTR sequence of FGFR1 mRNA. Mutation was generated on the FGFR1 mRNA 3′UTR sequence in the complementary site for the seed region of miR-216b. The wild type or mutant miR-216b-binding FGFR1 mRNA 3′UTR sequence was cloned into pMIR luciferase reporter. (E) The wild type (FGFR1/3′-UTR-WT) and mutant (FGFR1/3′-UTR-Mut) pMIR luciferase reporter were co-transfected into SMMC-7721 cells with miR-NC, miR-216b, miR-216b and pcDNA-NC or miR-216b and pcDNA/UCA1. The normalized luciferase activity in the control group was set as relative luciferase activity. qRT-PCR (F) and western blotting analyses (G) of the levels of FGFR1 mRNA and protein expression following treatment of SMMC-7721 cells with miR-NC, miR-216b, miR-216b + pcDNA-NC or miR-216b + pcDNA/UCA1, and HepG2 cells with siRNA-NC, siUCA1, pcDNA-NC or pcDNA/UCA1, GAPDH and β-actin were used as controls, respectively. The results were reproducible in three independent experiments, mean ± SD, **P < 0.01.

Mentions: As described above, UCA1 can inhibit miR-216b expression and function in HCC cells. We hypothesized that reduction of miR-216b might decrease repression to its mRNA targets, thereby further facilitated the malignant progression of HCC. Consequently, by performing a computational screen for genes with complementary sites of miR-216b in their 3′-UTR using online softwares including TargetScan (www.targetscan.org), miRanda (www.microrna.org) and miRBase (www.mirbase.org), we found that fibroblast growth factor receptor 1 (FGFR1) was a putative target of miR-216b. Mounting evidence has been reported that FGFR mediates FGF signaling in carcinogenesis and its expression is always dysregulated in many tumor tissues [26, 27]. We then detected the protein expression of FGFR1 by immunohistochemical staining in patients with HCC. As shown in Figure 6A, the immunostaining intensity of FGFR1 in HCC tissues was obviously higher than that of adjacent nontumourous tissues. Moreover, we analyzed FGFR1 protein expression in HCC tissues with different UCA1 levels. The low versus high UCA1 expression was defined as the median value of UCA1 level according to the cohort of tested patients. The levels of FGFR1 protein expression in high-UCA1 HCC tissues were drastically higher than that of low-UCA1 HCC tissues, P < 0.01 (Figure 6B). Meanwhile, a significant positive correlation was found between FGFR1 protein expression levels and UCA1 expression levels in HCC tissues (r = 0.7114, P < 0.0001); whereas, a significant negative correlation was found between FGFR1 protein expression levels and miR-216b expression levels in HCC tissues (r = −0.5040, P < 0.0001; Figure 6C). In addition, the expression of FGFR1 mRNA was also increased in HCC tissues than that in corresponding nontumourous tissues and an inverse correlation was found between FGFR1 mRNA and miR-216b expression levels in HCC tissues (r = −0.7094, P < 0.0001). On the other hand, a positive correlation between FGFR1 mRNA and UCA1 expression levels in HCC tissues was also noted (r = 0.6116, P < 0.0001, Supplementary Figure S2A–S2C). These data indicates that FGFR1 is co-expressed with UCA1 in majority HCC tissues and the interaction of UCA1, miR-216b and FGFR1 might be biologically significant in HCC.


Upregulated lncRNA-UCA1 contributes to progression of hepatocellular carcinoma through inhibition of miR-216b and activation of FGFR1/ERK signaling pathway.

Wang F, Ying HQ, He BS, Pan YQ, Deng QW, Sun HL, Chen J, Liu X, Wang SK - Oncotarget (2015)

Experimental verification the relationship between UCA1 and the miR-216b mRNA target, FGFR1(A) Representative immunohistochemical staining for FGFR1 from HCC tissues and corresponding nontumourous tissue. Upper: H&E staining; Lower: immunostaining (×200). (B) The statistical graph showed that immunohistochemistry (IHC) scores of FGFR1 in high-UCA1 HCC tissues were significantly higher than that of low-UCA1 HCC tissues. (C) The correlation analyses were performed between IHC scores of FGFR1 and the levels of UCA1 expression (left panel) or miR-216b expression (right panel) in HCC tissues. (D) Putative miR-216b-binding 3′UTR sequence of FGFR1 mRNA. Mutation was generated on the FGFR1 mRNA 3′UTR sequence in the complementary site for the seed region of miR-216b. The wild type or mutant miR-216b-binding FGFR1 mRNA 3′UTR sequence was cloned into pMIR luciferase reporter. (E) The wild type (FGFR1/3′-UTR-WT) and mutant (FGFR1/3′-UTR-Mut) pMIR luciferase reporter were co-transfected into SMMC-7721 cells with miR-NC, miR-216b, miR-216b and pcDNA-NC or miR-216b and pcDNA/UCA1. The normalized luciferase activity in the control group was set as relative luciferase activity. qRT-PCR (F) and western blotting analyses (G) of the levels of FGFR1 mRNA and protein expression following treatment of SMMC-7721 cells with miR-NC, miR-216b, miR-216b + pcDNA-NC or miR-216b + pcDNA/UCA1, and HepG2 cells with siRNA-NC, siUCA1, pcDNA-NC or pcDNA/UCA1, GAPDH and β-actin were used as controls, respectively. The results were reproducible in three independent experiments, mean ± SD, **P < 0.01.
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Figure 6: Experimental verification the relationship between UCA1 and the miR-216b mRNA target, FGFR1(A) Representative immunohistochemical staining for FGFR1 from HCC tissues and corresponding nontumourous tissue. Upper: H&E staining; Lower: immunostaining (×200). (B) The statistical graph showed that immunohistochemistry (IHC) scores of FGFR1 in high-UCA1 HCC tissues were significantly higher than that of low-UCA1 HCC tissues. (C) The correlation analyses were performed between IHC scores of FGFR1 and the levels of UCA1 expression (left panel) or miR-216b expression (right panel) in HCC tissues. (D) Putative miR-216b-binding 3′UTR sequence of FGFR1 mRNA. Mutation was generated on the FGFR1 mRNA 3′UTR sequence in the complementary site for the seed region of miR-216b. The wild type or mutant miR-216b-binding FGFR1 mRNA 3′UTR sequence was cloned into pMIR luciferase reporter. (E) The wild type (FGFR1/3′-UTR-WT) and mutant (FGFR1/3′-UTR-Mut) pMIR luciferase reporter were co-transfected into SMMC-7721 cells with miR-NC, miR-216b, miR-216b and pcDNA-NC or miR-216b and pcDNA/UCA1. The normalized luciferase activity in the control group was set as relative luciferase activity. qRT-PCR (F) and western blotting analyses (G) of the levels of FGFR1 mRNA and protein expression following treatment of SMMC-7721 cells with miR-NC, miR-216b, miR-216b + pcDNA-NC or miR-216b + pcDNA/UCA1, and HepG2 cells with siRNA-NC, siUCA1, pcDNA-NC or pcDNA/UCA1, GAPDH and β-actin were used as controls, respectively. The results were reproducible in three independent experiments, mean ± SD, **P < 0.01.
Mentions: As described above, UCA1 can inhibit miR-216b expression and function in HCC cells. We hypothesized that reduction of miR-216b might decrease repression to its mRNA targets, thereby further facilitated the malignant progression of HCC. Consequently, by performing a computational screen for genes with complementary sites of miR-216b in their 3′-UTR using online softwares including TargetScan (www.targetscan.org), miRanda (www.microrna.org) and miRBase (www.mirbase.org), we found that fibroblast growth factor receptor 1 (FGFR1) was a putative target of miR-216b. Mounting evidence has been reported that FGFR mediates FGF signaling in carcinogenesis and its expression is always dysregulated in many tumor tissues [26, 27]. We then detected the protein expression of FGFR1 by immunohistochemical staining in patients with HCC. As shown in Figure 6A, the immunostaining intensity of FGFR1 in HCC tissues was obviously higher than that of adjacent nontumourous tissues. Moreover, we analyzed FGFR1 protein expression in HCC tissues with different UCA1 levels. The low versus high UCA1 expression was defined as the median value of UCA1 level according to the cohort of tested patients. The levels of FGFR1 protein expression in high-UCA1 HCC tissues were drastically higher than that of low-UCA1 HCC tissues, P < 0.01 (Figure 6B). Meanwhile, a significant positive correlation was found between FGFR1 protein expression levels and UCA1 expression levels in HCC tissues (r = 0.7114, P < 0.0001); whereas, a significant negative correlation was found between FGFR1 protein expression levels and miR-216b expression levels in HCC tissues (r = −0.5040, P < 0.0001; Figure 6C). In addition, the expression of FGFR1 mRNA was also increased in HCC tissues than that in corresponding nontumourous tissues and an inverse correlation was found between FGFR1 mRNA and miR-216b expression levels in HCC tissues (r = −0.7094, P < 0.0001). On the other hand, a positive correlation between FGFR1 mRNA and UCA1 expression levels in HCC tissues was also noted (r = 0.6116, P < 0.0001, Supplementary Figure S2A–S2C). These data indicates that FGFR1 is co-expressed with UCA1 in majority HCC tissues and the interaction of UCA1, miR-216b and FGFR1 might be biologically significant in HCC.

Bottom Line: The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been recently shown to be dysregulated, which plays an important role in the progression of several cancers.Taken together, our data highlights the pivotal role of UCA1 in the tumorigenesis of HCC.Moreover, the present study elucidates a novel lncRNA-miRNA-mRNA regulatory network that is UCA1-miR-216b-FGFR1-ERK signaling pathway in HCC, which may help to lead a better understanding the pathogenesis of HCC and probe the feasibility of lncRNA-directed diagnosis and therapy for this deadly disease.

View Article: PubMed Central - PubMed

Affiliation: Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China.

ABSTRACT
The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been recently shown to be dysregulated, which plays an important role in the progression of several cancers. However, the biological role and clinical significance of UCA1 in the carcinogenesis of hepatocellular carcinoma (HCC) remain unclear. Herein, we found that UCA1 was aberrantly upregulated in HCC tissues and associated with TNM stage, metastasis and postoperative survival. UCA1 depletion inhibited the growth and metastasis of HCC cell lines in vitro and in vivo. Furthermore, UCA1 could act as an endogenous sponge by directly binding to miR-216b and downregulation miR-216b expression. In addition, UCA1 could reverse the inhibitory effect of miR-216b on the growth and metastasis of HCC cells, which might be involved in the derepression of fibroblast growth factor receptor 1 (FGFR1) expression, a target gene of miR-216b, and the activation of ERK signaling pathway. Taken together, our data highlights the pivotal role of UCA1 in the tumorigenesis of HCC. Moreover, the present study elucidates a novel lncRNA-miRNA-mRNA regulatory network that is UCA1-miR-216b-FGFR1-ERK signaling pathway in HCC, which may help to lead a better understanding the pathogenesis of HCC and probe the feasibility of lncRNA-directed diagnosis and therapy for this deadly disease.

No MeSH data available.


Related in: MedlinePlus