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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

UCA1 reduces miR-216b expression in HCC(A) Putative miR-216b-binding sequence of UCA1 RNA. Mutation was generated on the UCA1 RNA sequence in the complementary site for the seed region of miR-216b, as shown. A human UCA1 RNA containing wild type or mutant miR-216b-binding sequence was cloned into pMIR luciferase reporter. (B) The wild type (pMIR-UCA1-WT) and mutant (pMIR-UCA1-Mut) reporter plasmids were co-transfected into SMMC-7721 and HepG2 cells with miR-216b or negative control (miR-NC). The normalized luciferase activity in the control group was set as relative luciferase activity. (C) Cellular lysates from SMMC-7721 cells were used for RNA immunoprecipitation (RIP) with Ago2 antibody. Detection of Ago2 using IP-western (upper panel), and detection of UCA1 and miR-216b using qRT-PCR. RNA levels were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates (lower panel). (D) MiR-216b expression levels were analyzed in different liver cell lines by qRT-PCR and U6 was treated as internal control. (E) MiR-216b expression was examined by qRT-PCR and normalized to U6 expression in 98 pairs of HCC tissues (T) compared with corresponding nontumourous liver specimens (N). (F) The correlation analysis was performed between UCA1 expression levels and miR-216b expression levels in HCC tissues (n = 98). (G) miR-216b (1 μg) was co-transfected into SMMC-7721 and HepG2 cells with pcDNA-NC (empty vector, 1 μg) or pcDNA/UCA1 (1 μg, 2 μg, 4 μg). The expression of miR-216b was analyzed by qRT-PCR assay and U6 was used as an internal control. All experiments were at least repeated in triplicate, mean ± SD, **P < 0.01.
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Figure 4: UCA1 reduces miR-216b expression in HCC(A) Putative miR-216b-binding sequence of UCA1 RNA. Mutation was generated on the UCA1 RNA sequence in the complementary site for the seed region of miR-216b, as shown. A human UCA1 RNA containing wild type or mutant miR-216b-binding sequence was cloned into pMIR luciferase reporter. (B) The wild type (pMIR-UCA1-WT) and mutant (pMIR-UCA1-Mut) reporter plasmids were co-transfected into SMMC-7721 and HepG2 cells with miR-216b or negative control (miR-NC). The normalized luciferase activity in the control group was set as relative luciferase activity. (C) Cellular lysates from SMMC-7721 cells were used for RNA immunoprecipitation (RIP) with Ago2 antibody. Detection of Ago2 using IP-western (upper panel), and detection of UCA1 and miR-216b using qRT-PCR. RNA levels were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates (lower panel). (D) MiR-216b expression levels were analyzed in different liver cell lines by qRT-PCR and U6 was treated as internal control. (E) MiR-216b expression was examined by qRT-PCR and normalized to U6 expression in 98 pairs of HCC tissues (T) compared with corresponding nontumourous liver specimens (N). (F) The correlation analysis was performed between UCA1 expression levels and miR-216b expression levels in HCC tissues (n = 98). (G) miR-216b (1 μg) was co-transfected into SMMC-7721 and HepG2 cells with pcDNA-NC (empty vector, 1 μg) or pcDNA/UCA1 (1 μg, 2 μg, 4 μg). The expression of miR-216b was analyzed by qRT-PCR assay and U6 was used as an internal control. All experiments were at least repeated in triplicate, mean ± SD, **P < 0.01.

Mentions: Bioinformatics reveal UCA1 RNA contains one conserved target site of miR-216b. To confirm this possibility, the wild type sequence of UCA1 (UCA1-WT) or its mutant sequence (UCA1-Mut) (Figure 4A) was subcloned into the pMIR luciferase reporter and then co-transfected with miR-216b or miR-NC into SMMC-7721 and HepG2 cells. The relative luciferase activity of the pMIR-UCA1-WT was significantly decreased by 52.4% and 47.3% respectively, when miR-216b was co-transfected into SMMC-7721 and HepG2 cells. However, the luciferase activity of pMIR-UCA1-Mut was unaffected in both HCC cell lines by co-transfection with miR-216b (Figure 4B). It is well known that miRNAs may regulate their targets through forming RNA-induced silencing complex (RISC), moreover, recent studies have shown that lncRNAs can act as molecular sponges to regulate the miRNAs activity by associating with RISC [10, 11, 24]. To investigate whether both UCA1 and miR-216b might be in the RISC complex, RNA binding protein immunoprecipitation (RIP) experiments were performed on SMMC7721 cell extracts using antibodies against Ago2, a key component of the RISC complex. We confirmed that the Ago2 antibody precipitated the Ago2 protein from our cellular extract (Figure 4C, upper panel). Moreover, RNA levels of UCA1 and miR-216b in immunoprecipitates were determined by qRT-PCR. As expected, UCA1 and miR-216b were enriched 115-fold and 197-fold, respectively, in Ago2 pellets relative to control IgG immunoprecipitates. (Figure 4C, lower panel). Accordingly, our results suggest that UCA1 is present in Ago2-containing RISC, likely through association with miR-216b, in agreement with our bioinformatic analysis and luciferase assays.


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)

UCA1 reduces miR-216b expression in HCC(A) Putative miR-216b-binding sequence of UCA1 RNA. Mutation was generated on the UCA1 RNA sequence in the complementary site for the seed region of miR-216b, as shown. A human UCA1 RNA containing wild type or mutant miR-216b-binding sequence was cloned into pMIR luciferase reporter. (B) The wild type (pMIR-UCA1-WT) and mutant (pMIR-UCA1-Mut) reporter plasmids were co-transfected into SMMC-7721 and HepG2 cells with miR-216b or negative control (miR-NC). The normalized luciferase activity in the control group was set as relative luciferase activity. (C) Cellular lysates from SMMC-7721 cells were used for RNA immunoprecipitation (RIP) with Ago2 antibody. Detection of Ago2 using IP-western (upper panel), and detection of UCA1 and miR-216b using qRT-PCR. RNA levels were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates (lower panel). (D) MiR-216b expression levels were analyzed in different liver cell lines by qRT-PCR and U6 was treated as internal control. (E) MiR-216b expression was examined by qRT-PCR and normalized to U6 expression in 98 pairs of HCC tissues (T) compared with corresponding nontumourous liver specimens (N). (F) The correlation analysis was performed between UCA1 expression levels and miR-216b expression levels in HCC tissues (n = 98). (G) miR-216b (1 μg) was co-transfected into SMMC-7721 and HepG2 cells with pcDNA-NC (empty vector, 1 μg) or pcDNA/UCA1 (1 μg, 2 μg, 4 μg). The expression of miR-216b was analyzed by qRT-PCR assay and U6 was used as an internal control. All experiments were at least repeated in triplicate, mean ± SD, **P < 0.01.
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Figure 4: UCA1 reduces miR-216b expression in HCC(A) Putative miR-216b-binding sequence of UCA1 RNA. Mutation was generated on the UCA1 RNA sequence in the complementary site for the seed region of miR-216b, as shown. A human UCA1 RNA containing wild type or mutant miR-216b-binding sequence was cloned into pMIR luciferase reporter. (B) The wild type (pMIR-UCA1-WT) and mutant (pMIR-UCA1-Mut) reporter plasmids were co-transfected into SMMC-7721 and HepG2 cells with miR-216b or negative control (miR-NC). The normalized luciferase activity in the control group was set as relative luciferase activity. (C) Cellular lysates from SMMC-7721 cells were used for RNA immunoprecipitation (RIP) with Ago2 antibody. Detection of Ago2 using IP-western (upper panel), and detection of UCA1 and miR-216b using qRT-PCR. RNA levels were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates (lower panel). (D) MiR-216b expression levels were analyzed in different liver cell lines by qRT-PCR and U6 was treated as internal control. (E) MiR-216b expression was examined by qRT-PCR and normalized to U6 expression in 98 pairs of HCC tissues (T) compared with corresponding nontumourous liver specimens (N). (F) The correlation analysis was performed between UCA1 expression levels and miR-216b expression levels in HCC tissues (n = 98). (G) miR-216b (1 μg) was co-transfected into SMMC-7721 and HepG2 cells with pcDNA-NC (empty vector, 1 μg) or pcDNA/UCA1 (1 μg, 2 μg, 4 μg). The expression of miR-216b was analyzed by qRT-PCR assay and U6 was used as an internal control. All experiments were at least repeated in triplicate, mean ± SD, **P < 0.01.
Mentions: Bioinformatics reveal UCA1 RNA contains one conserved target site of miR-216b. To confirm this possibility, the wild type sequence of UCA1 (UCA1-WT) or its mutant sequence (UCA1-Mut) (Figure 4A) was subcloned into the pMIR luciferase reporter and then co-transfected with miR-216b or miR-NC into SMMC-7721 and HepG2 cells. The relative luciferase activity of the pMIR-UCA1-WT was significantly decreased by 52.4% and 47.3% respectively, when miR-216b was co-transfected into SMMC-7721 and HepG2 cells. However, the luciferase activity of pMIR-UCA1-Mut was unaffected in both HCC cell lines by co-transfection with miR-216b (Figure 4B). It is well known that miRNAs may regulate their targets through forming RNA-induced silencing complex (RISC), moreover, recent studies have shown that lncRNAs can act as molecular sponges to regulate the miRNAs activity by associating with RISC [10, 11, 24]. To investigate whether both UCA1 and miR-216b might be in the RISC complex, RNA binding protein immunoprecipitation (RIP) experiments were performed on SMMC7721 cell extracts using antibodies against Ago2, a key component of the RISC complex. We confirmed that the Ago2 antibody precipitated the Ago2 protein from our cellular extract (Figure 4C, upper panel). Moreover, RNA levels of UCA1 and miR-216b in immunoprecipitates were determined by qRT-PCR. As expected, UCA1 and miR-216b were enriched 115-fold and 197-fold, respectively, in Ago2 pellets relative to control IgG immunoprecipitates. (Figure 4C, lower panel). Accordingly, our results suggest that UCA1 is present in Ago2-containing RISC, likely through association with miR-216b, in agreement with our bioinformatic analysis and luciferase assays.

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