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Hypoxia-inducible MiR-182 promotes angiogenesis by targeting RASA1 in hepatocellular carcinoma.

Du C, Weng X, Hu W, Lv Z, Xiao H, Ding C, Gyabaah OA, Xie H, Zhou L, Wu J, Zheng S - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: We found that miR-182 was upregulated in the hypoxia-based microarray.We then revealed that miR-182 was also significantly increased in the HCC tissues compared to the corresponding normal tissues.In addition, the suppression of RASA1 phenocopied the pro-angiogenesis effects of miR-182.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, China.

ABSTRACT

Background: Hypoxia is a common feature of solid tumors, including HCC. And hypoxia has been reported to play an important role in HCC progression. However, the potential mechanism of miRNAs in hypoxia mediating HCC progression still remains unclear.

Methods: The HCC cells were cultured in the atmosphere of 1 % oxygen to induce hypoxia. The microRNA microarray was employed to search for the hypoxia-inducible miRNAs. RT-PCR, western blot and immunohistochemistry were used to detect the RNA and protein levels. HUVEC were applied to explore the angiogenesis level.

Results: We found that miR-182 was upregulated in the hypoxia-based microarray. We then revealed that miR-182 was also significantly increased in the HCC tissues compared to the corresponding normal tissues. In vitro capilliary tube formation assays showed that the miR-182 promoted angiogenesis. RASA1 was demonstrated as the direct target of miR-182. In addition, the suppression of RASA1 phenocopied the pro-angiogenesis effects of miR-182. Besides, RASA1 was also decreased in the hypoxia HCC cells while the inhibition of miR-182 partially restored the level of RASA1.

Conclusions: Our data showed that hypoxia regulated the expression of miR-182 and RASA1 to promote HCC angiogenesis.

No MeSH data available.


Related in: MedlinePlus

RASA1 was the direct target of miR-182 and the inhibition of RASA1 promoted angiogenesis. a miR-182 and its predicted binding sequence in the 3’UTR of RASA1. The mutant sequence was constructed by changing their complementary sites. b The SK-HEP-1 cells were co-transfected with miR-182 mimics or inhibitors or NC and 100 ng firefly luciferase reporter plasmid containing wild-type or mutant type 3’UTR of RASA1. After incubation for 48 h, the firefly luciferase activity of each sample was detected and normalized to the renilla luciferase activity. The data represent the mean ± SEM of triplicates. c The protein levels of RASA1 were examined by western blot after transfected with miR-182 mimics or inhibitors. d IHC assays were applied to explore the protein levels of RASA1 in 36 HCC tissues. The representative images were shown at the 400× magnification. The medium level of all 36 cases was chosen as the cut-off point for separating low-miR-182 (n = 18) and high-miR-182 (n = 18) groups. e Capillary tube formation assays was used to detect the angiogenesis effects of RASA1 suppression. The data represent the mean ± SEM of triplicates. (*P < 0.05, **P < 0.01)
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Fig3: RASA1 was the direct target of miR-182 and the inhibition of RASA1 promoted angiogenesis. a miR-182 and its predicted binding sequence in the 3’UTR of RASA1. The mutant sequence was constructed by changing their complementary sites. b The SK-HEP-1 cells were co-transfected with miR-182 mimics or inhibitors or NC and 100 ng firefly luciferase reporter plasmid containing wild-type or mutant type 3’UTR of RASA1. After incubation for 48 h, the firefly luciferase activity of each sample was detected and normalized to the renilla luciferase activity. The data represent the mean ± SEM of triplicates. c The protein levels of RASA1 were examined by western blot after transfected with miR-182 mimics or inhibitors. d IHC assays were applied to explore the protein levels of RASA1 in 36 HCC tissues. The representative images were shown at the 400× magnification. The medium level of all 36 cases was chosen as the cut-off point for separating low-miR-182 (n = 18) and high-miR-182 (n = 18) groups. e Capillary tube formation assays was used to detect the angiogenesis effects of RASA1 suppression. The data represent the mean ± SEM of triplicates. (*P < 0.05, **P < 0.01)

Mentions: It is well reported that the functions of miRNAs are mediated by their target genes. So we applied the database TargetScan and miRanda to search for the potential target genes of miR-182. Among the predicted genes, we focused on RASA1, because not only it was indicated by both databases (Fig. 3a), but also it has been reported to correlate with angiogenesis in a previous study [17]. The dual luciferase reporter results in SK-HEP-1 cells showed that the transfection of miR-182 mimics exerted repressive effects of luciferase activity of RASA1 3’UTR plasmid while the inhibition of miR-182 increased the luciferase activity (Fig. 3b). Moreover, the RT-PCR and western blot results in HCC cells lines indicated that overexpression of miR-182 reduced the mRNA level and protein level of RASA1 while the suppression of miR-182 revealed an opposite result (Fig. 3c and Additional file 1: Figure S1A). Based on the correlation in HCC cells, we then explore the level of RASA1 and the relationship between miR-182 and RASA1 in 36 HCC tissues. As shown in Fig. 3d and Additional file 1: Figure S1B, the expression of RASA1 was reduced in HCC tissues and the tissues with higher miR-182 levels tended to have the lower expression of RASA1. Furthermore, TCM from si-RASA1 group promoted the formation of capillary-like structure (Fig. 3e), which was consistent with miR-182 mimics transfection. Collectively, these findings imply that RASA1 is a direct target of miR-182 in both HCC cells and tissues.Fig. 3


Hypoxia-inducible MiR-182 promotes angiogenesis by targeting RASA1 in hepatocellular carcinoma.

Du C, Weng X, Hu W, Lv Z, Xiao H, Ding C, Gyabaah OA, Xie H, Zhou L, Wu J, Zheng S - J. Exp. Clin. Cancer Res. (2015)

RASA1 was the direct target of miR-182 and the inhibition of RASA1 promoted angiogenesis. a miR-182 and its predicted binding sequence in the 3’UTR of RASA1. The mutant sequence was constructed by changing their complementary sites. b The SK-HEP-1 cells were co-transfected with miR-182 mimics or inhibitors or NC and 100 ng firefly luciferase reporter plasmid containing wild-type or mutant type 3’UTR of RASA1. After incubation for 48 h, the firefly luciferase activity of each sample was detected and normalized to the renilla luciferase activity. The data represent the mean ± SEM of triplicates. c The protein levels of RASA1 were examined by western blot after transfected with miR-182 mimics or inhibitors. d IHC assays were applied to explore the protein levels of RASA1 in 36 HCC tissues. The representative images were shown at the 400× magnification. The medium level of all 36 cases was chosen as the cut-off point for separating low-miR-182 (n = 18) and high-miR-182 (n = 18) groups. e Capillary tube formation assays was used to detect the angiogenesis effects of RASA1 suppression. The data represent the mean ± SEM of triplicates. (*P < 0.05, **P < 0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493986&req=5

Fig3: RASA1 was the direct target of miR-182 and the inhibition of RASA1 promoted angiogenesis. a miR-182 and its predicted binding sequence in the 3’UTR of RASA1. The mutant sequence was constructed by changing their complementary sites. b The SK-HEP-1 cells were co-transfected with miR-182 mimics or inhibitors or NC and 100 ng firefly luciferase reporter plasmid containing wild-type or mutant type 3’UTR of RASA1. After incubation for 48 h, the firefly luciferase activity of each sample was detected and normalized to the renilla luciferase activity. The data represent the mean ± SEM of triplicates. c The protein levels of RASA1 were examined by western blot after transfected with miR-182 mimics or inhibitors. d IHC assays were applied to explore the protein levels of RASA1 in 36 HCC tissues. The representative images were shown at the 400× magnification. The medium level of all 36 cases was chosen as the cut-off point for separating low-miR-182 (n = 18) and high-miR-182 (n = 18) groups. e Capillary tube formation assays was used to detect the angiogenesis effects of RASA1 suppression. The data represent the mean ± SEM of triplicates. (*P < 0.05, **P < 0.01)
Mentions: It is well reported that the functions of miRNAs are mediated by their target genes. So we applied the database TargetScan and miRanda to search for the potential target genes of miR-182. Among the predicted genes, we focused on RASA1, because not only it was indicated by both databases (Fig. 3a), but also it has been reported to correlate with angiogenesis in a previous study [17]. The dual luciferase reporter results in SK-HEP-1 cells showed that the transfection of miR-182 mimics exerted repressive effects of luciferase activity of RASA1 3’UTR plasmid while the inhibition of miR-182 increased the luciferase activity (Fig. 3b). Moreover, the RT-PCR and western blot results in HCC cells lines indicated that overexpression of miR-182 reduced the mRNA level and protein level of RASA1 while the suppression of miR-182 revealed an opposite result (Fig. 3c and Additional file 1: Figure S1A). Based on the correlation in HCC cells, we then explore the level of RASA1 and the relationship between miR-182 and RASA1 in 36 HCC tissues. As shown in Fig. 3d and Additional file 1: Figure S1B, the expression of RASA1 was reduced in HCC tissues and the tissues with higher miR-182 levels tended to have the lower expression of RASA1. Furthermore, TCM from si-RASA1 group promoted the formation of capillary-like structure (Fig. 3e), which was consistent with miR-182 mimics transfection. Collectively, these findings imply that RASA1 is a direct target of miR-182 in both HCC cells and tissues.Fig. 3

Bottom Line: We found that miR-182 was upregulated in the hypoxia-based microarray.We then revealed that miR-182 was also significantly increased in the HCC tissues compared to the corresponding normal tissues.In addition, the suppression of RASA1 phenocopied the pro-angiogenesis effects of miR-182.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, China.

ABSTRACT

Background: Hypoxia is a common feature of solid tumors, including HCC. And hypoxia has been reported to play an important role in HCC progression. However, the potential mechanism of miRNAs in hypoxia mediating HCC progression still remains unclear.

Methods: The HCC cells were cultured in the atmosphere of 1 % oxygen to induce hypoxia. The microRNA microarray was employed to search for the hypoxia-inducible miRNAs. RT-PCR, western blot and immunohistochemistry were used to detect the RNA and protein levels. HUVEC were applied to explore the angiogenesis level.

Results: We found that miR-182 was upregulated in the hypoxia-based microarray. We then revealed that miR-182 was also significantly increased in the HCC tissues compared to the corresponding normal tissues. In vitro capilliary tube formation assays showed that the miR-182 promoted angiogenesis. RASA1 was demonstrated as the direct target of miR-182. In addition, the suppression of RASA1 phenocopied the pro-angiogenesis effects of miR-182. Besides, RASA1 was also decreased in the hypoxia HCC cells while the inhibition of miR-182 partially restored the level of RASA1.

Conclusions: Our data showed that hypoxia regulated the expression of miR-182 and RASA1 to promote HCC angiogenesis.

No MeSH data available.


Related in: MedlinePlus