Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia
AIM:To investigate the effect of specific silencing hypoxia inducible factor-1 alpha(HIF-1α)by small interference RNA(siRNA)on expression of vimentin in human uveal melanoma cell lines(OCM-1)under hypoxia, in order to discuss the role of HIF-1α on epithelial-mesenchymal transition(EMT)in uveal melan...
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| Format: | Article |
| Language: | English |
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Press of International Journal of Ophthalmology (IJO PRESS)
2014-03-01
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| Series: | Guoji Yanke Zazhi |
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| Online Access: | http://www.ies.net.cn/cn_publish/2014/3/201403008.pdf |
| _version_ | 1811284519229063168 |
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| author | Juan Zhao Zhan-Yu Zhou Shuang Wang Fu-Xiang Yuan Fa-Yan Lu |
| author_facet | Juan Zhao Zhan-Yu Zhou Shuang Wang Fu-Xiang Yuan Fa-Yan Lu |
| author_sort | Juan Zhao |
| collection | DOAJ |
| description | AIM:To investigate the effect of specific silencing hypoxia inducible factor-1 alpha(HIF-1α)by small interference RNA(siRNA)on expression of vimentin in human uveal melanoma cell lines(OCM-1)under hypoxia, in order to discuss the role of HIF-1α on epithelial-mesenchymal transition(EMT)in uveal melanoma.<p>METHODS: OCM-1 cells were cultured under normoxia and hypoxia <i>in vitro</i>. We added chemical hypoxia inducer cobalt chloride(CoCl<sub>2</sub>)into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment inside tumor to culture cells of hypoxic group. And hypoxic group cells were divided into five groups: simple hypoxic group, interference group, positive control group, negative control group and liposome group. SiRNA(including HIF-1α-siRNA, β-actin-siRNA and negative control)were designed and synthetized <i>in vitro</i>. Lipofectamine<sup>TM</sup> 2000 was taken to transfect siRNA into OCM-1 cells under hypoxia. RT-PCR and Western blot were used to check the expression status of HIF-1α and vimentin on mRNA and protein levels before and after hypoxia culture and cell transfection.<p>RESULTS: Compared with normoxia group, there was no obvious change on the expression level of mRNA of HIF-1α in simple hypoxia group(<i>P</i>>0.05), while the expression level of its protein increased obviously(<i>P</i><0.01); both mRNA and protein levels of vimentin were up-regulated(<i>P</i><0.01). Compared with other hypoxic groups, the expression level of mRNA of β-actin was down-regulated in positive control group(<i>P</i><0.01), which indicated that our operation of cell transfection was successful. In interference group, the expression of HIF-1α and vimentin were down-regulated obviously both on mRNA and protein levels(<i>P</i><0.01). There was no significant difference in the expression of HIF-1α and vimentin in negative control group and liposome group(<i>P</i>>0.05). <p>CONCLUSION: Hypoxia can up-regulate the expression of HIF-1α on protein level in OCM-1 cells, and activate the transcription of vimentin as the downstream gene of HIF-1α, up-regulate the expression of vimentin both on mRNA and protein levels. This hints that HIF-1α can regulate the EMT in uveal melanoma and plays an important role in the process of tumor invasion and metastasis. We successfully down-regulate the expression of HIF-1α and vimentin by transfecting OCM-1 with HIF-1α-siRNA. This suggests that suppression the expression of HIF-1α at the molecular level maybe can shut down the process of tumor invasion and metastasis and offer new directions for cancer treatment. |
| first_indexed | 2024-04-13T02:29:53Z |
| format | Article |
| id | doaj.art-f20d398d077a4113ab63f29313001974 |
| institution | Directory Open Access Journal |
| issn | 1672-5123 1672-5123 |
| language | English |
| last_indexed | 2024-04-13T02:29:53Z |
| publishDate | 2014-03-01 |
| publisher | Press of International Journal of Ophthalmology (IJO PRESS) |
| record_format | Article |
| series | Guoji Yanke Zazhi |
| spelling | doaj.art-f20d398d077a4113ab63f293130019742022-12-22T03:06:37ZengPress of International Journal of Ophthalmology (IJO PRESS)Guoji Yanke Zazhi1672-51231672-51232014-03-0114341141510.3980/j.issn.1672-5123.2014.03.08Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxiaJuan Zhao0Zhan-Yu Zhou1Shuang Wang2Fu-Xiang Yuan3Fa-Yan Lu4Department of Ophthalmology, the Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, ChinaDepartment of Ophthalmology, the Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, ChinaDepartment of Ophthalmology, the Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, ChinaDepartment of Ophthalmology, the Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, ChinaDepartment of Ophthalmology, the Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, ChinaAIM:To investigate the effect of specific silencing hypoxia inducible factor-1 alpha(HIF-1α)by small interference RNA(siRNA)on expression of vimentin in human uveal melanoma cell lines(OCM-1)under hypoxia, in order to discuss the role of HIF-1α on epithelial-mesenchymal transition(EMT)in uveal melanoma.<p>METHODS: OCM-1 cells were cultured under normoxia and hypoxia <i>in vitro</i>. We added chemical hypoxia inducer cobalt chloride(CoCl<sub>2</sub>)into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment inside tumor to culture cells of hypoxic group. And hypoxic group cells were divided into five groups: simple hypoxic group, interference group, positive control group, negative control group and liposome group. SiRNA(including HIF-1α-siRNA, β-actin-siRNA and negative control)were designed and synthetized <i>in vitro</i>. Lipofectamine<sup>TM</sup> 2000 was taken to transfect siRNA into OCM-1 cells under hypoxia. RT-PCR and Western blot were used to check the expression status of HIF-1α and vimentin on mRNA and protein levels before and after hypoxia culture and cell transfection.<p>RESULTS: Compared with normoxia group, there was no obvious change on the expression level of mRNA of HIF-1α in simple hypoxia group(<i>P</i>>0.05), while the expression level of its protein increased obviously(<i>P</i><0.01); both mRNA and protein levels of vimentin were up-regulated(<i>P</i><0.01). Compared with other hypoxic groups, the expression level of mRNA of β-actin was down-regulated in positive control group(<i>P</i><0.01), which indicated that our operation of cell transfection was successful. In interference group, the expression of HIF-1α and vimentin were down-regulated obviously both on mRNA and protein levels(<i>P</i><0.01). There was no significant difference in the expression of HIF-1α and vimentin in negative control group and liposome group(<i>P</i>>0.05). <p>CONCLUSION: Hypoxia can up-regulate the expression of HIF-1α on protein level in OCM-1 cells, and activate the transcription of vimentin as the downstream gene of HIF-1α, up-regulate the expression of vimentin both on mRNA and protein levels. This hints that HIF-1α can regulate the EMT in uveal melanoma and plays an important role in the process of tumor invasion and metastasis. We successfully down-regulate the expression of HIF-1α and vimentin by transfecting OCM-1 with HIF-1α-siRNA. This suggests that suppression the expression of HIF-1α at the molecular level maybe can shut down the process of tumor invasion and metastasis and offer new directions for cancer treatment.http://www.ies.net.cn/cn_publish/2014/3/201403008.pdfuveal melanomahypoxia-inducible factor-1 alphaVimentinepithelial-mesenchymal transitionRNA interferenceRNA interference |
| spellingShingle | Juan Zhao Zhan-Yu Zhou Shuang Wang Fu-Xiang Yuan Fa-Yan Lu Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia Guoji Yanke Zazhi uveal melanoma hypoxia-inducible factor-1 alpha Vimentin epithelial-mesenchymal transition RNA interference RNA interference |
| title | Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia |
| title_full | Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia |
| title_fullStr | Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia |
| title_full_unstemmed | Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia |
| title_short | Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia |
| title_sort | effect of specific silencing hif 1α on expression of vimentin in ocm 1 cells under hypoxia |
| topic | uveal melanoma hypoxia-inducible factor-1 alpha Vimentin epithelial-mesenchymal transition RNA interference RNA interference |
| url | http://www.ies.net.cn/cn_publish/2014/3/201403008.pdf |
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