Effect Of HIF-1α Gene Silencing On EMT In Malignant Melanoma Of The Human Choroid Cell

Objectives:To investigate the effect of specific silencing hypoxia inducible factor-1alpha (HIF-la) by small interference RNA (siRNA) on expression of Vimentin in malignant melanoma of the human choroid cell lines (OCM-1) under hypoxia, in order to discuss the role of HIF-1a on epithelial-mesenchymal transition (EMT) in malignant melanoma of the human choroid.Methods:Chemical hypoxia inducer Cobalt chloride (C0Cl2) were added into nutrient medium at the concentration of100μmol/L to simulate hypoxia microenvironment inside tumor. HIF-1α-siRNA were designed and synthetized in vitro. LipofectamineTM2000was taken to transfect siRNA into OCM-1cells under hypoxia. And hypoxic group cells were divided into five groups:simple hypoxic group, HIF-1a interference group, β-actin positive control group, negative control group and liposome group. RT-PCR and Western blot were used to check the expression status of HIF-la and Vimentin on mRNA and protein levels before and after hypoxia culture and cell transfection.Results:After hypoxia culture, OCM-1cells could transform from epithelial like into mesenchymal like in morphology. Compared with normoxia group, there was no obvious change on the expression level of mRNA of HIF-1a in simple hypoxia group (P>0.05), while the expression level of its protein increased obviously (P<0.01); both mRNA and protein levels of Vimentin were up-regulated (P<0.01). Compared with other hypoxic groups, the expression level of mRNA of β-actin was down-regulated obviously in positive control group (P<0.01), which indicated that our operation of cell transfection was successful. In HIF-1a interference group, the expression of HIF-1a and Vimentin were down-regulated obviously both on mRNA and protein levels (P<0.01). There was no obvious significance in the expression of HIF-lα and Vimentin in negative control group and liposome group (P>0.05).Conclusion:1. After hypoxia culture, OCM-1cells could transform from epithelial like into mesenchymal like in morphology, which indicated that hypoxia microenvironment can prompt EMT in OCM-1cells. 2. Hypoxia can up regulate the expression of HIF-1a on protein level in OCM-1cells, which indicated that hypoxia is a regulator in the expression of HIF-1α and that regulation mainly occurs at the level of post transcriptional.3. HIF-la can activate the transcription and expression of Vimentin, this hints that HIF-lα can regulate the EMT in OCM-1cells and plays an important role in the process of its invasion and metastasis.4. We use LipofectamineTM2000to transfect siRNA into OCM-1cells under hypoxia successfully.5. We down regulate the expression of HIF-la and Vimentin successfully by transfecting OCM-1with HIF-1α-siRNA, which further suggests that HIF-1a can regulate the expression of Vimentin and EMT,and that suppression the expression of HIF-lα at the molecular level maybe can shut down the process of tumor invasion and metastasis and offer new directions for cancer treatment.