Original Article
Effects of short hairpin RNA-mediated semaphorin 5A gene silencing on proliferation, metastasis and invasion of malignant melanoma cell line A375
Li Zhang, Yadong Li, Chen Chen, Lingjia Li, Yuyan Xie, Tongyun Liu, Wei Cun
Published 2016-08-15
Cite as Chin J Dermatol, 2016, 49(8): 573-577. DOI: 10.3760/cma.j.issn.0412-4030.2016.08.011
Abstract
ObjectiveTo study the effects of semaphorin 5A(SEMA5A)gene silencing by lentivirus-mediated short hairpin RNA(shRNA)on biological activity of malignant melanoma cell line A375.
MethodsTwo pairs of interference sequences for SEMA5A gene(shRNA1 and A375-shRNA2)and a pair of control interference sequences were designed to build lentiviral vectors, which were then transfected into HEK293T cells to gain lentivirus. A375 cells were divided into three groups: experimental group(A375-shRNA1 and A375-shRNA2 cells)transfected with the lentivirus containing shRNA1 or shRNA2, negative control group(A375-con cells)transfected with that containing the control shRNA, and blank control group(A375 cells)receiving no transfection. The A375 cells with stable knockdown of SEMA5A gene expression were screened by puromycin. Subsequently, reverse transcription-PCR and Western-blot analysis were performed to detect mRNA and protein expressions of Semaphorin 5A in these cells, and methyl thiazolyl tetrazolium(MTT)assay was applied to evaluate the growth of cells. The scratch assay and invasion assay were conducted to estimate migration and invasion ability of cells.
ResultsThe lentivirus containing the SEMA5A-targeting shRNAs or control shRNA was successfully transfected into A375 cells, and stably transfected cells were gained after puromycin selection. The expressions of semaphorin 5A mRNA and protein in the A375-shRNA2 cells were significantly reduced compared with those in the A375-con and A375 cells(all P < 0.05). MTT assay showed that the growth of A375-shRNA2 cells was significantly slower than that of A375-con and A375 cells(both P < 0.05), while there was no significant difference in the growth rate between A375-con and A375 cells(P > 0.05). The scratch assay showed that there was no obvious cell migration into the scratch in the experiment group, whereas the scratch was almost covered by cells in the negative control group and blank control group. The invasion assay showed that the number of A375-shRNA2 cells passing through the Transwell chamber was significantly smaller than that of A375 and A375-con cells(both P < 0.05), while there was no significant difference between that of A375 and A375-con cells(P > 0.05).
ConclusionThe silencing of SEMA5A gene by lentivirus-mediated shRNA could effectively down-regulate the expression of semaphorin 5A, and inhibit the growth, invasion and migration of A375 cells.
Key words:
Melanoma; Semaphorin 5A; Lentivirus; RNA, small interfering; Neoplasm invasiveness
Contributor Information
Li Zhang
Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming 650000, China
Yadong Li
Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650031, China
Chen Chen
Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650031, China
Lingjia Li
Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming 650000, China
Yuyan Xie
Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming 650000, China
Tongyun Liu
Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming 650000, China
Wei Cun
Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650031, China
