Constructing MiR-149 Suppression Lentivirus Vector And Investigating The Effect Of Low MiR-149 Expression On Proliferation And Migration Of Melanoma Cells

Objecfive Malignant melanoma is a highly aggressive skin cancer with high incidence worldwide.It originates from melanocytes.The incidence of melanoma is increasing worldwide,however,beyond surgical removal of the tumor,there is currently no curative therapy available,especially for its advanced stages.The molecular mechanisms leading to the development and progression of melanoma are the focus of intense investigation.Many studies have shown that miRNAs plays an important role in the development and progression of tumors,including melanoma.MiR-149 main as tumor suppressor genes in human cancer.This experiment that to construct miR-149 suppression lentivirus vector and investigate the effect of low miR-149 expression on proliferation and migration of melanoma cells.Methods Obtained by miRBase and NCBI databases for miR-149 sequence,the pair of primers were designed and synthesized.Double-stranded DNA oligonucleotide fragments were obtained by annealing response,the target gene was ligated with lentivirus vector FG12,the lentiviral plasmid was identified by double enzyme digestion and sequencing analysis.Lentivirus was generated by cotransfection with the above construct with packaging plasmids(p MDLg/p RRE?p RSV-Rev?p HCMV/G)into 293 T cell,lentivirus was harvested,the virus drops were measured.Getting multiplicity of infection,that is the ratio of virus and cell number,according to multiplicity of infection,lentivirus were used to infect Mel-RM cells,then real-time polymerase chain reaction(PCR)method was used to detect the miR-149 expression level.MTT assay was used to examine the cell proliferating capacity,cell migration assay was used to examine the cell migration.Results The successful construction of miR-149 recombinant lentivirus vectors was confirmed by DNA sequencing.Fluorescence microscope observation showed that the293 T packaging cells and melanoma cells expressed green fluorescence.The titer of concentrated virus was 3.2 × 1010TU/ml.Melanoma cells infected with lentiviruses,the expression level of miR-149 reduced effectively.MTT assay results showed that compared with the control group,experimental group cell proliferate more slowly.The migration results showed that experimental group cell migrated more slowly.Conclusion miR-149 suppression lentivirus vectors was successfully construct.Low miR-149 expression could suppress proliferation and migration of melanoma cells.This experiment for further study on the development of melanoma cells provide the basis.