Construction of Human Nasophayngeal Cancer Cells Stablely Expressing miR-18a and miR-18a siRNA / 中山大学学报(医学科学版)

ABSTRACT

[Objective]To construct miR-18a overexpression and inhibition lentivirus vectors and to determine their effects on human nasopharyngeal cancer(NPC)cell line CNE1 and CNE2.[Methods]Designed the primers for Real-time polymerase chain(PCR)reaction to obtain the miR-18a premature gene.The premature gene and the siRNA oligo-nucleutides of miR-18a were connected to the lentivirus vector GV369 and GV280,respectively.The construction vectors were confirmed by DNA sequencing.Then,293T cell was infected with the vectors plus Helper 1.0 and pHelper 2.0 vec-tors to obtain recombinant lentivirus vector for miR-18a overexpression and inhibition. The NPC cell line CNE1 and CNE2 were infected with the successful recombinant lentivirus vectors.Puromycin was added to select the positive infect-ed cells. PCR method was used to detect the miR-18a expression level after infecting the recombinant lentivirus vector into the NPC cell line.[Results]A recombinant lentivirus vector expressing miR-18a interference oligonucleutides was obtained and confirmed by DNA sequencing.The virus titer was 3×108TU/mL,and the expression of its target gene ATM was downregulated in CNE1 and CNE2.A recombinant lentivirus vector expressing miR-18a premature gene was obtained and confirmed by DNA sequencing. The virus titer was 3×108TU/mL,and the miR-18a was overexpressed in CNE1 (20.3 fold upregulation,P<0.01)and CNE2(122.5 fold upregulation,P<0.01),and its target gene ATM was downregu-lated.[Conclusions]The miR-18a overexpression and suppression lentivirus vectors are successfully constructed.These vec-tors could alter the expression level of miR-18a in NPC cell line significantly,and provide a stable cell line for functional studies in the future.