Construction of pri-mir-302/367 lentiviral vector and verification of its downstream regulatory genes

Abstract:

Objective：To construct inducible lentiviral vector containing human miR-302/367 and study its gene expression of miR-302a/b/c/d and miR-367 and downstream gene expression of OCT4，SOX2 and NANOG in HeLa cells. Methods：pri-mir-302/367 gene was amplified by PCR from human genomic DNA and was cloned into pLVX-TRE vector to construct the recombinant lentiviral vector. The ligation was analyzed by digestion and confirmed by sequencing. Then the recombinant vectors were transfected into 293FT cells and the lentiviral viruses were harvested from 293FT cells. After determining the titer，viruses were used to infect HeLa cells. RNA was extracted for real-time PCR to detect the expressions of miR-302/367 and its downstream genes and the expression were devict－ed into DOX+ group，DOX- group and blank group. Results：Digestion and sequencing demonstrated successful construction of recom－binant inducible lentiviral vector miR-302/367. The virus titers were 5×106 TU/ml. After 72 h introduction with DOX，the results of real-time PCR shown that miR-302/367 and pluripotent related genes（OCT4，SOX2 and NANOG） were significantly upregulated in DOX group compared with those in blank group and DOX- group（P＜0.05，miR-302a：PDOX vs. DOX-=0.032，PDOX vs. blank=0.048；miR-302b：PDOX vs. DOX-=0.001，PDOX vs. blank=0.001；miR-302c：PDOX vs. DOX-=0.000，PDOX vs. blank=0.000；miR-302d：PDOX vs. DOX-=0.002，PDOX vs. blank=0.047；miR-367：PDOX vs. DOX-=0.001，PDOX vs. blank=0.001；OCT4：PDOX vs. DOX-=0.000，PDOX vs. blank=0.001；SOX2：PDOX vs. DOX-=0.000，PDOX vs. blank=0.000；NANOG：PDOX vs. DOX-=0.000，PDOX vs. blank=0.000），while there was no significant difference between DOX group and blank group（P ＞0.05，miR-302a：Pblank vs. DOX-=0.057；miR-302b：Pblank vs. DOX-=0.832；miR-302c：Pblank vs. DOX-=0.445；miR-302d：Pblank vs. DOX-=0.979；miR-367：Pblank vs. DOX-=0.161；OCT4：P blank vs. DOX-=0.051；SOX2：Pblank vs. DOX-=0.060；NANOG：Pblank vs. DOX-=0.078）. Conclusions：The recombinant in－ducible lentiviral vector containing pri-mir-302/367 can be successfully constructed，which lay a solid foundation for further study the reprogramming of miR-302/367 in the following experiments.