pri-mir-302/367重组慢病毒载体的构建及其下游调节基因的鉴定

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

摘要:

目的：构建可诱导表达pri-mir-302/367 的慢病毒载体，并研究miR-302/367家族基因miＲ-302a/b/c/d和miＲ-367及其下游调节基因OCT4、SOX2和NANOG在 HeLa细胞中的诱导性表达。方法：以正常人基因组DNA为模板，PCR扩增pri-mir-302/367基因。经双酶切后连接入pLV-TRE质粒，构建pLV-TRE-302慢病毒重组体，酶切及测序予以鉴定。将重组质粒和辅助质粒共同转染293 FT细胞，并测定病毒滴度。用病毒转染HeLa细胞，强力霉素（doxycline，DOX）诱导后，通过real-time PCR测定miR-302/367家族基因及其下游调节基因的表达。实验分为诱导组（DOX+）、非诱导组（DOX-）和空白组（blank）。结果：酶切及测序证实重组慢病毒载体pri-mir-302/367构建成功，病毒滴度为5×106 TU/ml；DOX诱导72 h后，real-time PCR结果显示诱导组高于非诱导组和空白组（P＜0.05，其中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），而非诱导组与空白组相比差异无统计学意义（P ＞0.05，其中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：Pblank vs. DOX-=0.051；SOX2：Pblank vs. DOX-=0.060；NANOG：Pblank vs. DOX-=0.078）。结论：成功构建了携带人miR-302/367可控性慢病毒载体，为后续的重编程研究奠定了实验基础。

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.