Construction of the lentivirus vector containing the miR-424 gene and its influence on proliferation of cervical cancer cell line
10.3969/j.issn.1671-8348.2014.31.016
- VernacularTitle:miR-424慢病毒表达载体的构建及其对宫颈癌细胞增殖的影响
- Author:
Qing LI
;
Yuxin YANG
;
Nan DAI
;
Xiaoyan DAI
;
Tao REN
;
Dong WANG
;
Yi QING
- Publication Type:Journal Article
- Keywords:
Hela cells;
cell proliferation;
miR-424;
lentivirus expression plasmid
- From:
Chongqing Medicine
2014;(31):4165-4168
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the lentivirus vector containing the hsa‐miR‐424 gene ,and identify the expression level of miR‐424 in cells .Research the influence of hsa‐miR‐424 on proliferation of cervical cancer Hela cell line .Methods Using the human genomic DNA as template to design the upper and lower primers for synthesis of miR‐424 ,and amplifying the target fragment by polymerase chain reaction (PCR) .Recover the products and conduct sequencing after connecting it into the pMD18T vector .Ampli‐fy the product by PCR template as pMD18T‐miR424 ,and insert the fragment expressing pMD18T‐miR424 into the vector of pLen‐tis‐CMV‐GFP‐MCS‐PGK‐PURO after enzyme cutting to construct the pLentis‐CMV‐GFP‐miR424‐PGK‐PURO .Package the com‐pound with pMD2 .G and pSPAX2 in 293T cell to produce the lentivirus ,and using the supernatant containing lentivirus to infect the Hela cell line .Results The sequencing result proved the sequence of miR‐424 in plasmid vector was correct ,which proved the construction of lentivirus was successful and the target lentivirus was obtained .The expression of miR‐424 almost rise 60 times af‐ter infected the cervical cancer Hela cell by the carrier .The result of M TT method suggested :the cervical cancer Hela cell lines have slowed proliferation with infection miR‐424 lentivirus .Conclusion The miR‐424 lentivirus vector was constructed successfully and the high efficacy expression miR‐424 cell line was established and stable .The cervical cancer Hela cell were infected with the super‐natant containing lentivirus ,inhibited the proliferation of Hela cell successfully ,and laid a good foundation for subsequent research .
