Part:BBa_K1997007
let-7e
Let-7e is one of the founding members of the let7 family
Usage and Biology
let-7b and let-7e have the biggest difference with other let7, this two let7 have 4 different nucleotides with others.
There are 6 functional let7 target sites in the 3-prime UTR of HMGA2 (600698).1 The abnormal expression of let7b and let7e reduced HMGA2 expression and inhibit lung cancer cell from multiplication. Overexpression of the HMGA2 ORF without the 3-prime UTR rescued the growth-suppressive effect of let7 on lung cancer cells.1
People found a binding site for let7e and let7f in IL23R (607562). It has been proved that it is concerned with inflammatory bowel disease.2
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing
Sequencing
This part is sequenced as correct after construction.
PCR
Methods
The PCR is performed with Premix EX Taq by Takara.
F-Prime: 5’- GAATTCGCGGCCGCTTCTAGAATGC-3’
R-Prime: 5’- GGACTAGTATTATTGTTTGTCTGCC-3’
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.
Enzyme digestion test
Methods
After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with EcoRI and SpeI restriction endonuclease bought from TAKARA.
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
The result of the agarose electrophoresis was shown on the picture above.
Functional Test
In vitro transcription experiment of let-7e was performed.The Electrophoresis was performed on a 2％ Agarose glu.
The result of the agarose electrophoresis was shown on the picture below.
References
1.The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Lee YS, Dutta A. Genes Dev. 2007 May 1;21(9):1025-30. Epub 2007 Apr 16.
2.Cutting edge: a variant of the IL-23R gene associated with inflammatory bowel disease induces loss of microRNA regulation and enhanced protein production. Zwiers A, Kraal L, van de Pouw Kraan TC, Wurdinger T, Bouma G, Kraal G. J Immunol. 2012 Feb 15;188(4):1573-7. doi: 10.4049/jimmunol.1101494. Epub 2012 Jan 18.
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