shRNA for for knocking down long-chain non-coding RNAMIR205HG in targeted manner and application of shRNA
A targeted and sequence-based technology, applied in the direction of retroRNA virus, DNA/RNA fragments, applications, etc., can solve the problem that the effect and mechanism of breast cancer response to estrogen have not been reported, and achieve the promotion of ER protein degradation, inhibition of The effect of responsiveness
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Embodiment 1
[0031] Example 1: Construction of a MCF7 cell line that reduces the expression of lncRNA-MIR205HG
[0032] 1. Materials
[0033] 1.1 Plasmids and cells
[0034] The lentivirus packaging system used in the present invention is PLKO.1 vector plasmid, pMDG packaging plasmid, psPAX2 packaging plasmid, PRTR packaging plasmid; ER +Breast cancer cell line MCF7 and human embryonic kidney epithelial cells (293T) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. In the DMEM medium of penicillin (100 μg / mL) and penicillin (100 U / mL), the culture conditions were 37°C, 5% CO 2 .
[0035] 1.2 Reagents
[0036] Restriction endonucleases EcoRI and AgeI, T4 DNA ligase, and competent DH5α were purchased from FermentasMBI Company of the United States; Plasmid Mini Kit I was purchased from OMEGA Company of the United States; plasmids were transfected with Lipofectamine TM 2000 was purchased from Invitrogen Company of the United States; Taq DNA polymerase was purc...
example 2
[0058] Example 2: Validation of lncRNA-MIR205HG in MCF7-KD-MIR205HG cell line and normal MCF7 cell line
[0059] 1. Materials
[0060] 1.1 cells
[0061] The MCF7 cell culture method used in the experiment is the same as that in Example 1.
[0062] 1.2 Reagents
[0063] EDTA-containing trypsin digestion solution, purchased from Shanghai Beyond Biotechnology Research Institute; Reagent was purchased from Invitrogen Company of the United States; Reverse Transcription Kit (DDR037A) Nanjing Nuoweizan Biotechnology Co., Ltd. SYRB Premix Ex Taq for real-time quantitative PCR (polymerase chain reaction) TM (TliRNaseH Plus) kit was produced by Nanjing Novizan Biological Co., Ltd. Real-time PCR specific primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.
[0064] 2. Method
[0065] 2.1 Extraction of total RNA
[0066] Take the constructed MCF7 cell line as the object, culture it in a 60cm dish, and when the cells grow to 70%-80% density, digest the cells with...
Embodiment 3
[0075] Example 3: Application of LncRNA-MIR205HG in the Estrogen Response Process of MCF7 Cells
[0076] 1. Materials
[0077] 1.1 cells
[0078] The control group MCF7 cell line and MCF7-KD-MIE205HG cell line used in the experiment are the same as in Example 2.
[0079] 1.2 Reagents
[0080] Real-Time PCR reagents were the same as in Example 2; estrogen was purchased from Sigma; DMEM medium without phenol red was purchased from Gibco; CF serum was purchased from Gibco; EDTA-free trypsin was purchased from KGI; apoptosis , Periodic detection kits were purchased from KGI Company; CCK8 detection kits were purchased from Biyuntian Company.
[0081] 2. Method
[0082] 2.1 Real-Time PCR experiment
[0083] 2.1.1 Cell estrogen-free treatment and stimulation Take the established MCF7 cell line as the object, culture it in a 60cm dish, and replace it with phenol red-free DMEM containing 5% CF serum when the cell density reaches 30%-40%. Base treatment, change the medium every 12...
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