A kind of siRNA of silencing type IV collagen expression and its application
A collagen and silencing technology, applied in the field of RNA silencing, can solve the problems of systematic research on tumor infiltration and migration without tumor's own secretion of ECM, inhibition of tumor infiltration and migration, and lack of research reports.
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Embodiment 1
[0038] Example 1 Cell Culture and Treatment
[0039] Purpose of the experiment: To prepare cell samples for subsequent experiments.
[0040] Experimental materials: Liver cancer cell line SMMC-7721 was purchased from Subcomb Biotechnology Co., Ltd. (Shanghai, China); liver cancer cell line HepG2 and breast cancer cell line MDA-MB-231 were purchased from the American Germplasm Collection Center (ATCC, VA , United States) obtained. All cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, NY, USA).
[0041] Experimental procedure: All cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, containing 5% carbon dioxide concentration, under humid conditions.
Embodiment 2
[0042] siRNA preparation and transfection of embodiment 2 liver cancer cells
[0043] Purpose of the experiment: To prepare liver cancer cells knocked down by CoL4α1, PITPNM3, and Snail genes for subsequent experiments.
[0044] Experimental materials: HepG2 mediated by liposome-mediated liver cancer cell transfection, SMMC-7721, serum-free medium Opti-mem I (Invitrogen, CA, USA), Lipofectamine2000 reagent (Invitrogen, CA, USA), siRNA produced by Ribobio Synthesized by Science and Technology (Guangzhou, China), the base sequence is shown in Table 1 below.
[0045] Table 1 Oligonucleotide sequences of siRNAs
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[0048] Experimental steps:
[0049] 1) Inoculate cells into a 6-well plate one day before transfection, so that the cell density is about 60% on the day of transfection, and culture the cells in 10% FBS DMEM medium without antibiotics. Remove the medium before adding siRNA, and add 400ul serum-free DMEM to each well to remove the influence of se...
Embodiment 3
[0053] Example 3 CCL18 Promotes the Expression of Type IV Collagen in Tumor Cells
[0054] Purpose of the experiment: To detect the ability of the cells obtained in Examples 1 and 2 to express collagen IV under the action of CCL18, and to verify the inhibitory effect of siRNA on the expression of collagen IV in liver cancer cells mediated by CCL18.
[0055] Experimental materials: recombinant human CCL18 cytokine (PeproTech, NJ, USA), DMEM medium (DMEM, Gibco, NY, USA), fetal bovine serum (FBS, Gibco, NY, USA), rabbit anti-human Collagen IV polyclonal antibody (Cell Signaling technology, MA, U.S.), rabbit anti-human GAPDH polyclonal antibody (Cell Signaling technology, MA, U.S.), PVDF membrane (Millipore, MA, U.S.), protein marker (Thermo, MA, U.S.), skimmed milk powder (Erie, China), phenylmethylsulfonyl fluoride (PMSF, Shanghai, Shanghai, China), phosphatase inhibitor cocktail (China, Shanghai, China), RIPA cell lysate (China, Shanghai, China), ECL luminescent liquid (Thermo...
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