Small molecule noncoding RNA gene hsa-mir-101 and antineoplastic use thereof
A nucleic acid and drug technology, applied in the direction of anti-tumor drugs, DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of hsa-miR-101 and primary liver cancer research reports, and reduce proliferation and tumor formation Ability, Sensitivity-Promoting Effects
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Embodiment 1
[0014] Example 1: Detection of expression level of hsa-miR-101 in liver cancer tissue samples
[0015] Total RNA was extracted from liver cancer and non-cancerous liver tissues, RNA samples were mixed with RNA loading buffer at a ratio of 3:1, denatured at 80°C for 10 min, and then placed on ice for 5 min; Under the electric power of 10W, use 15% denatured polyacrylamide gel for about 40 minutes for electrophoresis separation; after the electrophoresis is completed, the corners of the gel are marked, and the gel and membrane are assembled in a semi-dry transfer tank in sequence, and the constant current 8mA / cm 2 Transfer to the membrane for 1 hour; remove the Hybond N+ nylon membrane and mark the corners, 70,000μJ / cm 2 UV cross-linking, and then dry-bake at 80°C for 2 hours; wet the nylon membrane with 2×SSC, spread it on the inner wall of the hybridization tube, drive away the air bubbles between the membrane and the tube wall, add the pre-hybridization solution, and put it ...
Embodiment 2
[0017] Example 2: Overexpression of hsa-miR-101
[0018] The small molecule RNA of hsa-miR-101 is introduced into the cells by inversion using Invitrogen’s transfection reagent Lipofectamine RNAi MAX. Taking 24-well plates as an example, other well plates can be scaled up according to the size of the wells. Or shrink: trypsinize cells, resuspend in DMEM without double antibody containing 10% FBS, make the cell density 3×10 4 cells / ml; dilute small molecule RNA in 100 μl opti-MEM, tap the tube wall to mix; add 1 μl Lipofectamine RNAi MAX to each tube, tap the tube wall to mix, and place at room temperature for 15 minutes; add RNAiMAX / siRNA diluent Into the 24-well plate, and then add 500 μl of cell diluent (to make the cell density about 30-50% confluent after 24 hours of transfection), mix well, culture in the incubator for a certain period of time, and then perform relevant function tests.
Embodiment 3
[0019] Example 3: Cell Apoptosis Detection
[0020] After the cells were treated as required, the culture supernatant was aspirated, fixed with 4% paraformaldehyde at room temperature for 15 min, stained with 1 μg / ml (final concentration) DAPI staining solution at room temperature for 5 min, and then observed under a Leica fluorescence microscope for nuclear morphology. Cells with chromatin condensation and DNA fragmentation are considered to be apoptotic cells, and the apoptosis rate is calculated according to the following method:
[0021] Apoptotic rate (%) = number of apoptotic cells / total number of cells (total number of cells ≥ 500)
[0022] We detected the effect of hsa-miR-101 on tumor cell apoptosis under the conditions of serum starvation and chemotherapy drug treatment, and found that the experimental group overexpressing hsa-miR-101 in HepG2 cells (ATCC, USA) was apoptotic after 48 hours of serum starvation. The cell death rate was ~2 times that of the negative co...
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