Applications of lncrna-d63785 and its interferents and products using it
A technology of lncrna-d63785 and siRNA-d63785, which is applied in the field of application and its products, can solve the problems that the pathogenesis of gastric cancer is not completely clear, and the prevention, diagnosis and treatment of gastric cancer cannot achieve satisfactory results, so as to promote cell Effects of apoptosis and inhibition of cell proliferation
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Embodiment 1
[0081] Example 1 lncRNA-D63785 expression detection
[0082] In this example, the clinical gastric cancer tissues and their paracancerous tissues were freshly isolated tumor tissues from No. 401 Hospital of the People's Liberation Army, which were cut into small tissue pieces and frozen at -80°C for later use.
[0083] Total RNA was extracted from gastric cancer tissue and its paracancerous tissues and cells using Trizol reagent method, and the expression level of lncRNA-D63785 was detected by real-time fluorescent quantitative PCR technology. The full length of lncRNA-D63785 amplified by PCR was 185bp, specifically SEQ ID NO.1 sequence shown.
[0084] The PCR reaction system is calculated as 50 μL, and the PCR conditions are as follows: 95°C for 3 min, one cycle; 95°C for 30 sec, 56°C for 30 sec, 72°C for 1 min, a total of 30 cycles; finally, 72°C for 5 min;
[0085] The PCR primers were designed as follows:
[0086] Upstream primer: 5'-TTGCTGCTGACACGCCG-3' (SEQ ID NO.3); ...
Embodiment 2
[0091] Example 2 The experiment of interfering substances of lncRNA-D63785 inhibiting the proliferation of gastric cancer cells
[0092] In this embodiment, the gastric cancer cell line BGC823 provided in Example 1 is randomly divided into three groups, which are respectively a blank group, a control group and an experimental group, and each group has three repetitions (the cell amount is about 1×10 6 ).
[0093] Among them, the blank group BGC823 was only cultured with normal cell culture medium.
[0094] In the experimental group, gastric cancer cells were transfected with lncRNA-D63785 interferor (transfected by liposome lipfectin2000 according to the kit operation instructions, purchased from Invitrogen Company), and normal medium was replaced after 4 hours of transfection (culture system: 200 μL) , continue to cultivate for 12h, 24h, 48h and 48h.
[0095] The treatment method of the control group was the same as that of the experimental group, only the interfering subst...
Embodiment 3
[0104] Example 3 The interference of lncRNA-D63785 inhibits the invasion and migration ability of gastric cancer cells
[0105] In this example, the Transwell method was used to detect the invasion ability of cells. In this embodiment, BGC823 is randomly divided into three groups, which are respectively blank group, control group and experimental group, and each group has three repetitions (the cell amount is about 1×10 6 ).
[0106] Among them, the blank group BGC823 was only cultured with normal cell culture medium.
[0107] The experimental group was coated with 50 mg / L Matrigel 1:8 dilution solution on the upper surface of the bottom membrane of the Transwell chamber, and air-dried at 4°C; after the lncRNA-D63785 interferor was treated for 12 hours, the cells were digested, and the cells were digested with PBS and serum-free medium successively. Wash once, suspend the cells with serum-free medium, count, and adjust the concentration to 2×10 5 / mL.
[0108] The treatmen...
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