Biomarker related to occurrence and development of gastric adenocarcinoma
A gastric cancer and drug technology, applied in the field of biomarkers, can solve the problem that the functional mechanism of 1ncRNA is not understood by humans
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Embodiment 1
[0057] Example 1 Screening of gene markers associated with gastric cancer
[0058] 1. Sample collection
[0059] The cancer tissues and corresponding paracancerous tissue samples of 4 cases of gastric adenocarcinoma were collected for high-throughput sequencing. All patients did not receive chemotherapy, radiotherapy or endocrine therapy before operation.
[0060] 2. RNA sample preparation and quality analysis
[0061] Total RNA was extracted using Tiangen Animal Tissue Total RNA Extraction Kit (catalogue number: DP431), and the steps are detailed in the instructions.
[0062] 1) Homogenization treatment
[0063] Add 300μl Lysis Solution RL for every 10-20mg tissue, and grind the tissue thoroughly with a grinding pestle; then add 590μl RNase-Free ddH to the homogenate 2 O and 10 μl Proteinase K, mix well and treat at 56°C for 10-20min.
[0064] 2) Centrifuge at 12,000rpm for 2-5min, and take the supernatant for the following operations.
[0065] 3) Slowly add 0.5 times th...
Embodiment 2
[0097] Example 2 QPCR sequencing to verify differential expression of RP11-320G24.1 gene
[0098] 1. The differential expression of the RP11-320G24.1 gene was verified by large sample QPCR of the cancer tissue samples and paracancerous tissue samples collected from 31 gastric adenocarcinoma patients according to the collection method in Example 1.
[0099] 2. RNA extraction
[0100] The total RNA was extracted using Tiangen Animal Tissue Total RNA Extraction Kit (Cat. No. DP431). See Example 1 for specific steps.
[0101] 3. QPCR
[0102] Primers were designed according to the gene sequences of RP11-320G24.1 and GADPH, the primer sequences are as follows:
[0103] RP11-320G24.1:
[0104] Forward primer: 5′-GTATAATCTGCCACTTCT-3′ (SEQ ID NO.1)
[0105] Reverse primer: 5'-GTCTTCCACATCATTCTA-3' (SEQ ID NO.2)
[0106] GAPDH:
[0107] Forward primer: 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO.3)
[0108] Reverse primer: 5'-GAGCCCCAGCCTTTCTCAT-3' (SEQ ID NO.4)
[0109] The Quant ...
Embodiment 3
[0114] Example 3 Silencing of RP11-320G24.1 and its effect on gastric adenocarcinoma cells
[0115] 1. Transient transfection
[0116] The siRNA interference fragment targeting RP11-320G24.1 gene was designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. The negative control is general siRNA-NC, RP11-320G24.1-siRNA group: 5′-AUAUACCUAAUUAUGUGUCAU-3′ (SEQ ID NO.5); 5'-GACACAUAAUUAGGUAUAUUU-3' (SEQ ID NO.6). Gastric adenocarcinoma MGC-803 cells were inoculated in six-well plates 24 hours before transfection. When the cell density reached 50%-70% confluence, the medium was replaced with serum-free medium. Mix the diluted interference fragment with Lipofectamine TM 2000 Liposomes were gently mixed and incubated at room temperature for 20 minutes to form a transfection complex; then the above mixture was added to the cell culture medium, mixed gently, and cultured in a 5% CO2, 37°C incubator for 6 to 8 hours Replace with complete medium. After 48h, the in...
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