Application of CLIC3 as lung adenocarcinoma diagnosis and treatment target
A lung adenocarcinoma and product technology, applied in the fields of treatment, prognosis prediction, and tumor diagnosis, can solve problems such as low diagnostic efficiency and failure to meet clinical needs
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Embodiment 1
[0065] Example 1 Screening of differentially expressed genes
[0066] 1. Materials:
[0067] The surgically resected cancer tissues and paracancerous tissues of 8 patients with primary lung adenocarcinoma were used as experimental samples. All cancer tissues were pathologically confirmed as lung adenocarcinoma. All patients with primary lung adenocarcinoma had not received radiotherapy and chemotherapy before operation, and the clinical data of all cases were complete.
[0068] 2. Obtaining tissue RNA
[0069] Total RNA was extracted from tissue samples, and the concentration and purity of the extracted RNA were detected by Nanodrop2000, the integrity of RNA was detected by agarose gel electrophoresis, and the RIN value was determined by Agilent2100. The total amount of RNA required for a single library construction is 5ug, the concentration is ≥200ng / μL, and the OD260 / 280 is between 1.8 and 2.2.
[0070] 3. Remove rRNA
[0071] A part (>24%) of the long non-coding RNAs i...
Embodiment 2
[0095] Example 2 Large sample verification screened out differentially expressed genes
[0096] Based on the results of the previous high-throughput transcriptome deep sequencing, we selected the CLIC3 gene for verification according to the size of the P value.
[0097] 1. Sample collection
[0098] According to the method of Example 1, 45 cases of lung adenocarcinoma tissues and corresponding paracancerous tissues were collected.
[0099] 2. Validation at the mRNA level
[0100] 2.1 Extract tissue RNA
[0101] Step is with embodiment 1.
[0102] 2.2 Reverse transcription
[0103] Reverse transcription using Primescript 1 st strand cDNA synthesis kit kit, the operation steps are as follows:
[0104] (1) Add the following reaction liquid in the microcentrifuge tube, as shown in Table 1:
[0105] Table 1 Reaction liquid
[0106]
[0107]
[0108] (2) Incubate at 70°C for 5 minutes, then rapidly cool to 4°C;
[0109] Add the following reagents into a microcentrifu...
Embodiment 3
[0133] Example 3 CLIC3 Gene Overexpression
[0134] 1. Plasmid construction
[0135] Amplification primers were designed according to the coding sequence of the CLIC3 gene, and the design of primers is well known to those skilled in the art. The coding sequence of the full-length CLIC3 gene was amplified from the cDNA library of adult fetal brain (clontech company, catalog number: 638831), the above cDNA sequence was inserted into the eukaryotic cell expression vector pcDNA3.1, and the obtained recombinant vector pcDNA3.1 was connected -CLIC3 was used in subsequent experiments.
[0136] 2. Culture and transfection of lung adenocarcinoma cells
[0137] 2.1 Cell culture
[0138] The lung adenocarcinoma cell line A549 was cultured in RPMI1640 medium and 10% fetal bovine serum.
[0139] 2.2 Cell transfection
[0140] (1) The day before transfection, 0.5-2*10 5 Tumor cells were suspended in 500 μl of antibiotic-free medium and seeded into 24-well culture plates.
[0141] (2)...
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