miRNAs for the diagnosis and treatment of hepatocellular carcinoma
A technology of hepatocellular carcinoma and reagents, applied in the field of miRNA
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Embodiment 1
[0050] Example 1 QPCR detection of miR-4772-3p expression in hepatocellular carcinoma
[0051] 1. Experimental materials
[0052] 1. Experimental reagents and instruments
[0053] miRNA first strand cDNA synthesis, biotechnology, catalog number B532451-0020,
[0054] miRNA fluorescence quantitative PCR kit, Shenggong, catalog number B532461-0002,
[0055] NanoVue Plus, BIOCHROM LTD, model number 28956057,
[0056] Fluorescence quantitative PCR instrument, Applied Biosystems, model ABI7300
[0057] 2. Experimental samples
[0058] Collect 32 cases of histopathologically diagnosed cancer tissues of patients with primary liver cancer and their corresponding> The 3cm para-cancer tissue samples, all samples were put into cryopreservation tubes within 30 minutes of surgical excision, put into liquid nitrogen tank quickly, and transferred to the laboratory -80℃ ultra-low temperature refrigerator.
[0059] Exclusion criteria: Having undergone anti-tumor treatment before surgery, suffering from othe...
Embodiment 2
[0093] Example 2 Verification of the diagnostic efficacy of biomarkers
[0094] 1. Data collection
[0095] The miRNA expression profile data of 342 cases of liver cancer tissues and 50 cases of adjacent tissues were collected from the TCGA database, and the expression level of miR-4772-3p in liver cancer tissues and adjacent tissues was analyzed.
[0096] 2. ROC curve analysis
[0097] Use the pROC package in R language to analyze receiver operating characteristics of miR-4772-3p, calculate the binomial accurate confidence space, and draw the ROC curve.
[0098] 3. Results
[0099] The ROC analysis results of miR-4772-3p are as follows figure 2 As shown, the AUC value of miR-4772-3p as the test result variable is 0.746, which has a higher area under the curve, indicating that the use of miR-4772-3p for the diagnosis of liver cancer has higher sensitivity and specificity, and has better Diagnostic efficiency.
Embodiment 3
[0100] Example 3 Detecting the expression level of miRNA in liver cancer cells
[0101] 1. Cell culture
[0102] Cultivation of human normal liver cell line L-02, low invasion and metastasis liver cancer cell line HepG2, and high invasion and metastasis liver cancer cell line Huh7. Cells are routinely cultured in 10% FBS, 100 units / ml penicillin and 100ug / ml chain In the 1640 cell culture medium of mycin, at 37℃, 5% CO 2 And cultivate under saturated humidity conditions. The cells were changed every 2d and passaged at 1:3.
[0103] 2. Extraction of cellular RNA
[0104] The TRIzol method was used to extract total cellular RNA.
[0105] Collect the cells in the log phase, add 1ml TRIzol, and mix well; use a 1ml syringe to repeatedly rupture the cells and cut the DNA, leave it at room temperature for 5 minutes, and the remaining steps are the same as in Example 1.
[0106] 3. Reverse transcription and fluorescence quantitative detection
[0107] The experimental process is the same as in ...
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