Application of mir-1292 and its target genes in the prevention and treatment of osteosarcoma metastasis
1. The technology of mir-1292 and osteosarcoma, applied in the field of molecular biology, can solve the problems affecting the expression of target genes
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Embodiment 1
[0062] Example 1 Collection of samples and extraction of total RNA
[0063] 5 patients with metastatic osteosarcoma and 10 normal controls. Patients with metastatic osteosarcoma (5 cases, A1-A5, see Table 1 for clinical information) and the control group (10 people) were required to fast for at least 12 hours, and 10ml of intravenous injection was drawn at room temperature from 7:00 to 8:00 the next morning. Blood was collected in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, PBMCs were extracted from peripheral blood mononuclear cells, 1ml Trizol reagent (Invitrogen Company) was added, mixed well, and the samples were stored at -80°C for RNA extraction. All blood samples and pathological results should be authentic and reliable, the study was approved by the ethics committee, and the patients gave informed consent.
[0064] RNA extraction standards: RNA purity: OD260 / 280≧1.8, 28S / 18S≧1; RNA integrity: RIN value≧7.0. RNA integrity detection method: Agilent 2100...
Embodiment 2
[0065] Example 2 Sequencing and data analysis
[0066] Sequencing: mRNA and miRNA were sequenced using llumina Hiseq2500 / Miseq second-generation high-throughput sequencing technology, and data processing was completed through the processes of removing joints, removing low-quality, and decontaminating to obtain the final data.
[0067] The miRNA and mRNA raw data were background-corrected by transcriptome data analysis software, and then the t-test was performed to obtain the P value, and then the Fisher test was used to combine the P values to screen for differentially expressed miRNA and mRNA. The p value was set < 0.01, and a total of 12 differentially expressed miRNAs were screened out, including 1 miRNA whose expression level was up-regulated (miR-1292-5p), and 11 miRNAs whose expression level was down-regulated. During the analysis process, the P value was set to <0.05, and a total of 425 differentially expressed mRNAs were screened out, including 324 genes with up-regu...
Embodiment 3
[0068] Example 3 Detection of miR-1292-5p and DST gene expression in osteosarcoma tissue by Real-time PCR
[0069] 1 sample collection:
[0070] The peripheral blood of 78 patients with metastatic osteosarcoma tumors and 53 healthy controls came from the hospital (collection time from January 2013 to February 2015).
[0071] 2 total RNA extraction:
[0072] Removal of RNase for related experimental items:
[0073] ① Rinse and soak all glassware with DEPC before use, 120°C for 20 minutes under high pressure, and 180°C for more than 2 hours.
[0074] ②Plastic utensils (such as: EP tubes / tips) need to be soaked in 0.1% DEPC water overnight before use, and then the liquid is controlled to dry, 120°C and high pressure for 20 minutes, and dried in an oven for later use.
[0075] leukapheresis
[0076] (1) Take 2m1 anticoagulated peripheral blood (blood collection time does not exceed 3h);
[0077] (2) Add an equal volume of sterile PBS to the peripheral blood and mix thoroughly...
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