MiR125 and use thereof in regulating protooncogene Pokemon expression

A proto-oncogene, a technology for use, applied in the field of biomedical therapy

Inactive Publication Date: 2015-08-19
HUAIYIN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It is far from enough to suggest many possible complex factors and the specific molecular mechanism in the pathogenesis of liver cancer. In view of the importance of Pokemon as a target gene, there is no report on whether miR-125 is involved in regulating the expression of Pokemon. The experimental results The acquisition will provide important guidance for the development of new drugs for liver cancer

Method used

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  • MiR125 and use thereof in regulating protooncogene Pokemon expression
  • MiR125 and use thereof in regulating protooncogene Pokemon expression
  • MiR125 and use thereof in regulating protooncogene Pokemon expression

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1: Construction of the dual fluorescent reporter gene carrier of Pokemon gene

[0020] A kit (Gibco) was used to extract total RNA from liver cancer cells and synthesize cellular cDNA. For specific operation methods, please refer to the kit instruction manual. The 3'UTR fragment containing the miR125 target of Pokemon gene mRNA was constructed, and the specific primer sequence was designed. The upstream primer sequence was: 5'-GGTCGCAGAAGGTGGAGA-3', and the downstream primer sequence was: 5'-GGTCGTAGTTGTGGGCAAA-3'. The PCR reaction system was set to 25 μL, including 1 μL template, 12.5 μL dNTP mix, 0.5 μL each of upstream primer and downstream primer (20 μmol / l), ddH 2 O 10.5 μL. The PCR reaction parameters were pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, and 30 cycles. The PCR fragment was cloned into the luciferase reporter gene vector pMIR-REPORT, identified by enzyme diges...

Embodiment 2

[0021] Example 2: Construction of liver cancer cell lines stably overexpressing miR-125

[0022] According to the cDNA sequence of human miR125 included in Genebank and the introduction of XhoI (5'), Eco R I (3') two enzyme cutting sites, designed two primers upstream and downstream: miR125 upstream primer sequence: 5'-CTATGTTTGAATGAGGCTTCAG-3'; miR125 downstream primer sequence: 5'-CGCGTCGCCGCGTGTTTAAACG-3'. Perform PCR reaction. The PCR reaction system is the same as in Example 1. The PCR reaction conditions are: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 60 s, 30 cycles. Using XhoⅠ, Eco R Ⅰ Restriction digestion of the target fragment and pEGFP-N3 vector for digestion and ligation, and sequencing confirmed that the sequence of the product pEGFP-miR125 was correct. The constructed pEGFP-miR125 and pEGFP-N3 empty plasmids were transfected into HepG2 cells that had been plated in a 6-well plate by liposome Lipof...

Embodiment 3

[0023] Example 3: Double fluorescent reporter gene experiments verify that Pokemon is the target gene of miR125

[0024] Liver cancer cell line HepG2-miR125 or HepG2 control cells that have stably overexpressed miR-125 were treated according to 1.5×10 5Inoculate each well into a 24-well plate and culture for 24 hours; use Lipofectamine2000 reagent to co-transfect pMIR-Pokemon-3'UTR or pMIR-control empty vector with control plasmid pRL-TK into HepG2-miR125 or HepG2-control cells; transfection After 48 h, the cell culture supernatant was aspirated and washed once with PBS; 200 μL cell lysate was added to each well, and centrifuged at 12,000 rpm for 10 min; 20 μL supernatant was mixed with 100 μL luciferase assay reagent II (Luciferase Assay Reagent II, LAR II) and mixed in TD-20 / 20 fluorometer detects firefly luciferase activity, records as the first reading, then adds 100 μL Stop & Glo reagent, mixes well within 10 min, and then takes the second reading to detect Renilla luci...

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Abstract

The invention discloses MiR125 and use thereof in regulating protooncogene Pokemon expression. The MiR125 is taken as a target spot for treating liver cancer; the target spot is used for preparing medicines for treating the liver cancer.

Description

technical field [0001] The invention relates to the field of biomedical treatment, in particular to the role of miR125 and the expression of proto-oncogene Pokemon regulated by miR125 in the treatment of liver cancer. Background technique [0002] Although radical resection and adjuvant therapy have been performed in early-stage cancer patients, they still have a high risk of recurrence and metastasis. Gene therapy will become a new tumor treatment method in addition to surgery, radiotherapy and chemotherapy, especially in improving the sensitivity of chemotherapy and radiotherapy, and reducing tumor recurrence and metastasis. An important part of comprehensive tumor treatment. The selection of target genes plays a crucial role in the efficacy of gene therapy. Among them, the proto-oncogene Pokemon is located upstream of many tumor suppressor genes and proto-oncogenes, and regulates the occurrence and development of tumors through the ARF-HDM2-p53 and Rb-E2F pathways, enab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7088A61K45/00A61P35/00A61P1/16
Inventor 孔晶柳晓平
Owner HUAIYIN INSTITUTE OF TECHNOLOGY
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