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Small interference RNA aimed at hsa_circ_0000478 gene and applications thereof

A technology encoding gene and colon cancer, applied in the field of small interfering RNA, can solve the problem of failure to clarify its pathogenesis, and achieve the effects of inhibiting cell invasion and migration, reducing proliferation rate and promoting early apoptosis

Active Publication Date: 2019-05-03
广州市番禺区中心医院
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although predecessors have done a lot of work on colon cancer, their pathogenesis has not yet been elucidated. Therefore, the research on the pathogenesis of colon cancer still needs to be further deepened to provide a basis for the diagnosis, prognosis and scientific treatment of colon cancer. Finally, it can effectively control the pathogenesis of colon cancer, improve the prognosis of patients, and reduce the economic burden of patients

Method used

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  • Small interference RNA aimed at hsa_circ_0000478 gene and applications thereof
  • Small interference RNA aimed at hsa_circ_0000478 gene and applications thereof
  • Small interference RNA aimed at hsa_circ_0000478 gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Screening of hsa_circ_0000478 high expression cell line

[0050] RT-PCR and the primers used are as follows:

[0051] hsa_circ_0000478 upstream primer F: 5'-cctattatgctctatcagcgct-3';

[0052] hsa_circ_0000478 downstream primer R: 5'-caatccggatggtcacagaa-3';

[0053] Product size: 127bp;

[0054] β-actin upstream primer F: 5'-CATGTACGTTGCTATCCAGGC-3';

[0055] β-actin downstream primer R: 5'-CTCCTTAATGTCACGCACGAT-3';

[0056] Product size: 250bp;

[0057] RT (reverse transcription) conditions: each take 5 μg of total cellular RNA and use Geneseed@Enzyme Mix (product of Invitrogen) to complete cDNA first-strand synthesis.

[0058] PCR conditions: Take 5 μL of the first strand of cDNA synthesized by reverse transcription as a template, 20 μL PCR reaction system: 0.5 μL of upstream primers, 0.5 μL of downstream primers, Geneseed@qPCR SYBE Green PCR Master Mix 10 μL, dH 2 O 4.0μL; PCR reaction conditions: pre-denaturation at 95°C for 5min; denaturation at 95...

Embodiment 2

[0060] Example 2 Detection of relative expression level of hsa_circ_0000478 after transient transfection of siRNA (si_circ_0000478) in LOVO cells

[0061] One day before transfection, 5*10 5 Cells were seeded on a 6-well plate with 2 mL of complete medium, and the confluency of the cells reached 70-90% before transfection. Cell grouping: blank control group (cells without any intervention), negative control group (only lipofectamine liposomes were added, purchased from thermofisher scientific company, model 11668027), and experimental group was added 2.5 μL siRNA (final concentration 50 nM) to 100 μL serum-free medium ), and mix gently. Mix the lipofectamine reagent evenly, dilute 4 μL lipofectamine reagent with 100 μL serum-free medium, mix gently, and place at room temperature for 5 minutes. The diluted siRNA and lipofectamine reagent were mixed, mixed gently, and left at room temperature for 20 minutes to form siRNA-lipofectamine complexes. Add 200 μL of plasmid-lipofect...

Embodiment 3

[0063] Example 3 Effect of si_circ_0000478 on LOVO proliferation

[0064] Cell grouping is the same as in Example 2. LOVO cells were 1×10 per well 4 Each cell was inoculated into a 96-well plate, and then continued to culture for about 24 hours, 48 ​​hours, and 72 hours. Add 50 μL of 1×MTT solution to each well, and place it in an incubator for 4 hours to reduce MTT to formazan. Aspirate the supernatant, add 150 μL DMSO (dimethyl sulfoxide) to each well to dissolve formazan, and shake well with a plate shaker. Measure the optical density of each well at 490nm with a microplate reader, and then calculate the inhibition rate of cell proliferation. Proliferation inhibition rate (%)=[(OD value of blank control group-OD value of experimental group) / OD value of blank control group]×100%.

[0065] After si_circ_0000478 transfected cells (24, 48, 72 hours), it can effectively inhibit the proliferation of LOVO cells, and the proliferation inhibition rates were (10.6±0.78%, 25.65±2.1...

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Abstract

The present invention discloses a small interference RNA aimed at hsa_circ_0000478 gene and applications thereof. The invention successfully obtains the siRNA of hsa_circ_0000478, and the transient transfection of LOVO cells can effectively interfere with the expression of the hsa_circ_0000478 gene, wherein the interference efficiency reaches (90.25+ / -0.96)%. The result of transfection of colon cancer LOVO cells shows that after being interfered, the hsa_circ_0000478 gene can significantly reduce the proliferation rate of LOVO cells, promote early cell apoptosis and inhibit cell invasion and migration. The present invention proves for the first time that the knockout of hsa_circ_0000478 expression has direct correspondence with the proliferation, invasion and migration of colon cancer cells, and the hsa_circ_0000478 gene can be used as a therapeutic target for colon cancer. The siRNA of the present invention is also expected to be applied to the treatment of colon cancer as a biotherapeutic technique.

Description

technical field [0001] The present invention relates to small interfering RNA technology, in particular to a small interfering RNA targeting hsa_circ_0000478 gene and its application. Specifically, it relates to an anti-colon cancer small interfering RNA (siRNA) targeting the hsa_circ_0000478 gene sequence, providing an anti-colon cancer hsa_circ_0000478 small interfering RNA sequence, which has targeted inhibition of colon cancer cell proliferation, invasion, migration, So as to achieve the effect of inhibiting the development and metastasis of colon cancer. Background technique [0002] Colon cancer is one of the most common malignant tumors of the digestive tract in the world, which seriously threatens human life and health. Its incidence rate ranks third among common malignant tumors, and its case fatality rate also ranks third among cancer deaths. The occurrence and development of colon cancer is a multi-step, multi-stage and multi-gene process, in which the abnormal e...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/113A61K31/713A61P35/00
Inventor 何金花黎毓光韩泽平左继东陈顺仪何梦玲
Owner 广州市番禺区中心医院
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