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Application of a nucleic acid molecule

A technology of nucleic acid molecules and nucleotide sequences, which is applied in the application field of nucleic acid molecules and can solve problems such as lack of research

Active Publication Date: 2020-11-03
THE SECOND AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, a large number of studies on the regulation of miRNAs on HMGB1 focus on bacterial sepsis, but in Candida albicans sepsis, there is still a lack of research.

Method used

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  • Application of a nucleic acid molecule
  • Application of a nucleic acid molecule
  • Application of a nucleic acid molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 mmu-miR-146b-5p directly regulates the target gene HMGB1

[0082] ①Bioinformatics prediction

[0083] The binding site between mmu-miR-146b-5p and HMGB1 3'-UTR region:

[0084] According to the bioinformatics software miRWalk, DIANA-microT, miRanda, MiRSystem, miRDB, miRMap, miRNAsMap, Pictar, PITA, RNA22, RNAhybrid and Targetscan, the miRNAs interacting with the 3'-UTR region of the HMGB1 gene were predicted.

[0085] ② Plasmid construction

[0086] Target gene HMGB1 3'-UTR region sequence:

[0087] Through the bioinformatics database NCBI, the sequence of the mouse HMGB1 3'-UTR region containing the predicted binding site (gttctc, 1327-1332 nucleotides) was obtained, and cDNA was synthesized as a template, named wt-HMGB1 3'-UTR, The sequence is shown in SEQ ID No.13.

[0088] Point mutation HMGB1 3'-UTR region sequence:

[0089] The target gene HMGB1 3'-UTR region binding site (gttctc, 1327-1332 nucleotides) predicted by mmu-miR-146b-5p was subjected to...

Embodiment 2

[0135] Example 2 mmu-miR-146b-5p inhibits the expression level of the target gene HMGB1

[0136] ①Transfection of mmu-miR-146b-5p

[0137] To prepare OligoRNA:

[0138] OligoRNA sequences were purchased from Shanghai Yingwei Jieji Biotechnology Co., Ltd., and the sequences are shown in Table 3:

[0139] table 3

[0140]

[0141] Cell processing and grouping:

[0142] Candida albicans stimulated transfected macrophages with mimic NC, mmu-miR-146b-5p mimic, inhibitor NC and mmu-miR-146b-5p inhibitor for 36 hours, and divided into normal, C.albicans, mimic NC, mmu-miR-146b -5pmimic, inhibitor NC and mmu-miR-146b-5p inhibitor groups, collect samples for follow-up experiments.

[0143] Transfection of mmu-miR-146b-5p:

[0144](1) The extracted mouse primary peritoneal macrophages were divided into 3×10 6 Inoculate each well in a 6-well plate, add 2 mL of complete medium to each well, and place in an incubator (37°C, 5% CO 2 ) to incubate for 12h;

[0145] (2) Before tran...

Embodiment 3

[0216] Example 3 mmu-miR-146b-5p can inhibit the translocation of HMGB1 in macrophages

[0217] Cell cytoplasmic protein and nuclear protein extraction:

[0218] According to the instructions of Jiangsu KGI Cytoplasmic and Nuclear Protein Extraction Kit, the BCA method was used for protein quantification, aliquoted and stored at -80°C.

[0219] Western blot experimental steps are as in Example 2.

[0220] Cell Laser Confocal Microscopy:

[0221] (1) Divide macrophages into 5×10 cells 5 Inoculate each well in a 24-well plate containing glass slides, add 1 mL of complete medium, and inoculate in an incubator (37°C, 5% CO 2 ) for 12 hours;

[0222] (2) OligoRNA was transfected into cells and incubated for 36 hours, and inactivated Candida albicans was stimulated for 36 hours;

[0223] (3) After the cell culture is over, wash with PBS solution (1min×3 times);

[0224] (4) Add 1mL 4% paraformaldehyde into each well, fix at room temperature for 30min;

[0225] (5) Washing wit...

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Abstract

The invention specifically relates to application of a nucleic acid molecule, belonging to the field of biotechnology. According to the invention, changes of the expression profile of miRNAs in the primary peritoneal macrophages of mice infected with Candida albicans are determined so as to obtain miRNAs capable of regulating a gene HMG1B, so the regulation effect of the miRNAs on the HMGB1 during Candida albicans infection can be investigated and a novel idea is provided for treatment of Candida albicans infection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a nucleic acid molecule. Background technique [0002] Deep fungal infection (deep fungal infection, DFI) is a serious infectious disease, which refers to the fungal infection disease caused by pathogenic fungi invading the subcutaneous tissue, mucous membrane and internal organs, and infecting organs. Among them, Candida albicans is one of the most common pathogens of human deep fungal infection. In recent years, due to the long-term abuse of broad-spectrum antibiotics, the increasing invasive operations such as built-in medical devices and organ transplantation, the morbidity and mortality of Candida albicans infection have continued to rise. Although many antifungal drugs have been developed, due to serious Toxic and side effects, as well as the continuous increase of fungal drug-resistant strains, make the treatment of DFI a thorny problem in clinical practice...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C07K14/47A61K48/00A61K31/711A61K31/7105A61P31/10
CPCA61K31/7105A61K31/711C07K14/47C12N15/113C12N2310/10
Inventor 孙航吴传新程静王云英王姣焦
Owner THE SECOND AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIV
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