The miRNA related to genome stability of human umbilical cord blood mesenchymal stem cells and its application

A technology of genome stability and quality stem cells, applied in the field of genetic engineering, can solve the problem of lack of quality assessment of molecular markers, achieve good clinical application value, broad application prospects, and reduce risks.

Active Publication Date: 2019-06-25
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is of great significance to evaluate the genome stability of human umbilical cord blood mesenchymal stem cells before large-scale subculture, but there is a lack of suitable molecular markers for quality assessment at this stage

Method used

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  • The miRNA related to genome stability of human umbilical cord blood mesenchymal stem cells and its application
  • The miRNA related to genome stability of human umbilical cord blood mesenchymal stem cells and its application
  • The miRNA related to genome stability of human umbilical cord blood mesenchymal stem cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Using bioinformatics technology to screen out miRNAs associated with genome stability of human umbilical cord blood mesenchymal stem cells

[0050] According to the genome stability of the mesenchymal stem cells after passage expansion (the 30th generation) (in the previous work, the inventors have mastered the information on the genome stability of the mesenchymal stem cells during the late passage, the article was published in Cell Death and Disease ( 2013) 4, e950), two groups of representative human umbilical cord blood mesenchymal stem cells (purchased from Tianjin Angsai Cell Genetic Engineering Co., Ltd.) were selected. A total of 6 cell lines in these two groups are all early passage (3rd passage) mesenchymal stem cells, and the genome stability of the late passage (30th passage) cells corresponding to the 3 lines in each group are normal (N), Moderate abnormality (M), severe abnormality (S). Perform miRNA-Seq sequencing on these 6 cell lines, combine...

Embodiment 2

[0053] Example 2 Correlation analysis of the miRNA screened in Example 1 and the genome stability of mesenchymal stem cells

[0054] According to the number of CNV (gene copy number variation) in the human umbilical cord blood mesenchymal stem cells after subculture, the genome abnormal value of the normal genome clones N1 and N2 in two independent experiments in Example 1 is defined as 0, and the genome is slightly abnormal The genome outlier value of clones M1 and M2 was 0.5, and the genome outlier value of clones S1 and S2 with severe genome abnormality was 1. Carry out Pearson correlation analysis (see Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E ), the calculated correlation coefficients were all greater than 0.85, and the p-values ​​were less than 0.01. This shows that the expression level of the miRNA screened in Example 1 is significantly positively correlated with the degree of abnormality of genome stability.

Embodiment 3

[0055] Example 3 Bioinformatics analysis of the relationship between miRNA and the genome stability of mesenchymal stem cells

[0056] Target genes were predicted for the 5 miRNAs screened in Example 1 through the TargetScanHuman7.0 database, and the 14 target gene features in the prediction results were weighted and scored, and the top 200 target genes with the highest scoring values ​​were screened out for follow-up Gene Ontology and Pathway and other functional analysis. It was found that the target genes of 4 miRNAs (miR-501-5p, miR-24-2-5p, miR-136-3p, and miR-127-3p) are involved in the regulation of cell cycle, apoptosis, etc. and are closely related to genome stability related molecular pathways. Therefore, they may affect the genomic stability of mesenchymal stem cells by targeting and regulating the expression level of target genes. miR-376a-5p, which is not involved in the regulation of cell cycle and apoptosis pathways, may affect the genome stability of mesenchy...

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Abstract

The invention provides an miRNA marker associated with genomic stability of human umbilical cord blood mesenchymal stem cells and a detection method of the miRNA marker. A new generation of high-throughput sequencing technology is used, in combination with a bioinformatic analysis method of a system, to screen and obtain the miRNA associated with genomic stability of human umbilical cord blood mesenchymal stem cells, wherein an RNA sequence is shown in SEQ IDNO.1-5. The invention also provides a specific primer for detecting the miRNA and a detection method of the specific primer, wherein conditions for the detection method of human umbilical cord blood mesenchymal stem cells are optimized. Through the detection of the specific miRNA marker provided by the invention, the genomic stability of human umbilical cord blood-derived mesenchymal stem cells after passage or amplification can be estimated effectively, to reduce costs and the risk of subsequent clinical application; the miRNA has excellent clinical application value and broad application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to miRNA molecular markers related to genome stability of human umbilical cord blood mesenchymal stem cells and applications thereof. Background technique [0002] miRNA is a type of endogenous, highly conserved, non-protein-coding small molecule RNA, usually 18-25nt in length. Studies have shown that: miRNA can be widely involved in various physiological and pathological regulatory processes such as developmental cycle, cell proliferation and differentiation, cell apoptosis, metabolism, neural regulation, tumorigenesis, and the interaction between virus and host. Abnormal genome stability phenomena such as gene fragment copy number variation, insertion, deletion, and chromosome number abnormalities in cells can cause abnormal expression or regulation of some miRNAs. [0003] Human umbilical cord blood mesenchymal stem cells are the most widely used and most promising t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12Q1/6876
CPCC12N15/113C12N2310/141C12Q1/6876C12Q2600/178
Inventor 方向东张昭军张倩韩忠朝
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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