Application of miR-26a in regulating germ cell apoptosis

A germ cell apoptosis, mir-26a technology, applied in the application field of miR-26a regulating germ cell apoptosis, can solve problems such as no related reports

Inactive Publication Date: 2019-08-16
ANHUI MINGSHAN ORGANIC AGRI TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] These findings suggest that miR-26a may play an important regulatory role in follicle development, but there is no relevant report on the study of miR-26a in porcine ovarian granulosa cells

Method used

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  • Application of miR-26a in regulating germ cell apoptosis
  • Application of miR-26a in regulating germ cell apoptosis
  • Application of miR-26a in regulating germ cell apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Bioinformatics prediction and conservative analysis

[0019] Download pig (Sus scrofa), human (Homosapiens), mouse (Mus musculus), cow (Bos taurus), goat (Capra hircus), monkey (Macacamulatta) from miRBase database (http: / / www.mirbase.org / ) , horse (Equus caballus), rat (Rattus norvegicus), zebrafish (Danio rerio), chicken (Gallus gallus) 10 species of miR-26a mature sequence, using MEGA6.0 software to analyze its conservation. The comparison of miR-26a mature sequences in different species is shown in the table below (the seed sequence is in the dotted box in the table below):

[0020]

[0021] It can be seen from the above table that the mature sequence of miR-26a is completely consistent in mammals and zebrafish, with 22 bases, and only one base U is missing at the 5' end of chicken, indicating that miR-26a has a higher sequence in vertebrates. of conservatism.

[0022] The porcine DHCR24 and Smad4 mRNA sequence information was queried according to the...

Embodiment 2

[0023] Embodiment 2 primer design and synthesis

[0024] The mature sequence of ssc-miR-26a (sequence number: MI0002429) was queried and downloaded from the latest miRBase22.1 database (www.mirbase.org). The miRNA primers were designed by the tailing method, and the downstream primers were universal primers. Tip GreenmiRNA Two-Step qRT-PCR SuperMix kit provided.

[0025] From the NCBI database, the mRNA sequences of porcine genes Smad4 (Gene ID: 397142), DHCR24 (Gene ID: 100628197), Caspase-3 (Gene ID: 397244), BCL-2 (Gene ID: 100049703) were downloaded. Use Primer Premier6.0 software to design primers for the mRNAs of these genes, and use Primer-BLAST in the NCBI platform (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC =BlastHome) Perform sequence alignment and specificity analysis on the designed primers.

[0026] Smad4, DHCR24, Bcl-2, Caspase-3, miR-26a and internal reference gene β-Actin and U6 primers were all synthesized by General Biosystems (An...

Embodiment 3

[0028] Example 3 Isolation, culture, inoculation and transfection of porcine ovary granulosa cells

[0029]Primary culture: (1) place the porcine ovary in preheated 37°C normal saline (adding 1% double antibody), and quickly transport it back to the laboratory; (2) place the ovary in preheated PBS (containing 1% double antibody ) was washed 3 times, and the follicular fluid from the medium-sized follicles with a diameter of 3-6 mm was extracted with a 1 mL sterile syringe; (3) the aspirated follicular fluid was placed in a 15 mL centrifuge tube with 5 mL of complete medium, and centrifuged at 1000 rpm for 5 min at room temperature; ) Discard the supernatant, resuspend with complete medium, centrifuge at 1000rpm for 5min, and repeat twice; (5) Discard the supernatant, resuspend with an appropriate amount of complete medium, inoculate in a 10cm cell culture dish; (6) place at 37°C , 5% CO 2 Cultivate in an incubator, observe the adherence after 48 hours, and remove the medium; ...

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Abstract

Studies show that miR-26b participates in the reproductive regulation of animals, miR-26b can regulate apoptosis of porcine ovarian granulosa cells by targeting Smad4 gene, miR-26a may further has thesimilar function, but studies on the miR-26a in germ cells are not reported. The invention discloses an application of the miR-26a in regulating germ cell apoptosis. The invention discloses that DHCR24 and Smad4 are target genes of the miR-26a. In porcine ovarian granulosa cells, the miR-26a negatively regulates the expression of the DHCR24 and the Smad4 at transcription level and translation level; miR-26a promotes apoptosis of ovarian granulosa cells by interfering with expression of the DHCR24 and the Smad4, and at the same time controls secretion of estradiol and progesterone by regulating steroid synthesis pathway and affecting hormone secretion.

Description

technical field [0001] The invention relates to the technical fields of cell engineering and genetic engineering, in particular to an application of miR-26a regulating germ cell apoptosis. Background technique [0002] In mammalian ovaries, folliculogenesis and atresia processes are tightly regulated by complex gene networks. Thus, miRNAs, which can originate from both cellular and extracellular sources, play a role in mediating these processes through extensive involvement in post-transcriptional mRNA regulation. Previous studies have shown that ovarian miRNAs play an important role in follicle and corpus luteum development, ovarian function, ovarian diseases and endocrine disorders, and miRNAs play a regulatory role by promoting granulosa cell apoptosis and inhibiting apoptosis. [0003] It has been reported that miR-26b is involved in the regulation of animal reproduction. miR-26b regulates the apoptosis of porcine follicular granulosa cells by targeting the Smad4 gene. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/87
CPCC12N15/87C12Q1/6883C12Q2600/178
Inventor 张晓东殷宗俊丁月云尚金男陶强强章会斌徐怡亮陈一歌朱卫华
Owner ANHUI MINGSHAN ORGANIC AGRI TECH DEV
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