CN112210554A - CNBP-mediated microRNA derived from pilose antler and application thereof - Google Patents
CNBP-mediated microRNA derived from pilose antler and application thereof Download PDFInfo
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Abstract
The invention relates to a micro RNA (microRNA) derived from pilose antler and mediated by CNBP (CNBP), and provides a factor which is derived from pilose antler cartilage of northeast red deer (Cervus elaphus) and has an inhibiting effect on cell migration, which is named as: the sequence of the microRNA PC-5p-7654 is CAGGAUUCCCUCAGUAAC, the sequence has the function of efficiently down-regulating the expression of the CNBP protein, and the cell migration can be inhibited.
Description
Technical Field
The invention relates to a small RNA (microRNA) for negatively regulating the migration of cells.
Background
In mammals, only antler horn is the only organ known to date to have the capacity of epimorphic regeneration, i.e. to regenerate completely after loss. In the regeneration period, the pilose antler extends 1-2cm from the top growing point per day on average, and finally stops automatically after the pilose antler grows rapidly for nearly one hundred days, so that the hairy skin can die and fall off slowly. The mechanism of antler regeneration is complex, and the antler regeneration mechanism is still an important research model of regeneration biology at present.
MicroRNA (miRNA) is an important gene expression negative regulatory factor which is endogenous and 18-24 nucleotides in length, and expresses the function of the gene by regulating the mRNA stability or translation efficiency of a target gene. It is derived from a long transcript with hairpin-like secondary structure and is cleaved by two endonucleases Drosha and Dicer to form a double-stranded miRNA. One strand forms the mature miRNA and the other strand degrades. Mature mirnas regulate target gene mrnas by binding to their 3' Untranslated Regions (UTRs) target sites. The selective activity of a miRNA depends on the seed sequence (seed region) of the miRNA complementary to this homologous mRNA (fig. 1). Because imperfect base pairing within miRNA seeds represents that this core 5-7 nucleotide sequence can potentially bind many mRNA sequences, one miRNA can simultaneously regulate the mRNA of multiple target genes. Research shows that miRNA plays a very key role in regulation in biological processes of proliferation, differentiation, organ morphogenesis, tumorigenesis and the like of a plurality of cells. miRNA plays a role in inhibiting oncogenes or tumors by inhibiting target genes, and at present, more and more miRNAs are proved to play a very important role in the processes of cancer occurrence and development, so miRNA can be used as a potential new target for treating cancers.
Cellular Nucleic Acid-Binding Protein (CNBP), also known as zinc finger Protein 9, is a seven highly conserved cysteine-histidine-cysteine zinc finger domain containing a 19kDa Protein and an arginine/glycine rich region. The first and second zinc fingers are highly similar to the arginine glycine-glycine (RGG) cassette found in RNA binding proteins. CNBPs are involved in a variety of biological processes, acting as nucleic acid chaperones and transcription factors. Several studies have shown that CNBP regulates the expression of macrophage colony stimulating factor (csf1), the c-myc protooncogene, components of the Wnt signaling pathway and proinflammatory cytokines. In addition, CNBP is involved in embryonic forebrain development and various human diseases including myotonic dystrophy (DM2) and sporadic inclusion body myositis (sbm). CNBP also promotes tumor growth through the translational up-regulation of ornithine decarboxylase, a regulator of polyamine synthesis, essential for cell proliferation.
Disclosure of Invention
The invention determines the negative regulatory factor which is derived from the cartilaginous antler of the northeast red deer (Cervus elaphus) and inhibits the cell migration through the mediation of CNBP, and determines the name and the sequence of the factor. The microRNA PC-5p-7654 (miRNA PC-7654 for short) screened from the microRNA database of the hairy antler cartilage tissue of the red deer is miRNA specifically expressed in the hairy antler cartilage and has the length of 18 nt. The sequence is shown as SEQ ID NO: shown at 1 (CAGGAUUCCCUCAGUAAC).
Cell migration detection of 293T, Hepg2 cells and experiments such as Western Blotting prove that miRNAPC-7654 can down-regulate the expression of cell proliferation key transcription factor CNBP (shown in figures 2 and 3) and can mediate inhibition of cell migration (shown in figure 4).
The invention has the following beneficial effects:
as a negative regulatory factor of gene expression, microRNA plays an important role in cell growth, organ development, individual growth and the progress of various diseases. The microRNA separated from the cartilaginous tissue at the top end of the rapidly growing pilose antler plays a very important role in regulating and controlling the regeneration and growth of the pilose antler.
Therefore, the miRNA PC-7654 is obtained from a microRNA sequencing result of the cartilaginous tissue at the top end of the antler of the newborn red deer in a rapid growth period, the transcription precursor of the miRNA PC-7654 is about 56nt in length, a hairpin structure can be formed, the full length of the mature miRNA is 18nt, and the miRNA has no homologous sequence in a miRBase database (Release 22.1). BLAST results show that there are no homologous sequences in the genomes of model species such as human, chimpanzee, mouse, rat, rabbit, dog, horse, cow, etc., whether full-length mature mirnas or their seed sequences.
The key factor CNBP for predicting the cartilaginous development of the antler is one of target genes of miRNA PC-7654 through TargetScan, miRNA PC-7654 experiments verify that the miRNA PC-7654 has a targeting effect on the CNBP, and the miRNA PC-7654 is proved to have the effect of efficiently down-regulating the CNBP protein in cells such as 293T, Hepg2 and the like (the experimental results are shown in figures 2 and 3), and the cell migration is inhibited (the experimental results are shown in figure 4).
The gene sequence screened by the invention is as follows:
name: MicroRNA PC-5p-7654
The sequence is as follows: CAGGAUUCCCUCAGUAAC are provided.
Drawings
FIG. 1 shows that the 3' UTR region of the CNBP mRNA of various models has a conserved site (underlined upper case sequence) that complementarily binds to the potential miRNA PC-7654seed sequence (lower case sequence).
FIG. 2 shows that miRNA PC-7654mimics down-regulate Firefly luciferase expression in 293T cells transfected with pMIR-REPORT-CNBP-3' UTR-WT vector. Respectively co-transfecting the miRNA PC-7654mimics, the pMIR-REPORT-CNBP-3 'UTR-WT and the pMIR-REPORT vector into a 293T cell, collecting protein after 36 hours and carrying out western detection, wherein the expression of Firefly luciferase in the cell transfected with the vector is obviously reduced compared with that of an original vector (pMIR-REPORT) without an inserted sequence because the pMIR-REPORT-CNBP-3' UTR-WT contains a binding site of a miRNA PC-7654seed sequence; the internal reference is beta-actin.
FIG. 3 shows that miRNA PC-7654mimics down-regulate the level of endogenous CNBP protein in Hepg2 cells. And transfecting the miRNA PC-7654mimics and NC with Hepg2 cells respectively, collecting protein after 36 hours, and performing western detection, wherein an internal reference is GAPDH.
FIG. 4 shows the number of cells migrating 24 hours after the detection of miRNA PC-7654mimics by the Trans-well method. Setting NC negative control group; cell migration was significantly inhibited in the experimental group. The experiment was repeated three times independently, averaged and SD values calculated, and t-test statistical differences (×.p < 0.001).
Detailed Description
The first embodiment is as follows: the embodiment of the CNBP-mediated microRNA derived from the pilose antler is named as: MicroRNA PC-5p-7654, the sequence of which is CAGGAUUCCCUCAGUAAC (shown as Seq ID No: 1).
The second embodiment is as follows: the application of CNBP-mediated microRNA derived from velvet antler inhibits the expression of cell nucleic acid binding protein (CNBP).
The third concrete implementation mode: the application of CNBP-mediated microRNA derived from velvet antler of the present embodiment has a biological function of mediating and inhibiting cell migration by inhibiting the expression of CNBP.
The invention is not limited to the above embodiments, and one or a combination of several embodiments may also achieve the object of the invention.
The beneficial effects of the present invention are demonstrated by the following examples:
the following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. In the following examples,% is by mass unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 acquisition Process of velvet antler cartilage miRNA PC-7654
1) Obtaining materials: the material used in the experiment is the cartilaginous tissue of the antler of the northeast red deer which is in the state of rapid growth for about 70 days. Immediately after the antler is sawed, the tissue at the top end of the antler is cut into slices with the thickness of less than 0.5cm by using sterilized scissors, immediately put into a protective solution (RNA later) filled with RNA degradation prevention, placed in a low-temperature storage box and taken back to a laboratory for storage.
2) Extraction of mRNA: separating cartilage Tissue of cornu Cervi Pantotrichum preserved in laboratory, weighing 30mg, quickly cutting into pieces in an ultraclean bench, placing in a sterile 1.5mL test tube, extracting total RNA in cornu Cervi Pantotrichum cartilage with DNeasy Blood & Tissue Kit (Cat.No.69504) of QIAGEN company (Germany), dissolving the total RNA in water of RNAse-free, and detecting the total RNA with high quality by spectrophotometry (OD260nm/OD280 nm: 1.74-1.83).
3) High-throughput sequencing of velvet antler cartilage small RNA: the extracted total RNA is delivered to United states of America company (Hangzhou) to carry out high-throughput sequencing on the velvet antler cartilage tissue small RNAs library through an Illumina miRNA Solexa sequencing platform, and then data processing is carried out by ACGT101-miR v4.2(LC Sciences) software.
4) Obtaining new microRNA (miRNA) of cartilaginous antler and miRNA PC-7654: comprehensively analyzing sequence original data obtained after Solexa deep sequencing of a smallerRNA library of cartilaginous tissues of the cartilaginous antler to obtain a new microRNA, miRNA PC-7654, wherein the sequence cannot be compared with miRNAs precursors (pre-miRNAs) of miRBase cattle, but the determined sequence (known mature body) can be compared with a genome, and the extended genome sequence can form a qualified hairpin structure.
5) Target gene prediction: one of the target genes of miRNA PC-7654 was predicted to be a cellular nucleic acid binding protein (CNBP) by TargetScan.
Example 2 miRNA PC-7654 can down-regulate expression of CNBP (cell migration-promoting key factor) at cellular level
1) Synthesis of miRNA PC-7654: the miRNA PC-7654 sequence is submitted to Shanghai Jima to synthesize mimics and corresponding Negative Control (NC).
2) Constructing a pMIR-REPORT-CNBP-3' UTR-WT vector: sequence alignment analysis shows that the 3' UTR region of CNBPmRNA of various models of organisms has potential complementary binding conserved sites (upper-case underlined sequences) with miRNA PC-7654seed sequences (lower-case sequences) (shown in figure 1). Thus, in this example, a 64bp fragment (Invtrogene, Shanghai) was synthesized based on the sequence of the 3 'UTR region of CNBP conserved in human, chimpanzee, rhesus monkey, mouse, rat, dog, cat, horse, cow, etc., and inserted into a pMIR-REPORT (Applied Biosystems, USA) vector to construct a wild-type vector pMIR-REPORT-CNBP-3' UTR-WT, wherein the insert comprises the complement of the seed sequence of miRNA PC-7654, and the sequence of the insert (as shown in Seq ID NO: 2) is as follows:
CCCTCCTTTTTCTGATTGATGGTTGTATTATTTTCTCTGAATCCTCTTCACTGGCCAAAGGTTG；
the pMIR-REPORT vector is designed by ABI company (USA) to insert a target sequence of miRNA into a multiple cloning site, and the luciferase protein Firefociferase reporter vector of pMIR-REPORT can be used for qualitatively and quantitatively measuring the functions of miRNA. When the miRNA PC-7654mimics and the pMIR-REPORT-CNBP-3' UTR-WT vector transfect cells together, if the expression of Firefly luciferase protein is reduced, the miRNA PC-7654 has a function of reducing the expression of the CNBP.
3) Cell culture: 293T cells and Hepg2 cells were purchased from Gilman Biotech (Shanghai) Inc., and the medium was DMEM (10569010, Gibco, USA) supplemented with 10% fetal bovine serum (110099141, Gibco, USA). Culturing at 37 deg.C under 5% carbon dioxide.
4) Transfection and Western detection: cells were cultured at 6X 105One cell/well is planted on a 6-well culture plate, the cell confluency reaches 60-70% after the culture is carried out for 24 hours, the mimics of the synthesized miRNA PC-2869 and the pMIR-REPORT-CNBP-3' UTR-WT vector are used as the mediation to transfect 293T cells, and the amount of the transfected recombinant plasmid is 1.5 ug/well. Cells were harvested at 36 hours, proteins were denatured, and 4% -12% PAGE precast gel (NP0335BOX, Invitrr)ogen, usa) electrophoresis. After the nitrocellulose membrane was transferred, it was blocked with TBST mixture of 5% skim milk powder for 1 hour, and then the nitrocellulose membrane was incubated in a Firefly Luciferase primary antibody (Sc74548, Santa Cruz Biotechnology, usa) (fig. 2) or CNBP primary antibody (Sc-515387Santa Cruz Biotechnology, usa) (fig. 3) for 1 hour, a fluorescently labeled secondary antibody (Licor Bioscience, usa) for 1 hour, and the membrane was scanned with β -actin (Sc47778, Santa Cruz Biotechnology, usa) or GAPDH (Sc47724, Santa Cruz Biotechnology, usa) as an internal reference, the Odyssey infrared laser imaging system, respectively. The detection result shows that the miRNA PC-7654 effectively reduces the expression of Firefly Luciferase or CNBP (shown in figure 2 and figure 3).
Example 3 MiRNA PC-7654 function of inhibiting cell migration mediated at cellular level by inhibiting expression of cellular nucleic acid binding protein (CNBP)
1) Cell culture and transfection: 293T cells were purchased from Gilman Biotech (Shanghai) Ltd, and the medium was DMEM (Gibco, USA), and the culture method was the same as 3 in example 2). Cells were transfected as in 4 of example 2).
2) Cell migration detection: suspending in water to a concentration of 1.0 × 106Mu.l (serum-free DMEM) of the cell suspension per ml was added to the upper chamber of a 24-well (8.0 μm pore size) cell culture insert transwell (353097, Corning, USA), 600 μ l of serum-free DMEM medium was added to the lower chamber of the transwell, after 6 hours of culture, the serum-free DMEM medium in the lower chamber was changed to 10% FBS-containing DMEM medium, and the cells in the upper chamber were transfected with 25pmol of miRNA PC-7654mimics and NC, respectively, and transfected with 4 in example 2). After 24 hours of transfection, the cells in the upper membrane layer were removed with a cotton ball, the cells migrated to the lower membrane layer were fixed with 4% paraformaldehyde (P0099, petunia, shanghai) and stained with 0.5% crystal violet (C0121, petunia, shanghai), washed with distilled water and dried, observed under a microscope (200 × multiple), and randomly selected 5 fields were photographed and counted. The cell migration results showed that the cell migration of Hepg2 of miRNA PC-2869mimics was inhibited compared to the control (NC), and that such inhibition of cell migration was mediated by inhibition of the expression of cellular nucleic acid binding protein (CNBP) (shown in FIG. 4).
Sequence listing
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccctcctttt tctgattgat ggttgtatta ttttctctga atcctcttca ctggccaaag 60
gttg 64
Claims (3)
1. The CNBP-mediated microRNA derived from the pilose antler is characterized by being named as: the sequence of the microRNA PC-5p-7654 is CAGGAUUCCCUCAGUAAC.
2. CNBP-mediated microRNA derived from velvet antler, characterized in that it inhibits cell migration.
3. The use of the velvet antler-derived CNBP-mediated microRNA according to claim 1, wherein the suppression of the cellular migration biological function is mediated by the inhibition of the expression of cellular nucleic acid binding protein (CNBP).
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| CN111690645A (en) * | 2019-03-13 | 2020-09-22 | 东北林业大学 | MicroRNA derived from cartilaginous antler and application thereof |
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