CN115927298A - Precursor of red deer miR PC-2869 and application thereof - Google Patents
Precursor of red deer miR PC-2869 and application thereof Download PDFInfo
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Abstract
The invention discloses a precursor of red deer miR PC-2869 and application thereof, relates to a precursor of small RNA (microRNA), and provides a factor which can generate mature miR PC-2869 of antler of northeast red deer (Cervus elaphus) and has an inhibitory effect on cells, wherein the factor is named as: the sequence of cel-mir PC-2869 is GTCCTTCCTGAGCCTTCTGTGGAGACCTAGTGCAAAAAAAGCCGACGCATTGGTTCAGTGGTAGAATTCTC, and the cel-mir PC-2869 has the function of efficiently reducing the expression of CDK8 protein and can inhibit cell proliferation.
Description
Technical Field
The invention relates to a precursor of small RNA (microRNA), which can generate mature miR PC-2869 and negatively regulate the proliferation of cells.
Background
microRNA (miRNA for short) is a conserved single-stranded non-coding RNA with the length of 19-24bp and a regulating function, and can identify and combine with a pairing region on mRNA, so that translation of the mRNA is inhibited or degradation of the mRNA is promoted, and expression of genes is negatively regulated. The transcription process of miRNA is generally completed under the participation of RNA polymerase II, and the transcription product is modified by capping and polyadenylation to form initial miRNA, namely pri-miRNA, and the typical animal pri-miRNA is composed of three parts of incompletely-matched stem, terminal loop and flanking sequence. The generation of miRNA is performed twice by sequence processing, the first of which occurs in the nucleus, and Drosha enzymes cleave the flanking sequences of pri-miRNA to generate a precursor miRNA, the pre-miRNA. In animal cells, pre-miRNA enters cytoplasm from nucleus under the combined action of exportin 5 and RNA-GTP, and the second processing process is started. The typical Dicer enzyme in the cytoplasm participates in a cleavage reaction that recognizes both ends of the pre-miRNA, completing a second cleavage of the stem and loop that may result in 1-2 different mature mirnas, each from one arm of the stem structure (-3 p and-5 p).
The microRNA PC-3p-2869 (miR PC-2869 for short) is miRNA expressed in deer antler cartilages of red deer (Cervus elaphus), and can negatively regulate and control various target genes, including EEF1A1, CDK8 and other genes. Among them, CDK8 is a member of the Cyclin-dependent kinase (CDK) family, belonging to the serine/threonine protein kinases. Most CDK proteins are involved in cell cycle regulation, in part transcriptional regulation, and very few other functions. The CDK8 protein is mainly involved in transcriptional control, can form a medium complex with cyclin C, MED12 and MED13, and the medium complex establishes a bridge for a transcription factor, a chromatin modifier, a promoter, an enhancer and RNA polymerase II (RNA Pol II) and plays a core role in the transcription process. CDK8 can participate in regulating and controlling signal pathways such as WNT/beta-catenin, JAK-STAT, TGF beta/BMP, notch and the like, thereby influencing the physiology of cells and the pathological process of some diseases. It has been shown that inhibition of CDK8 expression reduces the proliferation of colorectal cancer cells and reduces the number of cells that enter S phase from G1 phase. The miR PC-2869 can inhibit proliferation, migration and invasion of cells, has an important regulation and control effect in the rapid growth process of the pilose antler, and has application potential in the aspect of cancer treatment.
There are many ways in which miRNA overexpression can be achieved in cells, including the use of miRNA mimics, miRNA agomis, and the like. One effective way is to obtain a precursor sequence for expressing miRNA, and simulate the generation and processing and shearing processes of miRNA in cells, thereby achieving overexpression of miRNA in cells.
Disclosure of Invention
The invention determines a precursor which is derived from the red deer and can express and generate red deer mature miR PC-2869, and the mature product of the precursor can negatively regulate CDK8 gene expression and inhibit cell proliferation. The name and sequence of the precursor is also determined. According to the miRNA expression data of European red deer (Cervus elaphus hippophagus) genome and northeast red deer (Cervus elaphus xanthopygus) cartilage tissues, the miR PC-2869 precursor sequence is obtained through bioinformatics prediction and is named as cel-miR PC-2869. It is 80nt in length and is capable of forming a stable, typical stem-loop structure (shown in fig. 1), and the sequence is shown in Seq ID NO:1 (GTCCTTTCCTGAGCCACTTTTCTGTGGAGACCTAGGTGCATTCCAAAAGCC GACGCATTGGGTGGTTCAGTGGTAGAATTCTC).
After reverse transcription by stem loop method and quantitative PCR detection, the invention determines that the recombinant expression vector of cel-miR PC-2869 can express and process to generate mature miR PC-2869 (shown in figure 2) after transfecting cells.
The expression of the expression negative regulation miR PC-2869 target gene CDK8 of cel-miR PC-2869 is determined through a dual-luciferase report system and a western blot experiment (shown in figures 4 and 5).
The invention determines that the expression of cel-miR PC-2869 has the same biological function as miR PC-2869, namely the cell proliferation is inhibited through the proliferation experiment of MG63 cells (shown in figure 6).
The invention has the following beneficial effects:
according to the sequence of mature miR-PC-2869, the precursor sequence of miRNA is reversely predicted by utilizing the thermodynamic stability of RNA secondary structure and the stem-loop structure characteristic of miRNA precursor, and the method has a reference function for researching miRNA precursors of various animals.
The sequence and the structure of cel-miR PC-2869 are determined, the precursor sequence can be used for constructing various recombinant expression vectors, and the recombinant expression vectors can directly transfect cells or be packaged into virus particles to infect the cells, so that the rapid, high-quantity and stable expression of miR PC-2869 in the cells is realized. The invention increases an optional mode of over-expressing miR PC-2869, enhances the capacity of applying miR PC-2869, and widens the application prospect of miR PC-2869.
The invention determines that cel-miR PC-2869 not only has the capacity of generating mature miR PC-2869, but also has the same biological function as the mature miR PC-2869. The cel-miR PC-2869 can inhibit the expression of miR PC-2869 target gene CDK8 and can also inhibit the proliferation of cells, so the cel-miR PC-2869 also has the application potential of treating cancers.
The gene sequence of the invention is as follows
Name: cel-mir PC-2869
The sequence is as follows: GTCCTTCTCGAGCACTTTCTGTGGAGACCTAGGATTCCAAAAGCCG ACGCATTGGTGGTTCAGTGGTAGAATTCTC
Drawings
FIG. 1 is a stem-loop structure of cel-mir PC-2869. The sequence in light black is the mature miR PC-2869 sequence (CGCAUUGGGUGGUUCAGUGG).
FIG. 2 is a graph showing the expression level of cel-miR PC-2869 capable of expressing mature miR PC-2869. After the PLKO.1-cel-miR PC-2869 recombinant expression vector and the PLKO.1-puro empty vector are transfected into 293T cells respectively, the expression level of miR PC-2869 is analyzed through reverse transcription by a stem-loop method and quantitative PCR experiments. The stem-loop reverse transcription and quantitative PCR experiments showed that in 293T cells, the expression of mature miR PC-2869 by the PLKO.1-puro, PLKO.1-cel-miR PC-2869 recombinant expression vector was significantly increased relative to the empty vector, U6 was an internal control (p <0.5,; p <0.01,;).
FIG. 3 shows that miR PC-2869 has species conservation at the target site and flanking sequence on CDK8 mRNA. The target gene and the binding site of miR PC-2869 are predicted through a Targetscan website, the fact that CDK8 is the target gene of miR PC-2869 is shown, the sequence of the target binding site is very well conserved in human (Hsa), chimpanzee (Ptr), cattle (Bta), horse (Eca), cat (Fca) and dog (Fca), the lower case black sequence in the figure is the seed sequence (seed sequence) of miR PC-2869, and the lower case light black sequence is the pairing sequence of the miR PC-2869 seed sequence.
FIG. 4 shows that miR PC-2869 generated by expression of cel-miR PC-2869 can bind to a target site on CDK8 mRNA and negatively regulate the expression result of a luciferase reporter gene. After co-transfection of 293T cells with either the PLKO.1-cel-mir PC-2869 expression vector or the PLKO.1-puro empty vector and the CDK8-3 UTR wild-type (CDK 8-WT) or mutant (CDK 8-MUT) dual-luciferase reporter vector, the enzyme-linked immunosorbent assay showed that the luciferase reporter gene expression was significantly down-regulated in the CDK8-3'UTR wild-type + PLKO.1-cel-mir PC-2869 expression vector and the CDK8-3' UTR wild-type + PLKO.1-puro empty vector groups (p <0.5,; p <0.01,; etc.) relative to the CDK8-3'UTR mutant + PLKO.1-mir PC-2869 expression vector and the CDK8-3' UTR wild-type + PLKO wild-prKO 1-puro empty vector groups.
FIG. 5 is an expression diagram of miR PC-2869 produced by expression of cel-miR PC-2869 and capable of inhibiting endogenous CDK8 protein in MG63 cells. After MG63 cells are transfected with PLKO.1-cel-mir PC-2869 recombinant expression vector or PLKO.1-puro empty vector, the expression level of CDK8 protein in the cells is detected by western blot experiment, and the internal reference is GAPDH.
FIG. 6 shows the results of cell proliferation inhibition by cel-mir PC-2869. After MG63 cells are transfected by PLKO.1-cel-mir PC-2869 recombinant expression vector or PLKO.1-puro empty vector, the proliferation level of the cells is detected by CCK8 experiment. The PLKO.1-cel-mir PC-2869 recombinant expression vector group significantly down-regulated cell proliferation relative to the PLKO.1-puro empty vector group (p < 0.5; p < 0.01).
Detailed Description
The first embodiment is as follows: the precursor of miR PC-2869 of red deer in the present embodiment is named: cel-mir PC-2869, the sequence of which is
<xnotran> GTCCTTCCTGAGCCACTTTCTGTGGAGACCTAGTGCATTCCAAAAAGCCGACGCATTGG TGGTTCAGTGGTAGAATTCTC ( Seq ID No:1 ). </xnotran>
The second embodiment is as follows: the precursor of the stag miR PC-2869 and the application thereof can express and generate mature miR PC-2869.
The third concrete implementation mode: the precursor of the stag miR PC-2869 and the application thereof can negatively regulate and control the target gene CDK8 of miR PC-2869.
The fourth concrete implementation mode: the precursor of the stag miR PC-2869 and the application thereof have the function of inhibiting cell proliferation.
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,% by volume unless otherwise specified. The sequence orientations referred to in the examples below are all from 5 'to 3' by default. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1: acquisition of Cervus elaphus cel-mir PC-2869 sequence
The miR PC-2869 sequence is input into an online software Blast, nucleic acid Blast is carried out to determine the position of the miR PC-2869 on the genome of the red deer, and Blast results show that the miR PC-2869 sequence is located on chromosome 24496559-24496577 of the red deer. Selecting a nucleotide sequence with the length of about 300nt by taking miR PC-2869 as a center, putting the nucleotide sequence into RNAfold online software, predicting the secondary structure of the single-stranded RNA, continuously truncating and adjusting the length of sequences on two sides of miR PC-2869 according to a prediction result until a typical miRNA precursor stem-loop structure can be formed, taking the stem-loop structure as the miR PC-2869 precursor sequence, and determining that the miR PC-2869 is positioned on the 3' end arm of the stem-loop structure (see the orange nucleotide marked in figure 1).
Example 2: the cel-miR PC-2869 recombinant vector can express and produce mature miR PC-2869
(1) constructing a cel-mir PC-2869 recombinant vector: the upstream and downstream oligonucleotide strands of cel-mir PC-2869 (Hongxn technology, suzhou) were chemically synthesized, annealed, phosphorylated, and ligated into PLKO.1-puro vector (Clontech, USA) to obtain the recombinant expression vector PLKO.1-cel-mir PC-2869.
(2) And (3) cell culture: 293T cells were purchased from Huifei Biotech (Changsha) Ltd in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA). Standing at 37 deg.C, 5% CO 2 Culturing under the condition.
(3) Detecting the expression level of mature miR PC-2869 by stem-loop reverse transcription and quantitative PCR (polymerase chain reaction) experiments: the PLKO.1-cel-mir PC-2869 recombinant vector and the PLKO.1-puro empty vector were transfected into 293T cells, respectively, using lipo8000 (Biyunyan, shanghai) transfection reagent. Total cellular RNA (Invitrogen, USA) was extracted 24 hours later, and reverse transcription was performed on the total RNA using miRNA 1st Strand cDNA Synthesis kit (Novozam, nanjing), with reverse transcription primers miR-RT having the following sequence (Seq ID NO: 2):
GTCGTATCCAGTGCAGGGTCCGAGGTTCGCACTGGATACGACCCACTG. Then miRNA Universal was used
quantitative PCR analysis was performed with the qPCR Master Mix kit (Novozam, nanjing) to accurately quantify the expression levels of miR PC-2869 in both sets of transfected cells. The PCR upstream and downstream primers are as follows, and the upstream primer 2869-F is (as shown in Seq ID No: 3): GCGCGCATTGGTGGTT, the downstream primer 2869-R (shown as Seq ID No: 4) is: AGTGCAGGGTCCGAGGTAT. In this experiment, U6 was used as an internal reference, the primer sequences were as follows, and the upstream primer U6-F (shown as Seq ID No: 5) was: TGGAACGATACAGAGAATTAGCA, downstream primer U6-R (as shown in Seq ID No: 6) is: AACGCTTCACGAATTTGCGT. As a result, the large-scale expression of miR PC-2869 can be detected only in the cells transfected with PLKO.1-cel-miR PC-2869 recombinant vector.
Example 3: cel-mir PC-2869 negatively regulates the expression of CDK8 gene.
(1) Construction of psiCHECK2-CDK8-3' UTR expression vector: sequence alignment analysis revealed that the various animal CDK8 mRNA 3' UTR regions had potential miR PC-2869 seed sequence complementary binding sites (see FIG. 3). Thus, in this example, a 73bp fragment (shown in FIG. 3) was synthesized from the conserved CDK8 UTR region sequence of human, chimpanzee, bovine, horse, cat, and dog species and inserted into psiCHECK2 (Promega, USA) vector to construct a wild-type vector pSICHECK2-CDK8-3 UTR-WT (CDK 8-WT for short), wherein the insert comprises the complement of the seed sequence miR PC-2869, and the inserts (Seq ID NO: 7) are as follows:
TGTTGTGGATTTGCTACTTCCATAGTTACTTGACATGGTTCAGACTGACCAATGCATTTTT TTTCAGTGACAG. Accordingly, a mutant vector pSICHECK2-CDK8-3' UTR-MUT (CDK 8-MUT) was constructed in which the complementary region of the miR PC-2869 seed sequence was substitution mutated, and the inserts (as shown in Seq ID NO: 8) were as follows:
TGTTGTGGATTTGCTACTTCCATAGTTTACTTGACATGGTTCAGACTGACAACTTCATTTT TTTCAGTGACAG。
(2) Cell culture: 293T and MG63 cells were purchased from Fenghui Biotech (Changsha) Ltd, the medium was DMEM (Gibco, USA), and 10% fetal bovine serum (Gibco, USA) was added to each of them. Standing at 37 deg.C, 5% CO 2 Culturing under the condition.
(3) And (3) dual-luciferase detection: 5X 10 4 293T cells were seeded in 24-well plates, 293T cells were co-transfected with PLKO.1-cel-mir PC-2869 recombinant expression vector and pSICHECK2-CDK8-3' UTR-WT as experimental groups; 293T cells co-transfected with PLKO.1-cel-mir PC-2869 recombinant expression vector and pSICHECK2-CDK8-3' UTR-MUT as a control; 293T cells co-transfected with PLKO.1-puro vector and pSICHECK2-CDK8-3' UTR-WT were also used as a control. Cells were harvested after 24 hours and luciferase activity was detected using a dual luciferase reporter assay kit (petunia, shanghai). The results showed that the relative expression level of renilla luciferase in the experimental group was significantly reduced compared to the two control groups (fig. 4), indicating that the mature miR PC-2869 produced by cel-miR PC-2869 was functional and able to bind to the site on CDK 8' utr, thus negatively regulating luciferase reporter gene expression.
(4) Western blot detection of endogenous CDK8 protein levels: 2X 10 5 MG63 cell line of (1) mu.g of PLKO.1-cel-mir PC-2869 recombinant vector or PLKO.1-puro empty vector was transfected into MG63 cells in a 6-well plate using lipo8000 reagent (Bilun sky, shanghai), and after 36 hours, total protein was extracted and subjected to western blot assay, and its protein expression level was detected using CDK8 antibody (A5081, bimake, USA), with an internal reference being GAPDH protein (sc-47724, santa Cruz, USA).
Example 4: cell proliferation inhibition by cel-mir PC-2869
(1) Cell culture and transfection: MG63 cells were purchased from Biotech (Shanghai) Co., ltd, and the culture medium was DMEM (Gibco, USA), and the culture method was the same as that of example 2 (3). The cells were transfected as in (4) of example 2.
(2) And (3) detecting cell proliferation activity: after MG63 cells were transfected with PLKO.1-cel-mir PC-2869 or PLKO.1-puro empty vector, the cell proliferation level was examined using CCK-8 (Dojindo, japan) kit, and the change in cell number was examined at 5 time points, respectively. The results of the assay showed that proliferation of cells in the PLKO.1-cel-mir PC-2869 transfected group was inhibited compared to the control (PLKO.1-puro transfected cells) (shown in FIG. 6).
Sequence listing
<110> northeast university of forestry
Precursor of miR PC-2869 of red deer and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 80
<212> DNA
<213> northeast red deer (Cervus elaphus)
<400> 1
gtccttcctg agccactttc tgtggagacc tagtgcattc caaaaagccg acgcattggt 60
ggttcagtgg tagaattctc 80
<210> 2
<211> 50
<212> DNA
<213> Artificial sequence (")
<400> 2
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacccactg 50
<210> 3
<211> 16
<212> DNA
<213> Artificial sequence (")
<400> 3
gcgcgcattg gtggtt 16
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (")
<400> 4
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence (")
<400> 5
tggaacgata cagagaagat tagca 25
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (")
<400> 6
<210> 7
<211> 73
<212> DNA
<213> Artificial sequence (")
<400> 7
tgttgtggat ttgctacttc catagtttac ttgacatggt tcagactgac caatgcattt 60
ttttcagtga cag 73
<210> 8
<211> 73
<212> DNA
<213> Artificial sequence (")
<400> 8
tgttgtggat ttgctacttc catagtttac ttgacatggt tcagactgac aacttcattt 60
ttttcagtga cag 73
Claims (4)
1. A precursor of red deer miR PC-2869 is characterized in that the precursor is named as: cel-mir PC-2869, which has the sequence of GTCCTTCCTGAGCACTTTCTGTGGAGACCTATTCAAAAAAGCCGACGATTGGTTCAGTGGTAGAATTCTC.
2. A precursor of red deer miR PC-2869 and application thereof are characterized in that the precursor can express and generate red deer mature miR-PC-2869.
3. A precursor of red deer miR PC-2869 and application thereof are characterized in that a mature product miR-PC-2869 generated by the precursor can inhibit cell proliferation.
4. The precursor of elk miR PC-2869 and the use thereof according to claim 1, characterized in that the completion of a cellular proliferative biological function is inhibited by inhibiting the expression of a cell Cycle Dependent Kinase (CDK) 8.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006107826A2 (en) * | 2005-04-04 | 2006-10-12 | The Board Of Regents Of The University Of Texas System | Micro-rna's that regulate muscle cells |
| CN111690645A (en) * | 2019-03-13 | 2020-09-22 | 东北林业大学 | MicroRNA derived from cartilaginous antler and application thereof |
| CN112301000A (en) * | 2020-11-16 | 2021-02-02 | 吉林大学 | Reporter cell line capable of converting stress signal into EGFP fluorescent signal |
-
2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006107826A2 (en) * | 2005-04-04 | 2006-10-12 | The Board Of Regents Of The University Of Texas System | Micro-rna's that regulate muscle cells |
| CN111690645A (en) * | 2019-03-13 | 2020-09-22 | 东北林业大学 | MicroRNA derived from cartilaginous antler and application thereof |
| CN112301000A (en) * | 2020-11-16 | 2021-02-02 | 吉林大学 | Reporter cell line capable of converting stress signal into EGFP fluorescent signal |
Non-Patent Citations (1)
| Title |
|---|
| YANXIA CHEN等: "Deep sequencing identifies conserved and novel microRNAs from antlers cartilage of Chinese red deer (Cervus elaphus)", GENES GENOM, vol. 37, 31 December 2015 (2015-12-31), pages 419 - 427, XP035966785, DOI: 10.1007/s13258-015-0270-9 * |
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