MiR-301a simulant for targeted inhibition of IL-4 / 13A gene expression of pseudosciaena crocea and application of miR-301a simulant

Abstract

The invention relates to the field of molecular biotechnology and genetic engineering, and discloses a miR-301a simulant for inhibiting IL-4 / 13A gene expression of large yellow croaker and application of the miR-301a simulant. The miR-301a simulant comprises a double-stranded RNA (Ribonucleic Acid); wherein the sequence of the positive-sense strand of the double-stranded RNA is 5 '-GUGCAAUAGUAUUGUCAAAGCA-3', and the sequence of the antisense strand of the double-stranded RNA is 5 '-UGCUUUGCAAUACUUUGCAC-3'. Researches find that the mRNA expression level of the IL-4 / 13A gene of the pseudosciaena crocea can be remarkably reduced, T and B lymphocyte proliferation of the pseudosciaena crocea can be inhibited and adaptive immune response of the pseudosciaena crocea can be weakened by transfecting the miR-301a simulant in head kidney leukocytes of the pseudosciaena crocea. Therefore, the miR-301a simulant can be used for related research on IL-4 / 13A and adaptive immune response of the large yellow croaker, and has potential application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of molecular biology and genetic engineering, and discloses a miR-301a mimic for targeted inhibition of IL-4 / 13A gene expression in Pseudosciaena crocea and application thereof. BACKGROUND

[0002] Interleukins are a class of small molecular polypeptides secreted by immune cells such as mononuclear macrophages, T lymphocytes, B lymphocytes and some non-immune cells such as endothelial cells and fibroblasts, and the molecular weight thereof is usually 10-30 kDa. Interleukins activate intracellular JAK-STAT, NF-κB, MAPK and other signaling pathways by binding to specific receptors on the surface of target cells, regulate gene expression, and thus coordinate innate and adaptive immune responses. Interleukin 4 / 13A (IL-4 / 13A) in Pseudosciaena crocea is a pleiotropic cytokine that can regulate the host's immune response to pathogenic infection by promoting T cell proliferation and differentiation and B cell proliferation.

[0003] MicroRNAs (miRNAs) are a kind of non-coding RNAs about 22 nt long widely existing in eukaryotes. Most miRNAs can specifically pair with the bases in the 3' non-coding region (UTR) of target genes completely or partially, and then realize the post-transcriptional degradation or translation inhibition of target genes. miRNAs are widely involved in life processes of organisms such as growth and development, cell apoptosis, angiogenesis and inflammatory response. At present, there is no miRNA mimic applied to regulate IL-4 / 13A gene expression and adaptive immune response in Pseudosciaena crocea. SUMMARY

[0004] The present application provides a miR-301a mimic for targeted inhibition of IL-4 / 13A gene expression in Pseudosciaena crocea and application thereof.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The miR-301a mimic comprises double-stranded RNA, wherein the sequence of the sense strand of the double-stranded RNA is 5'-GUGCAAUAGUAUUGUCAAAGCA-3', and the sequence of the antisense strand is 5'-UGCUUUGACAAUACUAUUGCAC-3'.

[0006] The miR-301a mimic is used for preparing a preparation for inhibiting IL-4 / 13A gene expression in Pseudosciaena crocea.

[0007] The miR-301a mimic is used for preparing a medicine for inhibiting T lymphocyte proliferation in Pseudosciaena crocea.

[0008] The application of the miR-301a mimics in the preparation of a drug for inhibiting the proliferation of B lymphocytes of Pseudosciaena crocea.

[0009] The application of the miR-301a mimics in the research on adaptive immune response of Pseudosciaena crocea.

[0010] The present application has the following advantages: The present application provides a miR-301a mimics capable of targeting IL-4 / 13A gene of Pseudosciaena crocea for the first time, which can effectively reduce the transcription level of IL-4 / 13A gene when being transferred into the head kidney leukocytes of Pseudosciaena crocea, and can also inhibit the proliferation of T cells and B cells of Pseudosciaena crocea, thereby inhibiting the adaptive immune response of Pseudosciaena crocea. The above results show that the miR-301a mimics can be used for the research on IL-4 / 13A and acquired immune response of Pseudosciaena crocea. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 : miR-301a mimics inhibits the activity of pmiRGLO-IL-4 / 13A-3'UTR reporter gene.

[0012] Figure 2 : Change of mRNA level of IL-4 / 13A gene in head kidney leukocytes of Pseudosciaena crocea after transfection of miR-301a mimics.

[0013] Figure 3 : Change of T cell and B cell proliferation ratio in head kidney leukocytes of Pseudosciaena crocea after transfection of miR-301a mimics. DETAILED DESCRIPTION

[0014] In order to make the technical solutions and advantages of the present application clearer, the following embodiments are used to specifically explain the content of the present application, but should not be regarded as limiting the scope of the present application.

[0015] In the following embodiments, the methods used are conventional methods, and the materials used are commercially available, unless otherwise specified.

[0016] In the following embodiments, the sequence of the sense strand of the miR-301a mimics used is 5'-GUGCAAUAGUAUUGUCAAAGCA-3', and the sequence of the antisense strand is 5'-UGCUUUGACAAUACUAUUGCAC-3'.

[0017] In the following embodiments, the sequence of the sense strand of the NC mimics used is 5'-UUCUCCGAACGUGUCACGU-3', and the sequence of the antisense strand is 5'-ACGTGACACGUUCGGAGAA-3'.

[0018] The sequence of the miR-301a is: 5'-ACGAAACUGUUAUGAUAACGUG-3'.

[0019] The binding site of the miR-301a in the 3'UTR region (SEQ ID NO. 1) of the IL-4 / 13A gene mRNA (GenBank accession number: XM_027273581.1) of the Pseudosciaena crocea is predicted by using the bioinformatics software miRanda and PITA, and the sequence of the binding site is: 5'-UUGGAGCGAUUACAUAUUGCAG-3'.

[0020] By changing 7 continuous bases in 3'UTR of IL-4 / 13A gene mRNA which are complementary to miR-301a, the mutant fragment (TGAGCCAACTGTCATCAGCTCACTTCACTGTTCATTTTTCACTGTTCATTTTTTCATCTTCTGAGGTGGTATAATTGACAATGACAAATAACAAATTATTGGAGGATATGAATATAGAATTTATTGTATGATTTGTTCTATAAAATAAGTCGTTTTTTCTTTATGGATTGTTTTTTTTTTTTTTGGAGCGATTACAGCGGATCGATTTGTACAGGTATTGTGTTCATTTCCTGTCCAGCACCCAGCACAGTTCAATATGTCAAACATGTAATAAATAGTCGTTTAACCGATATTTAGTTTCATCTGCACGGTTAGCAGTTTTTAGTCAGCACAGCGATCACTATAGCAACTTCATGAAGGTTCATGAACAGCTTTCCAAGTGAAAACATAAAAGCTGTTGTGATCCTCATATGAGCACTATCAAATACACCTTCATATTCACTGTTTTATTGTATTTAACAGAGTGTTAATATAAATATATTATGGCTGTTTTCTGTATTTGAATGATATATTTATTGTAATCCTCTCATGCAGCATTGAAATAAAAGATATACTTTTATCAC) was inserted into the Nhe I and Xho I sites of pmirGLO vector, and the mutant plasmid pmirGLO-IL-4 / 13A-3'UTR-MUT was constructed.The 3'UTR fragment of the Large Yellow Croaker IL-4 / 13A gene mRNA (TGAGCCAACTGTCATCAGCTCACTTCACTGTTCATTTTCACTGTTCATTTTTTCATCTTCTGAGGTGGTATAATTGACAATGACAAATAACAAATTATTGGAGGATATGAATATAGAATTTATTGTATGATTTGTTCTATAAAATAAGTCGTTTTTTCTTTATGGATTGTTTTTTTTTTTTTTGGAGCGATTACATATTGCAGATTTGTACAGGTATTGTGTTCATTTCCTGTCCAGCACCCAGCACAGTTCAATATGTCAAACATGTAATAAATAGTCGTTTAACCGATATTTAGTTTCATCTGCACGGTTAGCAGTTTTTAGTCAGCACAGCGATCACTATAGCAACTTCATGAAGGTTCATGAACAGCTTTCCAAGTGAAAACATAAAAGCTGTTGTGATCCTCATATGAGCACTATCAAATACACCTTCATATTCACTGTTTTATTGTATTTAACAGAGTGTTAATATAAATATATTATGGCTGTTTTCTGTATTTGAATGATATATTTATTGTAATCCTCTCATGCAGCATTGAAATAAAAGATATACTTTTATCAC) was inserted into the Nhe I and Xho I sites of the pmirGLO vector to construct the wild plasmid pmirGLO-IL-4 / 13A 3'UTR-WT.

[0021] The logarithmic phase HKT-293T cells were inoculated into a 24-well plate at a density of 2x10 5 cells per well, and cultured to 70%-80% confluence. Then, the miR-301a mimic and the wild plasmid pmirGLO-IL-4 / 13A 3'UTR-WT or the mutant plasmid pmirGLO-IL-4 / 13A-3'UTR-MUT were co-transfected into the cells, wherein the final concentration of the miR-301a mimic was 50 nm / well, and the final concentration of the plasmid was 200 ng / well. After 36 h of culture, the luciferase activity of the cell samples was detected by using a dual luciferase reporter system detection kit, and the Renilla luciferase activity was used as an internal reference. The results are shown in Table 1. Figure 1As shown, the miR-301a mimic can significantly inhibit the luciferase activity of pmirGLO-IL-4 / 13A 3'UTR-WT, but has no significant effect on the luciferase activity of pmirGLO-IL-4 / 13A-3'UTR-MUT. This result shows that the miR-301a mimic can bind to the 3'UTR region of the large yellow croaker IL-4 / 13A gene, thereby inhibiting the expression of the upstream gene.

[0022] Example 2: miR-301a mimic inhibits IL-4 / 13A gene mRNA level in large yellow croaker head kidney leukocytes 6 The miR-301a mimic was transfected into the large yellow croaker head kidney leukocytes, and the final concentration of the miR-301a mimic was 50 nm / well, and the cells were incubated in a 28℃, 5% CO2 incubator. NC mimic (NC miRics) was used as a control. After 24 h, total RNA was extracted from the large yellow croaker head kidney leukocytes, and the extracted RNA was reverse transcribed into cDNA using an RNA reverse transcription kit. The cDNA was used as a template for a fluorescent quantitative PCR reaction. The PCR reaction system was as follows: cDNA template 0.2 μL, 10 μM of upper and lower primers each 0.2 μL, SYBR Green Mix 10 μL, and sterile water 9.6 μL. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 57℃ annealing for 20 s, 72℃ extension for 20 s, 40 cycles, and finally 60-95℃ melting curve stage. The mRNA levels of endogenous miR-301a and IL-4 / 13A were detected, and U6 was used as the miRNA internal reference, and β-actin was used as the mRNA internal reference. The primer sequences used in the fluorescent quantitative PCR reaction are shown in Table 1. The results are shown in Figure 2 As shown, after transfection of the miR-301a mimic, the miR-301a level in the large yellow croaker head kidney leukocytes was significantly up-regulated, and the large yellow croaker IL-4 / 13A gene mRNA was significantly down-regulated, with a down-regulation amplitude of about 60%, indicating that the miR-301a mimic inhibits the large yellow croaker IL-4 / 13A gene mRNA level.

[0023] Table 1 Primers used in fluorescent quantitative PCR experiment Example 3: miR-301a mimics inhibit T cell and B cell proliferation in large yellow croaker head kidney leukocytes Leukocytes from the head kidney of yellow croaker were isolated using the same method as above, incubated with hydroxyfluorescein diacetate succinimidyl ester (CFSE) in the dark for 10 min, and then seeded into 24-well plates at 5 × 10 cells per well. 5 Cells were cultured in an incubator at 28°C and 5% CO2. After 2 hours, cells were transfected with miR-301a mimics or NC mimics at a final concentration of 50 nM and cultured at 28°C and 5% CO2 for another 24 hours. Cells were then stimulated with PBS or phytohemagglutinin (PHA) at a final concentration of 1 μg / mL for 48 hours. The stimulated cells were incubated with anti-Zap70 or anti-large yellow croaker IgM antibodies (dilution ratio 1:500) at 4°C in the dark for 30 minutes, washed twice with PBS containing 2% bovine serum albumin (BSA), and then goat anti-rabbit IgG antibodies conjugated to Alexa Fluor 647 (dilution ratio 1:1000) were added. The cells were incubated at 4°C in the dark for 1 hour, washed twice with PBS containing 2% BSA, and analyzed for Zap70 by flow cytometry. + T cells and IgM + The proliferation percentage of B cells. Figure 3 As shown, whether in the PBS control group or the PHA treatment group, after transfection with miR-301a mimics, Z ap70 + T cells and IgM + The proportion of proliferating cells in B cells was significantly lower than that in the random miRNA (NC mimic) transfection group, indicating that miR-301a mimics weakened the promoting effect of PHA on T cell and B cell proliferation by targeting large yellow croaker IL-4 / 13A, thereby weakening the adaptive immune response of large yellow croaker.

Claims

1. A miR-301a mimic, characterized in that: The miR-301a mimic comprises double-stranded RNA; wherein the sense strand sequence of the double-stranded RNA is: 5'-GUGCAAUAGUAUUGUCAAAGCA-3', and the antisense strand sequence is: 5'-UGCUUUGACAAUACUAUUGCAC-3'.

2. Use of the miR-301a mimic according to claim 1 in the preparation of a preparation for inhibiting the expression of IL-4 / 13A gene mRNA in large yellow croaker.

3. Use of the miR-301a mimetic according to claim 1 in the preparation of a drug for inhibiting the proliferation of T lymphocytes of large yellow croaker.

4. Use of the miR-301a mimetic according to claim 1 in the preparation of a medicament for inhibiting the proliferation of B lymphocytes of large yellow croaker.

5. Use of the miR-301a mimics according to claim 1 in the study of adaptive immune response in large yellow croaker.

Images