Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, fall within the scope of the present invention.
In the invention, miR-378a-5p mimics (a simulator) represents a simulator of miR-378a-5p, is synthesized by simulating miR-378a-5p in a living body by using a chemical synthesis method, and can enhance the expression quantity of miR-378a-5p. NC (negative control) of miR-378a-5p mimics represents a negative control of miR-378a-5p mimics, i.e., a sequence similar in structure to miR-378a-5p mimics but not meaningless, without over-expression of miR-378a-5p. The transfection of miR-378a-5p mimics with its corresponding NC was performed in order to exclude the effect of transfection of such sequences on the experimental results.
MiR-378a-5p mimics used in the invention is double-stranded:
The sense strand sequence is CUCCUGACUCCAGGUCCUGUGU;
The antisense strand sequence is ACAGGACCUGGAGUCAGGAGUU.
In the invention, miR-378a-5p inhibitor represents an inhibitor of miR-378a-5p, and is a chemically modified inhibitor specifically aiming at miR-378a-5p in cells. NC of miR-378a-5pinhibitor represents a negative control of miR-378a-5p inhibitor, namely a sequence similar to miR-378a-5pinhibitor in structure but nonsensical, and does not influence expression of miR-378a-5 p.
The miR-378a-5p inhibitor used in the invention is single-chain ACACAGGACCUGGAGUCAGGAG.
In the present invention, the NC of miR-378a-5p is an artificially designed double-stranded miRNA molecule, and does not target any known human, mouse and rat genes.
In the present invention, siRNA-NC is a common negative control that has no homology to the sequence of the target gene and has no homology to other genes in the target cell.
The above mimics, inhibitors or NC's may be designed and obtained by those skilled in the art without specific explanation based on conventional knowledge.
The invention is further illustrated by the following examples.
Example 1
In the embodiment, the effect of miR-378a-5p on initiating CD4+ T cell differentiation is verified by using mouse spleen initiating CD4+ T cells, and the targeting relationship between miR-378a-5p and a target gene BRAF is verified.
1. Isolation and culture of mouse spleen-initiated CD4+ T cells:
(1) Grinding spleen, namely dissecting and killing a mouse, taking the spleen aseptically, placing the mouse in a 70 mu m filter membrane, adding sample diluent, lightly grinding the mouse, finally preparing 5ml of cell suspension, and blowing and uniformly mixing the mouse and the sample diluent;
(2) Separating, namely adding 5ml of mouse spleen lymphocyte separating liquid into a 15ml centrifuge tube, tilting the centrifuge tube, slowly adding the cell suspension of the last step into the centrifuge tube twice, centrifuging for 20min at 450xg and 22 ℃;
(3) Cleaning, namely separating the solution into 4 layers after centrifugation, discarding the upper liquid layer, carefully sucking the white membrane layer into another clean centrifuge tube, adding 10ml of cleaning solution, 350xg, and centrifuging at 22 ℃ for 10min, and cleaning twice;
(4) Counting, namely finally, resuspending the count by using a complete culture medium;
(5) After resuspension counting, 40. Mu.L of magnetic bead sorting buffer, 10. Mu.L of magnetic bead sorting buffer was added per 1X 107 cellsCD4+ T Cell Biotin-Antibody Cocktail, incubated at 4℃for 5min in the absence of light;
(6) 20. Mu.L of magnetic bead sorting buffer, 20. Mu.L of Anti-biotin MicroBeads, 10. Mu.L of CD44 microblades were added to each 1X 107 cell, and incubated at 4℃for 10min in the absence of light;
(7) Fixing a sorting column to prepare sorting, operating the process on ice, diluting the incubated cell suspension, firstly wetting the sorting column, then adding a certain amount of magnetic bead sorting buffer, adding the cell suspension for multiple times, and collecting the cell suspension by using a 15ml centrifuge tube;
(8) After sorting, 350Xg,4 ℃, centrifuging for 10min, and then re-suspending with 1640 complete medium for later use.
2. Transfection of miR-378a-5p mimics, inhibitor:
(1) Firstly, dissolving miR-378a-5p mimics, miR-378a-5p inhibitor and negative control according to the specification to form a storage solution with the concentration of 20 mu M;
(2) Anti-CD3 coated in 24-well plates the previous day was then discarded, and initial CD4+ T cells from magnetic bead sorting were plated in well plates (400. Mu.L/well);
(3) The transfection mimic concentration was calculated and the transfection system was formulated as follows in tables 1 and 2:
table 1 transfection System of miR-378a-5p mimics
TABLE 2 transfection of miR-378a-5p inhibitor
(4) After preparing a transfection system, lightly flicking and uniformly mixing fingers, standing at room temperature for 8min, dripping the mixture into a corresponding pore plate according to groups, and placing the mixture into a 37 ℃ and 5% CO2 incubator for culture;
(5) At the time of culturing for 6 hours, cytokines that differentiated the primary CD4+ T lymphocytes in Th1 and Th2 directions were added, respectively, as shown in tables 3 and 4:
TABLE 3Th1 polarization factor
TABLE 4Th2 polarization factor
(6) Culturing is continued for 48 hours, and the cells are harvested for subsequent experiments.
3. Transfection of siRNA-BRAF, OE-BRAF:
The target gene of miR-378a-5p is predicted through a bioinformatics website, and the target gene is proved to be BRAF by the double-luciferase reporter gene. The target genes of miR-378a-5p are predicted by TARGETSCAN, MIRWALK, MIRDB pieces of bioinformatics software, and through double-luciferase report analysis, the BRAF wild type 3' UTR region is controlled by miR-378a-5p, while the mutation is not controlled by miR-378a-5 p.
The interference fragments of the target gene BRAF are respectively siRNA-BRAF-972, siRNA-BRAF-1341 and siRNA-BRAF-1845/siRNA-NC, and the interference efficiency is detected by qRT-PCR. And (3) transfecting the siRNA into a mouse spleen initialized CD4+ T cell, and finally screening out the siRNA-BRAF-1341 fragment with the best interference effect by utilizing a qRT-PCR technology to carry out subsequent experiments. The sequence of the synthesized interference fragment is as follows, siRNA-BRAF-972:sense:5'-CCAACUUGAUUUGCUGUUUTT-3';
Anti-sense:5′-AAACAGCAAAUCAAGUUGGTT-3′;
siRNA-BRAF-1341:Sense:5′-CCAACUGAUGCGCUGUCUUTT-3′;
Anti-sense:5′-AAGACAGCGCAUCAGUUGGTT-3′;
siRNA-BRAF-1845:Sense:5′-GCCACAACUGGCAAUUGUUTT-3′;
Anti-sense:5′-AACAAUUGCCAGUUGUGGCTT-3′。
the above-mentioned interference fragment and control fragment siRNA-NC were synthesized by Shanghai Ji Ma pharmaceutical technologies Co.
Packaging target gene BRAF over-expressed slow virus. Firstly, downloading CDS sequences of BRAF genes at NCBI, delivering Feng Hui to biologically construct BRAF over-expression plasmids, inserting the BRAF over-expression plasmids into pLVX-EGFP-IRES-PURO, taking empty vector as a reference, carrying out plasmid amplification and plasmid extraction, packaging recombinant plasmid lentivirus to obtain the BRAF over-expression lentivirus, and verifying transfection efficiency through qRT-PCR.
SiRNA-BRAF-1341 and OE-BRAF are transfected into mouse spleen initiating CD4+ T cells, and the influence of target gene BRAF on initiating CD4+ T cell differentiation is studied. The siRNA-BRAF transfection system is shown in Table 5.
Table 5siRNA-BRAF transfection system:
Transfection of OE-BRAF 1.5 mu L Lipo 3000 is added per 100 mu L virus, mixed by flick and then kept stand for 10min, then added into cells, cultured for 6h, added with corresponding polarization factors, and further cultured for 48h for subsequent experiments.
4. Cell total RNA extraction and qRT-PCR embodiment:
In the invention, qRT-PCR detection of target genes and miRNA are respectively adoptedBioscience Co LtdOne STEP RT QPCR KIT (Sybr Green) and Ji Ma company miRNA qRT-PCR TBKit。
The total RNA extraction step is described in TRIZOL operating instructions of Thermofisher, and the specific extraction steps are as follows:
(1) Adding 1mL TRIZOL per 1×107 cells, blowing with a pipette until the liquid is clear and no cell mass exists, mixing for about 10 times, and standing at room temperature for 10min;
(2) Adding 200 μl (1/5 volume of the total amount) of chloroform in dark place, vigorously mixing for 15s, and standing at room temperature for 5min;
(3) Centrifugation at 12000Xg,4℃for 15min, carefully aspirate the upper aqueous layer (approximately 400-500. Mu.L, which is contraindicated for aspiration of the middle layer) into a fresh 1.5mL EP tube;
(4) Adding isopropyl alcohol with equal volume, blowing and mixing uniformly, and standing at room temperature for 10min;
(5) Centrifuging at 4deg.C, 12000Xg, and 10min, discarding supernatant (RNA forms white gel-like particles at bottom);
(6) 1mL of precooled 75% alcohol (used as prepared) is added;
(7) Centrifuging at 4 ℃ for 5min at 7500xg, discarding the supernatant (leaving a point at the bottom, sucking the supernatant out by a 10 mu L pipette), and drying by a super clean bench for 5min;
(8) Adding 20-30 mu L of enzyme-free water, taking 1 mu L of RNA to detect purity and content, and using the rest for reverse transcription.
QRT-PCR was performed according to the real-time fluorescent quantitative PCR reagent specifications, the reaction system is shown in Table 6, and the reaction conditions are shown in Table 7.
TABLE 6qRT-PCR reaction System
Table 7qRT-PCR reaction conditions:
The experimental result is used for detecting the content of the sample gene or miRNA by a Ct value comparison method, and the specific calculation formula is as follows:
gene relative expression level=2- { < (test group objective gene Ct value) - (test group internal gene Ct value) > (control group objective gene Ct value) - (control group internal gene Ct value) >
Wherein, the reference gene is used as reference for GAPDH for gene detection and U6 for miRNA detection.
The results are shown in FIG. 1-FIG. 5:
As can be seen from panel A in FIG. 1, the expression of miR-378a-5p was elevated 48h after transfection of miR-378a-5p mimics at different concentration gradients into mouse spleen naive CD4+ T cells, but the expression of miR-378a-5p was no longer increased with increasing concentration when the transfection concentration reached 60 nM.
As can be seen from the graph B in FIG. 1, the miR-378a-5p inhibitor with different concentration gradients is reduced in the expression of miR-378a-5p after being transfected into the initial CD4+ T cells of the spleen of the mouse for 48 hours, wherein the reduction effect is obvious at the concentration of 100nM, and the reduction effect is not obvious at the concentration of 150nM, and the method has no statistical significance.
FIG. 2 shows the differentiation of mice spleen-initiated CD4+ T cells after transfection of 60nM miR-378a-5p mimics or 100nM miR-378a-5pinhibitor to reflect the effect of miR-378a-5p on initiating CD4+ T cell differentiation. As can be seen from FIG. 2, transfection of 60nM miR-378a-5p mimics promoted expression of the Th 1-type cell-representing factor IFN-gamma and transcription factor T-bet (panels A and B) and inhibited expression of the Th 2-type cell-representing factor IL-4 and transcription factor GATA-3 (panels E and F), and transfection of miR-126a-5p inhibitor inhibited expression of the Th 1-type cell-representing factor IFN-gamma and transcription factor T-bet (panels C and D) and promoted expression of the Th 2-type cell-representing factor IL-4 and transcription factor GATA-3 (panels G and H).
FIG. 3 shows the interference effects of different interference fragments siRNA-BRAF-972, siRNA-BRAF-1341 and siRNA-BRAF-1845/siRNA-NC and OE-BRAF. As can be seen from panel B, the interference effect of the siRNA-BRAF-1341 fragment is the best.
FIG. 4 is a graph showing the differentiation of mouse spleen-initiated CD4+ T cells after transfection of OE-BRAF and siRNA-BRAF-1341. As can be seen from FIG. 4, after transfection of siRNA-BRAF-1341, the levels of the Th1 type cytokine IFN-gamma and transcription factor T-bet mRNA are significantly increased (panels C and D), while the levels of the Th2 type cytokine IL-4 and transcription factor GATA-3mRNA are significantly decreased (panels G and H), after transfection of OE-BRAF, the levels of the Th1 type cytokine IFN-gamma and transcription factor T-bet mRNA are significantly decreased (panels A and B), while the levels of the Th2 type cytokine IL-4 and transcription factor GATA-3mRNA are significantly increased (panels E and F).
FIG. 5 shows the result of the function recovery of miR-378a-5p target gene BRAF. As can be seen from fig. 5, after co-transfection of miR-378a-5p and BRAF in mouse spleen-initialized CD4+ T cells, the promotion of differentiation of miR-378a-5 p-initiated CD4+ T cells toward Th1 direction can be restored by BRAF.
5、Western blot
(1) SDS-PAGE gels were prepared. Formulated as in table 8.
TABLE 8SDS-PAGE gel composition
(2) Adding samples according to the sequence of a marker, a sample 1 and a sample 2, wherein the voltage is set to be 80V, when bromophenol blue runs to about 1 cm below the separation gel, the voltage is adjusted to be 120V, continuing electrophoresis for about 2 hours, and when bromophenol blue runs to the bottom of the glass plate, stopping electrophoresis;
(3) Transferring membrane, namely firstly placing PVDF membrane in methanol to activate for about 8-10 minutes, then immersing a membrane transferring clamp, filter paper and the like in membrane transferring liquid pre-cooled in advance, peeling SDS-PAGE gel, clamping the membrane transferring clamp according to the sequence of a red plate, a sponge cushion, the filter paper, the PVDF membrane, the gel, the filter paper, the sponge cushion and a blackboard, placing the membrane transferring clamp in a membrane transferring groove, switching on a power supply of an electrophoresis apparatus, setting constant current 250mA for 2 hours, and starting membrane transferring;
(4) Sealing, namely preparing 5% skimmed milk, placing the skimmed milk on a shaking table at normal temperature, and sealing for 2 hours at 65 rpm/min;
(5) Washing the membrane with TBST, at 110rpm/min, repeating for three times each for 6 min;
(6) An antibody is prepared in advance, a PVDF membrane is placed in the prepared antibody, and the PVDF membrane is incubated by a shaking table at 4 ℃ overnight;
(7) Washing the membrane, namely recovering the primary antibody, and washing the membrane by TBST three times for 6min each time;
(8) Secondary antibody incubation, namely preparing the secondary antibody in advance, placing the PVDF film into the prepared antibody, and incubating for 2 hours on a shaking table at normal temperature;
(9) Washing the membrane, namely recovering the secondary antibody, and washing the membrane by TBST for three times, wherein each time is 6min;
(10) And (3) exposure, namely preparing ECL chemiluminescent liquid, photographing through an imaging system, storing a result and analyzing data.
The results are shown in fig. 6 and 7:
as can be seen from FIG. 6, the transfection of miR-378a-5p mimics can promote the expression of the Th1 cell representative factor IFN-gamma and inhibit the expression of the Th2 cell representative factor IL-4, and the transfection of miR-378a-5p inhibitor can inhibit the expression of the Th1 cell representative factor IFN-gamma and promote the expression of the Th2 cell representative factor IL-4.
As can be seen from FIG. 7, after transfection of siRNA-BRAF-1341, the expression of the Th1 type cytokine IFN-gamma protein is obviously increased, while the expression of the Th2 type cytokine IL-4 is obviously decreased, and after transfection of OE-BRAF, the expression of the Th1 type cytokine IFN-gamma protein is obviously decreased, while the expression of the Th2 type cytokine IL-4 is obviously increased. The above findings indicate that BRAF inhibits the differentiation of mouse spleen naive CD4+ T cells in Th1 direction, promoting their differentiation in Th2 direction.
6. Flow cytometry
(1) 1 Μl (24 well plate) cell stimulation cocktail and protein transport inhibitor (500×) was added before harvesting the cells for 5h;
(2) The cell quantity of each tube is regulated to be 1 multiplied by 106, and the tube is resuspended by buffer 2;
(3) Surface dyeing, namely adding PerCP-Cy5.5-CD3 and APC-CD4 into the mixture, mixing the mixture uniformly by vortex, and incubating the mixture for 30min at 4 ℃ in a dark place;
(4) Washing, namely adding 1mL buffer 2,350xg,4 ℃ into each tube, and centrifuging for 8min;
(5) Fixing, namely adding 600 mu L of 4% paraformaldehyde into each tube, and fixing for 15min at normal temperature in a dark place;
(6) Washing, namely adding 1mL buffer 2,350xg,4 ℃ into each tube, and centrifuging for 8min;
(7) 1mL buffer 3 is added to each tube, and the membrane is broken overnight at 4 ℃ in the dark;
(8) Intracellular staining, namely adding 100 mu L buffer 3 into each tube for re-suspension after centrifugation for 8min at 350xg and 4 ℃ in the next day, adding 2 mu L PE-IFN-gamma or PE-IL-4, and incubating for 30min at 4 ℃ in the dark;
(9) Washing, namely adding 1mL buffer 2,350xg,4 ℃ into each tube, and centrifuging for 8min;
(10) And (3) loading the sample, namely, re-suspending the sample by using about 350 mu L buffer 2, and loading the sample after filtration.
The results are shown in FIG. 8-FIG. 10:
as can be seen from FIG. 8, the transfection of miR-378a-5p mimics can promote the expression of the Th1 cell representative factor IFN-gamma and inhibit the expression of the Th2 cell representative factor IL-4, and the transfection of miR-378a-5p inhibitor can inhibit the expression of the Th1 cell representative factor IFN-gamma and promote the expression of the Th2 cell representative factor IL-4.
As can be seen from FIG. 9, after transfection of siRNA-BRAF-1341, the expression of the Th1 type cytokine IFN-gamma protein is obviously increased, while the expression of the Th2 type cytokine IL-4 is obviously decreased, and after transfection of OE-BRAF, the expression of the Th1 type cytokine IFN-gamma protein is obviously decreased, while the expression of the Th2 type cytokine IL-4 is obviously increased. The above findings also demonstrate that BRAF inhibits differentiation of mouse spleen naive CD4+ T cells in Th1 direction, promoting differentiation in Th2 direction.
FIG. 10 is a result of the recovery of the function of the miR-378a-5p target gene BRAF. As can be seen from fig. 10, after co-transfection of miR-378a-5p and BRAF in mouse spleen-initialized CD4+ T cells, the promotion of differentiation of the initialized CD4+ T cells to Th1 direction caused by miR-378a-5p can be restored by BRAF.
Example 2
In the embodiment, the targeting relationship between miR-378a-5p and BRAF is detected through a double-luciferase reporter gene.
Referring to the Dual-Luciferase ReporterAssay System kit from Promega, the specific steps are as follows:
(1) 293T cells and plasmids of interest, which are divided into 96-well plates, are prepared in advance for transfection, preferably until the cell density reaches 50% -70%.
(2) Mu.L of DMEM was thoroughly mixed with 0.16. Mu.g of Dlk1-3UTR target plasmid and 5pmol of mmu-miR-378a-5p/NC, and then left at room temperature (solution A), after which 10. Mu.L of DMEM was thoroughly mixed with 0.3. Mu.L of transfection reagent (0.8 mg/ml) (solution B), and left at room temperature for 5min.
(3) And (3) fully and uniformly mixing the solution A and the solution B, and standing at room temperature for 20min.
(4) Cells were exchanged for fresh medium prior to transfection, and the transfection mixture was added to mix. Culturing at 37deg.C with 5% CO2.
(5) Fresh medium was exchanged after 6h of transfection and cell detection was collected after 48h of transfection.
(6) The ratio of the expression quantity of the Renilla luciferase to the firefly luciferase is the relative activity of the Renilla luciferase, namely the activity of the corresponding target gene (three repeats).
Experimental results:
Construction of the BRAF wild-type (wt) 3' UTR and 3' UTR containing the sequence mutation of the predicted binding site into psiCHECK-2, by double luciferase reporter assay, it was found that the BRAF wild-type 3' UTR region was regulated by miR-378a-5p, whereas the mutant was not regulated by miR-378a-5 p. As shown in fig. 11.
Example 3
This example demonstrates the expression of miR-378a-5p and BRAF after rEg.P29 immunization of mice.
(1) Expression and purification of rEg.P29. The recombinant strain preserved in the laboratory in earlier stage is selected, induced and amplified for culture, and then purified by utilizing an affinity chromatography method.
(2) The experimental animals were grouped, immunized and obtained by randomly dividing 18 female Balb/c mice of 6-8 weeks of age into three groups (6 in each group) of PBS group, adjuvant group and rEg.P29+ adjuvant group. The immunization protocol was PBS group (100. Mu.L PBS), adjuvant group (50. Mu.L PBS+50. Mu.L adjuvant), rEg.P29+adjuvant group (20. Mu.g.P29+50. Mu.L PBS+50 MLFCA), immunization 2 times, 1 week apart, two times each with three subcutaneous injections into the abdomen, wherein the first adjuvant was Freund's complete adjuvant and the second adjuvant was Freund's incomplete adjuvant. After 1 week of the second immunization, the magnetic beads sorted spleen CD4+ T cells and RNA and protein were extracted.
(3) RNA expression conditions of miR-378a-5P and BRAF are detected by using a qRT-PCR technology, and BRAF protein expression conditions are detected by using a Western blot technology, so that the result is shown in figure 12, compared with PBS and an adjuvant group, miR-378a-5P expression in a P29+ adjuvant group is obviously increased, and the expression level of BRAF is obviously reduced.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.