CN116650513A - Application of miR-29 in preparation of preparation for regulating cell energy metabolism and cell stem property - Google Patents
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Abstract
The invention provides an application of miR-29 in preparation of a preparation for regulating cell energy metabolism and cell stem property, wherein the miR-29 can be knocked out to improve mitochondrial oxidative phosphorylation level of mouse intestinal stem cells by activating fatty acid oxidation, so that the intestinal stem cell stem property is inhibited; meanwhile, the inhibition of miR-29 in a humanized colorectal tumor cell line can promote the oxidative phosphorylation level mediated by fatty acid oxidation by up-regulating target gene Hnf4g, so as to inhibit the expansion of intestinal tumor cells, and the inhibition of miR-29 or up-regulating the target gene thereof can play an anticancer role by remodelling the energy metabolism of tumor cells, thereby providing a theoretical basis for preparing medicines for regulating the energy metabolism of stem cells.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to the field of relationship between miRNAs and metabolic pathways.
Background
With the development of modern science and technology, nucleic acid-based drugs are being developed and utilized. microRNA (miRNA) is a non-coding small RNA consisting of 20-24 nucleotides that functions primarily by binding to the mRNA of the target gene, inhibiting its transcription or inducing its degradation. Many mirnas have been shown to be involved in regulating cancer genes and are widely used in the treatment of various diseases such as IBD. In the earlier study, miR-29 can promote the repair of the radiation injury of the small intestine of a mouse, the radiation injury repair process mainly depends on regeneration and dedifferentiation of intestinal stem cells, so that the miR-29 is presumed to possibly regulate the renewal and differentiation fate of the intestinal stem cells, and simultaneously, hnf4g is also found to be a target gene of miR-29, and Hnf4g is a transcription factor involved in regulating fatty acid oxidation, so that miR-29 can regulate mitochondrial fatty acid oxidation of the intestinal stem cells, however, no clear evidence exists for regulating energy metabolism and dryness of the intestinal stem cells by miR-29, and no report exists on the application of miR-29 in medicines for regulating cell energy metabolism.
Disclosure of Invention
The technical problem to be solved by the application is to provide the application of miR-29 in medicines for regulating stem cell energy metabolism, discuss the action mode and the action effect of miR-29 on stem cell energy metabolism, and then develop the preparation by taking miR-29 or a target gene thereof as a target point and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
On one hand, the application adopts CRISPR/Cas9 technology and prokaryotic injection to construct miR-29ab1 knockout mice, andLgr5mating and breeding of mice with-eGFP-IRES-CreERT 2 transgenic mode to obtain Lgr5 + The intestinal stem cells are fluorescently marked on the mice, and the oxidation activity and the energy metabolism level of the fatty acid of the intestinal epithelial cells are evaluated from the aspects of metabolic products, related enzyme expression, mitochondrial respiration and the like by separating the intestinal stem cells of the mice through a flow type, separating and culturing intestinal organoids; the intestinal stem cell stem property of the miR-29ab1 knockout mice is evaluated through stem cell number and in combination with cell cycle analysis.
On the other hand, in Lgr5 + Liposome transfection of miR-29a and/or miR-17 a in human colorectal tumor cell lineThe way of 29b inhibitor inhibits miR-29a/b function, and the cellular fatty acid oxidation and oxidative phosphorylation levels are evaluated by measuring substrate metabolism levels, related enzyme expression, ATP content and mitochondrial respiration; assessing intestinal tumor cell dryness by cell staining and cell cycle analysis, and verifying whether energy metabolism participates in dryness regulation by using a fatty acid oxidation activator; meanwhile, the miR-29a/b target gene Hnf4g is overexpressed, and whether the regulation of Hnf4g can regulate the energy metabolism and the dryness of stem cells is verified.
Specifically, the invention provides the following technical scheme:
in a first aspect, there is provided an application of miR-29a or miR-29b in preparation of a preparation for promoting intestinal cell stem property; specifically, miR-29a or miR-29b promotes intestinal cell stem property by inhibiting Hnf4g expression.
In a second aspect, there is provided an application of miR-29a or miR-29b in preparation of a preparation for inhibiting energy metabolism of intestinal tumor cells; in particular, the energy metabolism includes fatty acid oxidative activity, mitochondrial maximum respiratory availability, and/or Etomoxir sensitivity.
In a third aspect, there is provided an use of an inhibitor of miR-29a or miR-29b in the preparation of a formulation for inhibiting intestinal tumour cell stem activity; specifically, miR-29a or miR-29b promotes intestinal cell stem property by inhibiting Hnf4g expression.
In a fourth aspect, the use of an inhibitor of miR-29a or miR-29b in the preparation of a formulation that promotes energy metabolism of an intestinal tumor cell line; in particular, the energy metabolism includes fatty acid oxidative activity, mitochondrial maximum respiratory availability, and/or Etomoxir sensitivity.
In a fifth aspect, there is provided an application of an Hnf4 g-related biological agent in preparing a drug for promoting energy metabolism of intestinal cells or inhibiting stem cell properties of intestinal cells, the Hnf4 g-related biological agent comprising: 1) Nucleic acid molecules encoding Hnf4 g; 2) Hnf4g protein; 3) An expression cassette comprising 1) said nucleic acid molecule; 4) A vector comprising 3) said expression cassette; 5) A cell comprising 1) said nucleic acid molecule or 3) said expression cassette or 4) said vector; 6) A formulation that promotes expression of Hnf4 g; preferably, the preparation of 6) promoting the expression of Hnf4g is an inhibitor of miR-29a or miR-29 b.
Drawings
FIG. 1 shows a schematic diagram of intracellular fatty acid metabolic pathways.
FIG. 2miR-29ab1 -/- Changes in mRNA levels of fatty acid oxidation-related enzymes in the mouse intestinal crypt.
FIG. 3miR-29ab1 -/- Changes in maximum respiration availability and etoloxir sensitivity of mice intestinal organoids: the A maximum respiratory availability is obviously increased; b: etoloxir sensitivity increases significantly.
FIG. 4miR-29ab1 -/- The number of stem cells in the intestinal organoids of mice varies.
FIG. 5 shows changes in the fatty acid metabolism efficiency of LS174T cell lines following transfection of miR-29a/b inhibitors.
FIG. 6 LS174T cell line granule energy metabolism changes after transfection of miR-29a/b inhibitors.
FIG. 7 LS174T cell line cell stem changes after transfection of miR-29a/b inhibitors.
FIG. 8 LS174T cell line Hnf4g was altered at the mRNA and protein levels after transfection of miR-29a/b inhibitors.
FIG. 9 overexpression of Hnf4g causes fatty acid oxidation in colorectal tumor cells, mitochondrial energy metabolism, and changes in cell stem properties.
FIG. 10 activation of fatty acid oxidation results in increased differentiation of LS174T cell lines and dry changes
Detailed Description
EXAMPLE 1 Regulation of fatty acid oxidation by MiR-29 deficiency in intestinal Stem cells
As shown in FIG. 1, fatty acid is transported into cell mitochondria from CPT1A, beta-oxidation occurs under the action of enzymes such as ACADM, acetyl-carnitine is produced during the time, part of the final product acetyl-CoA synthesizes ketone body under the action of HMGCS2, and part of the final product acetyl-CoA enters TCA circulation and ATP is produced through oxidative phosphorylation. The level of fatty acid oxidation was therefore explored through three aspects of product, related enzyme expression and productivity.
1. Animal experiment
C57BL/6N strain WT (wild type) mice and miR-29ab1 -/- (miR-29 ab1 whole body knockout) mice are fed into SPF-class animal houses, the room temperature is 20-22 ℃, the humidity is 50-60%,12 h light/dark cycles and free drinking water are adopted, and the mice are fed with normal feed. Mating with Lgr5-eGFP-IRES-CreERT2 transgenic mice to obtain the final products, namely, miR-29ab1+/+ mice and Lgr5-eGFP, and miR-29ab 1-/-mice.
2. Mouse intestinal crypt separation
(1) The mice were euthanized, the whole small intestine was dissected out, the washed-out sections of intestine were cut longitudinally, and rinsed several times in pre-chilled PBS.
(2) The round slide was sanded at 45 ° to scrape the inner wall surface of the small intestine and remove the fluff.
(3) The intestines were placed in a 50 mL centrifuge tube containing 20 mL pre-chilled PBS, the centrifuge tube 100 was shaken, the turbid liquid was poured out, fresh pre-chilled PBS was added, and the above procedure was repeated 10 times.
(4) After the washing, a new 20. 20 mL precooled PBS was added, 200. Mu.L of 0.5M EDTA solution was added, and the mixture was shaken on a horizontal shaker at room temperature for 15 min.
(5) The digestate was poured out, loaded into 10 mL fresh pre-chilled PBS, transferred through a 70 μm cell sieve to a fresh 50 mL centrifuge tube under vigorous shaking 100, repeated 3 times, and concentrated in the same centrifuge tube.
(6) Centrifuge at 200 Xg for 5 min, pour out the liquid and precipitate as mouse intestinal crypt.
3. Fatty acid oxidative activity assay
(1) The mouse intestinal crypts were extracted and the pellet was resuspended in RPMI sugarless medium and 400 μl/well added to a 24-well plate.
(2) mu.L of 20 mM [ U ] 13 C]Mixing the marked palmitic acid mother solution with 100 μl of RPMI sugarless medium, adding 100 μl/well into the crypt suspension, mixing uniformly, and adding 37 ℃ and 5% CO 2 Culture 1 h.
(3) The crypts were collected into a 1.5 mL centrifuge tube, centrifuged at 200 Xg for 5 min at 4℃and the pellet was stored at-80 ℃.
(4) Extracting metabolite from the crypt precipitate with 25% methanol, 25% acetonitrile and 50% water, swirling at 4deg.C for 10 min, centrifuging at 12000 rpm for 10 min at 4deg.C, and vacuum centrifuging and drying the supernatant.
(5) The precipitate was redissolved with 100. Mu.L of chromatographic grade water, 1. Mu.L of sample was taken and analyzed by LC-MS detection. A ZIC-pHILIC 2.1 x 150 mm (5 μm) column was used, QExactive Orbitrap mass spectrometer.
(6) The flow rate of the liquid chromatography mobile phase is 0.15 mL/min, and the ultraviolet detection wavelengths are 245 nm and 210 nm. Mobile phase A was 20 mM NH 4 HCO 3 + 0.1% NH 3 ·H 2 O, wherein the mobile phase B is acetonitrile, the mobile phase A is 20 percent of the mobile phase A+80 percent of the mobile phase B in 0-20 min, and the mobile phase A is 80 percent of the mobile phase A+20 percent of the mobile phase B in 20-30 min.
(7) The mass spectrum adopts an MRM scanning mode, the spraying voltage is 4.5 kV (neg) and 5.5 kV (pos), the temperature is 500 ℃, and the air curtain is 20 psi,GS1 55 psi,GS2 55 psi.
(8) Detection of acetylcarnitine, citric acid, [ M+2] acetylcarnitine, [ M+2] citric acid.
4. Small intestine crypt Lgr5 + Altered gene expression profiles at cellular mRNA levels
(1) To the crypt pellet isolated from the Lgr5-eGFP mice was added 2 mL single cell digests for resuspension, with a water bath at 37 ℃ for 4 min.
(2) The pre-chilled PBS was supplemented to 10 mL, transferred through a 40 μm cell sieve to a new 50 mL centrifuge tube, and digestion was stopped by adding 5% fetal bovine serum.
(3) Centrifugation at 500 Xg for 5 min, pouring off the liquid, resuspending the pellet with 1. 1 mL pre-chilled PBS, transferring to a 1.5 mL centrifuge tube, and obtaining a single cell suspension.
(4) 10. Mu.L of 7-AAD was added, mixed well and kept away from light on ice for 15 min.
(5) Centrifugation was performed at 500 Xg for 5 min at 4℃and the pellet was resuspended in PBS pre-chilled by 1 mL and transferred to a flow tube through a 40 μm cell sieve.
(6) Flow-sorting of the Lgr5high7-AAD-, lgr5negative 7-AAD-cells was performed by receiving 100. Mu.L of lysate RL and storing it at-80 ℃.
(7) And extracting total RNA of the cells by using a micro RNA extraction kit.
(8) The mRNA was inverted to cDNA using a reverse transcription kit.
(9) The gene expression is detected by adopting a real-time fluorescence quantitative PCR method, and the primer sequences are as follows:
Lgr5
F：5’-ACATTCCCAAGGGAGCGTTC-3’（SEQ ID NO:1）
R：5’-ATGTGGTTGGCATCTAGGCG-3’(SEQ ID NO:2)
Olfm4
F：5’-CAGCCACTTTCCAATTTCACTG-3’(SEQ ID NO:3)
R：5’-GCTGGACATACTCCTTCACCTTA-3’(SEQ ID NO:4)
Ascl2
F：5’-AAGCACACCTTGACTGGTACG-3’(SEQ ID NO:5)
R：5’-AAGTGGACGTTTGCACCTTCA-3’(SEQ ID NO:6)
Cpt1a
F：5’-CCATGAAGCCCTCAAACAGATC-3’(SEQ ID NO:7)
R：5’-ATCACACCCACCACCACGATA-3’(SEQ ID NO:8)
Acadm
F：5’-AGGGTTTAGTTTTGAGTTGACGG-3’(SEQ ID NO:9)
R：5’-CCCCGCTTTTGTCATATTCCG-3’(SEQ ID NO:10)
Hmgcs2:
F：5’-GAAGAGAGCGATGCAGGAAAC-3’(SEQ ID NO:11)
R：5’-GTCCACATATTGGGCTGGAAA-3’(SEQ ID NO:12)
as shown in the figure 2 of the drawings, 13 c-labeled palmitic acid enters small intestine crypt cells and is oxidized by fatty acid to generate 13 C-labeled acetylcarnitine and is produced after entering the TCA cycle 13 C-labeled citric acid, miR-29ab1 -/- Acetyl carnitine and citric acid in mouse intestinal crypt 13 The increase in the C-labelling rate proves that the oxidation activity of the fatty acid is increased. Lgr5 in a flow sort nest high Intestinal stem cells, found miR-29ab1 -/- mRNA level gene expression of fatty acid oxidation related enzyme of the intestinal stem cells of mice is up-regulated, which proves that the fatty acid oxidation activity rises in the intestinal stem cells.
5. Isolated culture of intestinal organoids
(1) Extracting small intestine crypts of mice under aseptic condition, adding 10 mL precooled DMEM/F12 culture medium to resuspend and precipitate, taking out 1 ten thousand crypts/cell mass according to counting result, and transferring to a 15 mL centrifuge tube.
(2) Centrifugation at 200 Xg for 5 min, supernatant removed, pellet resuspended in 60. Mu.L GM medium pre-chilled and transferred to a 1.5 mL centrifuge tube.
(3) 150. Mu.L of Matrigel was pipetted with a 200. Mu.L sterile gun head pre-chilled at-20℃and added to a 24-well plate pre-warmed at 37℃at 50. Mu.L/Kong Dian after being mixed with the resuspended crypt under pipetting 50.
(4) Pouring the inoculated pore plate into a 37 ℃ incubator for 15 min, taking out, adding 450 mu L of a 37 ℃ preheated IOGM mouse small intestine organoid culture kit culture medium (GM culture medium) into each pore, and directly placing the mixture into 37 ℃ and 5% CO 2 Culturing in incubator for about 5-7 days, during which organoid formation is observed with microscope every day.
(5) The culture medium was aspirated during passaging, 1 mL pre-chilled DMEM/F12 medium was added to each well, and matrigel was completely dispersed under 20 puffs.
(6) Transferring into 15 mL centrifuge tube, centrifuging at 200 Xg for 5 min, re-suspending with GM culture medium, blowing 200 to obtain crypt suspension, inoculating into 24-well plate with the same operations (3) and (4), and culturing for 3-4 days for continuous passage.
6. Intestinal organoid mitochondrial energy metabolism level detection
(1) Media were prepared (4 ℃ C. For one day):
test medium: 10 mM glucose, 5 mM sodium pyruvate, 2 mM glutamine were added to SeaHorse basal medium.
(2) Organoids were cultured in 24-well plates 36 h, 4-well organoids were inoculated evenly into 20 wells of a Seahorse 24-well plate, 3 μl was inoculated per well, and culture was continued by adding 200 μl of organoid medium for 30 h.
(3) The 12-24 h hydrated probe card before the machine is put in a common incubator at 37 ℃ without adding CO 2 )。
(4) The medium was removed, rinsed with 500. Mu.L mitochondrial pressure test medium, then 525. Mu.L mitochondrial pressure test medium per well was added, and incubated in a common 37℃incubator (without additional CO 2 ) And (5) standing for 1 to h.
(5) Drugs were sequentially added to the probe wells at 75 μl per well, with the upper left well being 40 μM oligomycin, the upper right well being 18 μM FCCP, and the lower left well being 40 μM rotenone.
(6) And (3) loading, namely selecting a mitochondrial pressure test program and modifying the program: basal, oligomycin, FCCP, rotenone, 3 cycles of mixing for 4 min, waiting for 0 min, and measuring for 2 min, except for the 1 st cycle of oligomycin addition, which is mixing for 5 min, waiting for 10 min, and measuring for 2 min, to obtain change of mitochondrial Oxygen Consumption Rate (OCR), since mitochondrial respiratory chain is coupled with oxidative phosphorylation, basal OCR level is affected by oxidative phosphorylation level, FCCP is uncoupler, oxygen consumption rate is not affected by oxidative phosphorylation after addition, maximum respiration capacity of mitochondria is reflected, and the ratio of Basal OCR in maximum OCR is maximum respiration availability to reflect oxidative phosphorylation level.
7. Intestinal organoid fatty acid oxidative energy level detection
(1) Media were prepared (4 ℃ C. For one day):
restriction medium: 0.5 mM glucose, 0.5 mM carnitine, 1 mM glutamine, 50 ng/mL EGF, 100 ng/mL Noggin, 500 ng/mL R-spondin 1, 1.333% B-27,1% N-2 were added to SeaHorse basal medium.
Test medium: 2.5 mM glucose, 0.5 mM carnitine was added to SeaHorse basal medium.
(2) Organoids were cultured in 24-well plates 36 h, 4-well organoids were inoculated evenly into 20 wells of a Seahorse 24-well plate, 3 μl was inoculated per well, and culture was continued by adding 200 μl of organoid medium for 30 h.
(3) The 12-24 h hydrated probe card before the machine is put in a common incubator at 37 ℃ without adding CO 2 )。
(4) The organoid medium was replaced with 200. Mu.L of restriction medium for continued culture 6 h
(5) The medium was removed, rinsed with 500. Mu.L of test medium, then 405. Mu.L of mitochondrial pressure test medium per well was added, and incubated in a common 37℃incubator (without additional CO 2 ) Standing for 45 min.
(6) 45. Mu.L of a palmitic acid solution or BSA solution of medium 1 mM was added thereto, and the mixture was allowed to stand still at 37℃for 15 minutes.
(7) Drugs were added sequentially to the probe wells at 75 μl per well, with 280 μM etonox or test medium in the upper left well, 40 μM oligomycin in the upper right well, 18 μM FCCP in the lower left well, and 40 μM rotenone in the lower right well.
(8) And (3) loading, namely selecting a mitochondrial pressure test program and modifying the program: basal→etodoxir/medium→oligomycin→FCCP→rotenone, 3 cycles are performed per step, each cycle including mixing for 4 min, waiting for 0 min, measuring for 2 min, except for adding oligomycin for 5 min, waiting for 10 min, measuring for 2 min, to obtain mitochondrial OCR changes.
(9) Etomir is a fatty acid oxidation inhibitor, and the degree of decrease in mitochondrial oxygen consumption rate after etomir addition (etomix sensitivity) reflects the contribution of fatty acid oxidation to oxidative phosphorylation.
The SeaHorse results in FIG. 3 show that miR-29ab1 -/- The maximum respiration availability of mitochondria of the intestinal organoids of mice is increased, the oxidative phosphorylation level is increased, meanwhile, the etonox ir sensitivity is obviously increased, the contribution degree of fatty acid oxidation to oxidative phosphorylation is proved to be increased, and miR-29ab1 is knocked out to activate fatty acid oxidation of intestinal stem cells so as to promote the energy metabolism level.
Example 2: regulating and controlling effect of miR-29 deletion on stem cell stem property of intestinal tract
1. Mouse intestinal organoid stem cell Activity
(1) Isolated culture of Lgr5-eGFP; miR-29ab1, using the method described in example 1 +/+ Mice and Lgr5-eGFP; miR-29ab1 -/- Intestinal organoids of mice.
(2) On day 5 of culture, the organoid was observed and photographed for Lgr5-GFP signal using a fluorescence microscope, and the number of intestinal stem cells was evaluated.
2. Mouse intestinal crypt cell cycle analysis
(1) WT mice and miR-29ab 1-/-mice intestinal crypt single cells were obtained by the method in example 1.
(2) 1 mL pre-cooling 70% ethanol was added dropwise, gently blown, and left at-20℃for 2 h.
(3) Centrifugation was performed at 1000 Xg for 5 min at 4℃and 1 mL of pre-chilled PBS was used for washing once, propidium iodide staining solution 0.5 mL was added in the dark, water bath was performed at 37℃in the dark for 30 min, and the reaction was stopped on ice.
(4) Through a 40 μm cell sieve, and transferred to a flow tube.
(5) Flow assays, detecting about 488 nm (PE) fluorescence.
(6) Cell cycle was analyzed with Flowjo software. The fluorescence intensity is proportional to the DNA content, and the cells in the G0/G1 phase all contain single DNA, while the fluorescence intensity of the cells in the G2/M phase is twice that of the cells in the G0/G1 phase, and the fluorescence intensity in the S phase is between the two.
FIG. 4 shows the results of miR-29ab1 -/- The number of stem cells in the intestinal organoids of the mice is reduced, and the G0/G1 phase of cells in the division retardation state in the crypt is increased, namely the active state of the cells is reduced, which indicates that the miR-29ab1 is knocked out to reduce the stem cell stem property of the intestinal stem cells.
Example 3: inhibiting regulation and control effect of miR-29 on energy metabolism of intestinal tumor cells
1. Human Lgr5 + Colorectal tumor cell line LS174T culture and transfection
(1) MEM medium containing 1 mM sodium pyruvate and 10% fetal bovine serum is used at 37deg.C and 5% CO 2 LS174T cell line was cultured.
(2) After 24 h of adherent growth in a 12-well plate, the medium was replaced with 900 μl of pre-warmed Opti-MEM medium.
(3) mu.L of transfection reagent Lipofectamine 2000 was added to 50. Mu.L of Opti-MEM, 2. Mu.L of miRNA inhibitor mother liquor (20. Mu.M) was added to another 50. Mu.L of Opti-MEM, and the mixture was allowed to stand at room temperature for 5 min.
Note that: for cotransformation of miR-29a/b inhibitor, 2. Mu.L of mother liquor or 4. Mu.L of NC inhibitor are added respectively as control.
(4) Mixing the culture medium containing the transfection reagent and the inhibitor, and standing at room temperature for 20 min.
(5) 100. Mu.L of the mixture was added dropwise to the cell culture medium (final concentration 40 nM).
(6) 37℃、5% CO 2 After 6 h culture, the medium was changed to 1 mL complete medium.
The miR-29 inhibitor is a chemically modified RNA single-strand, and the nucleic acid sequence of the RNA single-strand is as follows (m represents methylation modification):
miR-29a inhibitor
mUmAmAmCmCmGmAmUmUmUmCmAmGmAmUmGmGmUmGmCmUmA (SEQ ID NO:21)
miR-29b inhibitor
mAmAmCmAmCmUmGmAmUmUmUmCmAmAmAmUmGmGmUmGmCmUmA (SEQ ID NO:22)
2. Evaluation of fatty acid metabolism efficiency of cell lines
(1) After transfection, the medium was changed to basal medium containing 100. Mu.M BOTIBY fluorescently labeled palmitic acid.
(2) After incubation of 12 h, the medium was removed, washed with pre-warmed PBS, and replaced with complete medium.
(3) After 24. 24 h culture, the medium was removed and fixed with 4% paraformaldehyde cell fixative at room temperature for 15 min in the absence of light.
(4) Wash 5 min x 3 times with PBS, add DAPI dye and incubate 5 min in the dark.
(5) Washing with PBS for 5 min×3 times, taking out the slide, dripping anti-fluorescence quenching sealing liquid, reversely buckling on the slide glass, observing with a fluorescence microscope, and shooting. The smaller the remaining fluorescent material, the faster the consumption of palmitic acid, reflecting the fatty acid metabolism efficiency.
3. Altered mRNA level gene expression profile in cell lines
(1) Cell total RNA was extracted 24 h after cell transfection by TRIzol method.
(2) The mRNA was inverted to cDNA using a reverse transcription kit.
(3) The gene expression is detected by adopting a real-time fluorescence quantitative PCR method, and the primer sequences are as follows:
Cpt1a
F：5’-TCCAGTTGGCTTATCGTGGTG-3’(SEQ ID NO:13)
R：5’-TCCAGAGTCCGATTGATTTTTGC-3’(SEQ ID NO:14)
Acadm
F：5’-ACAGGGGTTCAGACTGCTATT-3’(SEQ ID NO:15)
R：5’-TCCTCCGTTGGTTATCCACAT-3’(SEQ ID NO:16)
Hmgcs2
F：5’-GCCCAATATGTGGACCAAACT-3’(SEQ ID NO:17)
R：5’-GAAGCCCATACGGGTCTGG-3’(SEQ ID NO:18)
4. altered expression of fatty acid oxidation-related enzyme protein levels in cell lines
(1) After cell transfection 48 h was lysed with loading buffer and cellular proteins were extracted, metal bath at 100 ℃ for 10 min.
(2) 10% of separation gel and 5% of concentrated gel are prepared, and protein samples or pre-dyed protein markers are added for SDS-PAGE. The constant pressure is 80 to V for 30 min, and then the constant pressure is 120 to V for about 1 to h.
(3) Constant flow 200 mM ice bath transfer 2 h.
(4) The PVDF membrane was blocked with skim milk at room temperature for 2 h on a shaking table and incubated overnight at 4 ℃.
(5) PBST was washed 5 min X5 times, secondary antibody diluted 1:5000 with blocking solution and incubated 1 h on a shaker at room temperature.
(6) PBST was washed 5 min×5 times, ECL luminescence was drop-applied to the film and photographed by exposure with a gel imager.
The results are shown in FIG. 5, and the LS174T cell line fatty acid metabolism efficiency is improved after the miR-29a/b inhibitor is transfected, and the mRNA and protein level expression of the fatty acid oxidation related enzyme are both up-regulated, so that the miR-29a/b is proved to be inhibited to activate the fatty acid oxidation of colorectal tumor cells.
5. Cell line ATP content assay
(1) After cell transfection 48 h the medium was removed and washed 3 times with PBS.
(2) mu.L of ATP detection lysate was added to each well, cells were scraped into the lysate on ice with a cell scraper and transferred to a 1.5 mL centrifuge tube.
(3) Centrifuging at 12000 Xg for 15 min at 4deg.C, transferring supernatant to obtain sample, and placing on ice.
(4) 100 mu L/well working solution is added into a black 96-well plate, and the plate is kept stand for 5 min at room temperature in a dark place.
(5) The light-shielding condition is maintained, and 20 mu L of standard curve sample or sample to be tested is added into each hole.
(6) And (3) measuring a Relative Luminescence Unit (RLU) by using a multifunctional enzyme-labeled instrument, substituting a standard curve, and calculating the ATP content in the sample to be measured.
(7) Protein concentration was determined using BCA kit and ATP content was calculated for each g of protein.
6. Cell line granule energy metabolism level determination
(1) Media were prepared (4 ℃ C. For one day):
test medium: 10 mM glucose, 1 mM sodium pyruvate, 2 mM glutamine were added to SeaHorse basal medium.
(2) After completion of cell transfection, the cells were digested, centrifuged, resuspended in 1 well of 1 mL complete medium, plated evenly into 10 wells of a SeaHorse 24 well plate with 200. Mu.L of each well and cultured for 48 h.
(3) The 12-24 h hydrated probe card before the machine is put in a common incubator at 37 ℃ without adding CO 2 )。
(4) The medium was removed, rinsed with 500. Mu.L mitochondrial pressure test medium, then 525. Mu.L mitochondrial pressure test medium per well was added, and incubated in a common 37℃incubator (without additional CO 2 ) And (5) standing for 1 to h.
(5) Drugs were sequentially added to the probe wells at 75 μl per well, with 12 μM oligomycin in the upper left well, 9 μM FCCP in the upper right well, and 10 μM rotenone in the lower left well.
(6) And (3) loading, namely selecting a mitochondrial pressure test program and modifying the program: basal-oligomycin-FCCP-rotenone, each step is subjected to 3 cycles, each cycle comprises mixing for 3 min, waiting for 2 min, and measuring for 3 min to obtain mitochondrial OCR, wherein the ratio of the basic OCR in the maximum OCR is the maximum respiration availability, so as to reflect the oxidative phosphorylation level.
The result shows that the ATP content of LS174T cell line is increased after the miR-29a/b inhibitor is transfected, and the oxidative phosphorylation level is increased, which indicates that the miR-29a/b is inhibited to promote mitochondrial energy metabolism of colorectal tumor cells. (the results are shown in FIG. 6).
Example 4: inhibiting miR-29 pair for regulating intestinal tumor cell stem property through energy metabolism
1. Cell line cell cycle analysis
The cell fraction of each cycle of the LS174T cell line was determined by the method described in example 2.
2. Differentiation of cell lines
(1) Cells were inoculated on cell slide, after transfection, 48 h medium was removed, washed with pre-warmed PBS, 4% paraformaldehyde cell fixative was added, and the cells were fixed on a shaker at room temperature for 15 min.
(2) Washing with pure water for 5 min, adding Alcian blue dye, and incubating for 20 min at room temperature.
(3) Washing for 5 min with tap water, adding nuclear solid red dye, and incubating for 5 min at room temperature.
(4) Adding pure water, washing for 1 min, naturally airing, taking out the climbing slices, sealing the slices in a neutral manner, and observing and shooting by a fluorescence microscope. The LS174T cell line can differentiate into a goblet cell line, and the Alcian blue+ fraction is mucin secreted by goblet cells, so the Alcian blue+ area ratio indicates that the LS174T cell line has a higher degree of differentiation, the fewer Lgr5+ cells, the lower the dryness.
FIG. 7 shows that the proportion of cells in the LS174T cell line at the G0/G1 stage, which stops dividing, is increased after the miR-29a/b inhibitor is transfected, and the differentiation degree is increased, which shows that the miR-29a/b inhibition reduces the colorectal tumor cell stem property.
Example 5: upregulation of miR-29 target gene Hnf4g for regulating intestinal tumor cell stem by activating fatty acid oxidation-mediated energy metabolism
1. Cell line Hnf4g expression
LS174T cell line Hnf4g was assayed for mRNA and protein expression by the method of example 3.
Hnf4g
F：5’-CCTCCTCGCTTTCAGCAAACT-3’(SEQ ID NO:19)
R：5’-CGGTCAAGGCAGCAATCATGT-3’(SEQ ID NO:20)
The results show that LS174T cell line Hnf4g is up-regulated at both mRNA level and protein level after transfection of miR-29a/b inhibitor, and that inhibition of miR-29a/b activates its target gene Hnf4g in colorectal tumor cell lines (results are shown in FIG. 8).
2. Overexpression of Hnf4g by cell line
(1) After cells had grown in 12-well plates with adherence at 24 h, the medium was replaced with 500. Mu.L of pre-warmed Opti-MEM medium.
(2) mu.L of transfection reagent Lipofectamine 2000 was added to 250. Mu.L of Opti-MEM, and 2. Mu.g of pcDNA3.1 (+) plasmid (empty plasmid) or pcDNA-Hnf4g of plasmid was added to another 250. Mu.L of Opti-MEM and allowed to stand at room temperature for 5 min.
(3) The medium containing the transfection reagent and plasmid was mixed and allowed to stand at room temperature for 20 min.
(4) mu.L of the mixture was added dropwise to the cell culture medium (final concentration 2. Mu.g/mL).
(5) 37℃、5%CO 2 After 6 h culture, the medium was changed to 1 mL complete medium.
3. Effect of overexpression of Hnf4g on energy metabolism and dryness of intestinal tumor cells
(1) The expression of 48 h after transfection of the plasmid was evaluated by the method of example 3 on the level of Hnf4g and fatty acid oxidation-related enzyme protein.
(2) The change in ATP content and mitochondrial oxidative phosphorylation levels of 48 h LS174T cell line after plasmid transfection was evaluated by the method in example 3.
(3) Differentiation of the LS174T cell line 48 h after transfection of the plasmid was evaluated as described in example 4.
The results of FIG. 9 show that the overexpression of Hnf4g can cause oxidation activation of fatty acid in colorectal tumor cells, promote mitochondrial energy metabolism, inhibit cell stem property and have the same effect as miR-29a/b inhibition.
4. Regulation and control of intestinal tumor cell stem property by inhibiting energy metabolism change caused by miR-29 or over-expression of Hnf4g
(1) Expression of LS174T cell line fatty acid oxidation-related enzyme protein levels after 48 h treatment with 1 μm fatty acid oxidation activator GW501516 was evaluated as in example 3.
(2) LS174T cell line differentiation after 48 h treatment with 1. Mu.M of the fatty acid oxidation activator GW501516 was evaluated as in example 4.
The results of FIG. 10 show that activating fatty acid oxidation results in increased LS174T cell line differentiation and reduced stem, demonstrating the regulatory effect of inhibiting miR-29 or over-expressed Hnf4 g-mediated intestinal tumor cell fatty acid oxidation activation parameters and their inhibition of cell stem.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Claims (10)
- Application of miR-29a or miR-29b in preparation of preparation for promoting intestinal cell dryness.
- 2. The use of claim 1, wherein miR-29a or miR-29b promotes intestinal cell stem performance by inhibiting Hnf4g expression.
- Application of miR-29a or miR-29b in preparation of preparation for inhibiting energy metabolism of intestinal tumor cells.
- 4. Use according to claim 3, wherein said energy metabolism comprises fatty acid oxidative activity, mitochondrial maximum respiratory availability and/or Etomoxir sensitivity.
- Application of miR-29a or miR-29b inhibitor in preparation of preparation for inhibiting intestinal tumor cell stem property.
- 6. The use of claim 5, wherein miR-29a or miR-29b promotes intestinal cell stem performance by inhibiting Hnf4g expression.
- Application of miR-29a or miR-29b inhibitor in preparation of preparation for promoting energy metabolism of intestinal tumor cell line.
- 8. The use according to claim 7, wherein said energy metabolism comprises fatty acid oxidative activity, mitochondrial maximum respiratory availability and/or Etomoxir sensitivity.
- An application of an Hnf4g related biological agent in preparing an agent for promoting energy metabolism of intestinal cells or inhibiting stem cell activity of intestinal cells, wherein the Hnf4g related biological agent comprises: 1) Nucleic acid molecules encoding Hnf4 g; 2) Hnf4g protein; 3) An expression cassette comprising 1) said nucleic acid molecule; 4) A vector comprising 3) said expression cassette; 5) A cell comprising 1) said nucleic acid molecule or 3) said expression cassette or 4) said vector; 6) A formulation that promotes expression of Hnf4 g.
- 10. The use of claim 9, wherein the formulation of 6) that promotes expression of Hnf4g is an inhibitor of miR-29a or miR-29 b.
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| CN102146412A (en) * | 2011-01-11 | 2011-08-10 | 中山大学 | Small molecular non-coding RNA gene hsa-miR-29b and use thereof |
| CN104619353A (en) * | 2011-12-13 | 2015-05-13 | 俄亥俄州国家创新基金会 | Methods and compositions related to miR-21 and miR-29a, exosome inhibition, and cancer metastasis |
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| CN116059231A (en) * | 2022-11-01 | 2023-05-05 | 中国农业大学 | Application of miR-29 in preparation of anti-radiation medicine |
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| CN102146412A (en) * | 2011-01-11 | 2011-08-10 | 中山大学 | Small molecular non-coding RNA gene hsa-miR-29b and use thereof |
| CN104619353A (en) * | 2011-12-13 | 2015-05-13 | 俄亥俄州国家创新基金会 | Methods and compositions related to miR-21 and miR-29a, exosome inhibition, and cancer metastasis |
| EP3597739A1 (en) * | 2018-07-19 | 2020-01-22 | Korea Advanced Institute of Science and Technology | Composition for regulating cancer cell division or differentiation comprising setdb1 or a setdb1 inhibitor |
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