CN114574489B - MicroRNA regulated and controlled by docosahexaenoic acid and application thereof - Google Patents
MicroRNA regulated and controlled by docosahexaenoic acid and application thereof Download PDFInfo
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Abstract
The invention discloses microRNA regulated and controlled by docosahexaenoic acid and application thereof, and belongs to the technical field of molecular biology. Docosahexaenoic acid has the effect of relieving liver injury caused by oxidative stress, but at present, how to rapidly and accurately evaluate the protective effect of the docosahexaenoic acid on liver injury in early stage is yet to be established. According to the invention, microRNA miR-101-3p is used as an early molecular detection marker for protecting liver injury by docosahexaenoic acid, so that the liver injury protection effect of the docosahexaenoic acid is rapidly and accurately evaluated in the early stage of liver injury. The application direction of microRNA miR-101-3p is expanded, and a basis is provided for the establishment of an evaluation method for protecting liver injury by docosahexaenoic acid.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to microRNA regulated and controlled by docosahexaenoic acid and application thereof.
Background
Causes of liver damage generally include infection, autoimmunity, drugs and poisons, alcohol factors, genetic metabolism, and liver damage secondary to other systemic diseases. In recent years, the diversity of life forms and the influence of the surrounding environment have resulted in the superposition of various factors, the change of disease spectrums, and the increasing number of the incidences of difficult and complicated liver diseases. The clinical manifestations of liver injury caused by different causes are various, laboratory examination and imaging examination lack specificity, and the difficulty in diagnosing the cause of liver injury is also increased.
Oxidative stress is one of the important factors inducing liver injury. Oxidative stress is the damage to tissues caused by the imbalance of the in vivo oxidation system and the antioxidant system, which results in the production of excessive high-activity molecules, such as Reactive Oxygen Species (ROS), in the body. Hydrogen peroxide is one of the important species of active oxygen, which is capable of penetrating the cell membrane to form reactive oxygen radicals, causing oxidative damage. The excessive generation of ROS can damage the cell structure, and cause the oxidative damage related diseases such as senile dementia, tumor, inflammation and the like. Research on oxidative stress injury and protection of tissues and organs has been attracting attention. The w-unsaturated fatty acid refers to unsaturated fatty acid containing more than two double bonds, including alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and the like, is essential fatty acid for human body, and has physiological functions of resisting oxidation, inflammation and cancer. Wherein docosahexaenoic acid is an important constituent of cephalin, and plays an important role in nutrition, development, health and the like of human body. It has been found that docosahexaenoic acid has a liver injury reducing effect caused by oxidative stress. However, at present, how to rapidly and accurately evaluate the protective effect of the docosahexaenoic acid on liver injury in early stage is yet to be established.
MicroRNA (miRNA) is an endogenous, non-coding small RNA with regulatory functions found in eukaryotes, which is about 18 to 24 nucleotides in size. miRNAs regulate the physiological and pathological processes of the organism by either completely or incompletely pairing with the 3' -UTR (3 ' -untranlated regions,3' -UTR) base of the target mRNA, regulating translation of the target mRNA or promoting its degradation. Recently, research shows that miR-101-3p is involved in the development of tumors such as gastric cancer, ovarian cancer, liver cancer and breast cancer, but whether the miR-101-3p can be used as an early molecular diagnostic marker for reducing liver injury by docosahexaenoic acid has not been reported.
The metabolic enzyme in HepG2 cells is kept relatively stable, the phenotype and the interior of the cells are not changed due to the increase of passage times, and the contained bioconversion metabolic enzyme is homologous to normal human liver parenchymal cells. Therefore, hepG2 is often used in ideal cell lines for in vitro hepatocyte metabolism and genetic experimental studies. The cell modeling adopts the currently accepted modeling mode of hydrogen peroxide induced liver injury, and a stable cell model is established, thus providing a foundation for subsequent experimental study.
Disclosure of Invention
The invention aims to solve the problems and provide a microRNA regulated and controlled by docosahexaenoic acid and application thereof. According to the invention, the microRNA miR-101-3p is used as an early molecular diagnosis marker for protecting liver injury by docosahexaenoic acid, so that the application direction of the microRNA miR-101-3p is expanded, and a basis is provided for establishing an evaluation method for protecting liver injury by docosahexaenoic acid.
In order to achieve the above purpose, the invention adopts the following technical scheme:
One of the objects of the present invention is: providing a microRNA regulated by docosahexaenoic acid: the microRNA is miR-101-3p, and the nucleotide sequence is 5'-UACAGUACUGUGAUAACUGAA-3'.
Another object of the invention is: the microRNA regulated and controlled by the docosahexaenoic acid is used as an early molecular diagnosis marker in the detection of the liver injury protected by the docosahexaenoic acid.
Further, the application comprises the following steps:
(1) Constructing a hydrogen peroxide induced liver injury cell model, calculating the ratio of miR-101-3p/U6 expression in the hydrogen peroxide induced liver injury cell model by using U6 as an internal reference and carrying out real-time fluorescence quantitative PCR, and marking the ratio as A;
(2) The hydrogen peroxide induced liver injury cell model is treated by adopting docosahexaenoic acid, and the ratio of miR-101-3p/U6 expression quantity in the treated cell model is calculated by real-time fluorescence quantitative PCR and is marked as a;
(3) Setting the ratio of miR-101-3p/U6 in normal cells without induced injury to be 1.0, and evaluating the capacity of docosahexaenoic acid in resisting hydrogen peroxide to induce liver injury according to the following formula; the capacity of resisting hydrogen peroxide to induce liver injury R= |A-a|/1.0X100%.
A third object of the present invention is to: an early molecule detection reagent for protecting liver injury by docosahexaenoic acid is provided, wherein the detection reagent comprises microRNA regulated by the docosahexaenoic acid.
The invention establishes a hydrogen peroxide induced liver injury cell model, and adopts docosahexaenoic acid to treat the hydrogen peroxide induced liver injury cell model, and real-time fluorescence quantitative PCR calculates the ratio of miR-101-3p/U6 expression in the cell model before and after treatment, and the result shows that the proliferation rate of HepG2 cells and the miR-101-3p expression after hydrogen peroxide treatment are obviously reduced, and the ratio of the proliferation rate of the HepG2 cells and the miR-101-3p/U6 after hydrogen peroxide treatment can be obviously improved. miR-101-3p can be used as a miRNA marker for evaluating the hydrogen peroxide-induced liver injury resistance of docosahexaenoic acid, and the protection effect of the docosahexaenoic acid can be evaluated rapidly and accurately in the early stage of liver injury.
The beneficial effects of the invention are as follows:
According to the invention, microRNA miR-101-3p is used as an early molecular detection marker to perform early and rapid molecular detection of liver injury, so that the protection effect of docosahexaenoic acid can be rapidly and accurately evaluated in the early stage of liver injury. The application direction of microRNA miR-101-3p is expanded, and a basis is provided for the establishment of an evaluation method for protecting liver injury by docosahexaenoic acid.
Drawings
FIG. 1 effect of hydrogen peroxide at different concentrations on HepG2 cell proliferation rate. * P < 0.01, n=3 compared to control group.
FIG. 2 effect of docosahexaenoic acid on HepG2 cell proliferation rate. The "+" in the figure indicates that the corresponding substance is added; "-" indicates that no corresponding substance was added. * P < 0.01, n=3 compared to control group.
FIG. 3 relative expression level of docosahexaenoic acid in HepG2 cell miR-101-3 p. The "+" in the figure indicates that the corresponding substance is added; "-" indicates that no corresponding substance was added. * P < 0.01, n=3 compared to control group.
Detailed Description
The technical scheme of the invention is further described through specific examples. The present invention is not limited to the following examples. The starting materials employed in the present invention are commercially available or may be synthesized by methods known in the art.
Experimental samples and reagents:
The human liver cancer cell line HepG2 is purchased from a Shanghai cell bank of China academy of sciences; docosahexaenoic acid, sigma, usa; qPCR primers, guangzhou Ruibo biotechnology Co., ltd; TRIzol, thermo Fisher, usa; M-MLV REVERSE TRANSCRIPTASE, promega, USA; SYBR Premix Ex Taq II fluorescent quantitative kit, dajiara company.
EXAMPLE 1 construction of Hydrogen peroxide-induced liver injury model
MTT method for detecting influence of hydrogen peroxide on proliferation rate of HepG2 cells
HepG2 cells were seeded at a density of 5X 10 4/mL per well of 100. Mu.L in 96-well plates, 6 wells were repeated, incubated in a 37℃5% CO 2 incubator for 24. 24 h to a cell density of about 80%, DMEM medium (DMEM high sugar medium containing 10% (V/V) fetal bovine serum and penicillin/streptomycin (1% (V/V)) was discarded, DMEM medium containing different concentrations (100. Mu.M, 200. Mu.M, 400. Mu.M and 800. Mu.M) of hydrogen peroxide was added to the experimental wells, after 4 h, all DMEM medium was discarded, 15. Mu.L of MTT solution (0.5 mg/mL) and 100. Mu. LDMEM medium were added to each well, after 4. 4 h was incubated, 150. Mu.L of dimethyl sub-a (DMSO) was added to each well for termination of incubation, 10. 10 min was continuously shaken to allow the formazan crystals to be sufficiently dissolved, and the absorbance (OD) values of the solutions were measured at nm wavelength, respectively. Wells were not treated with hydrogen peroxide, and were subjected to subsequent detection with MTT at the same time as the control wells.
The formula is:
。
As a result, as shown in FIG. 1, hydrogen peroxide was toxic to HepG2 cells, and at a hydrogen peroxide concentration of 200. Mu.M or more, the proliferation rate of HepG2 cells was reduced by about 30%, and at a hydrogen peroxide concentration exceeding 200. Mu.M, the proliferation rate was further significantly reduced. In a subsequent experiment, a hydrogen peroxide-induced liver injury model was constructed with hydrogen peroxide at a concentration of 200. Mu.M.
EXAMPLE 2 protection of Hydrogen peroxide-damaged HepG2 cell model by docosahexaenoic acid (DHA)
Three groups of cells were set up: a negative control group, a hydrogen peroxide damage model sample group and a hydrogen peroxide damage plus DHA intervention sample group; wherein: the negative control group is untreated HepG2 cells cultured by adopting DMEM culture solution; the hydrogen peroxide damage model sample group is prepared by adding 200 mu M hydrogen peroxide into DMEM culture solution to culture 4h HepG2 cells; the hydrogen peroxide damage plus DHA intervention sample group is HepG2 cells which are cultured by adding 100 mu M DHA in DMEM culture solution to cultivate the HepG2 cells 48 h and then adding 200 mu M hydrogen peroxide to cultivate the HepG2 cells 4 h.
1. MTT method for detecting influence of DHA on proliferation rate of hydrogen peroxide induced liver injury HepG2 cells
The effect of DHA on the proliferation rate of hydrogen peroxide-induced liver injury HepG2 cells was examined by MTT method, and the result is shown in FIG. 2, wherein the protection of DHA increases the proliferation rate of cells by 36% after hydrogen peroxide treatment; DHA is shown to have a protective effect on hydrogen peroxide-induced damage.
2. Real-time quantitative PCR (polymerase chain reaction) detection of influence of DHA (docosahexaenoic acid) on miR-101-3p/U6 ratio in hydrogen peroxide damage HepG2
(1) Primer design
Searching a miRBase database for the sequence of miR-101-3 p: 5'-UACAGUACUGUGAUAACUGAA-3', and then designing primers. The primer sequences are specifically as follows:
Upstream primer of miR-101-3 p: 5'-GCCGCCACATGTGAGGCAAGG-3' the process of the preparation of the pharmaceutical composition,
Downstream primer of miR-101-3 p: 5'-AATTGAAAAAAAAAAGTGATTATTAT-3'.
(2) MiR-101-3p expression level detection
Extracting total RNA of the HepG2 cell by using a TRIzol Reagent; reverse transcription of RNA using M-MLV REVERSE TRANSCRIPTASE to synthesize a cDNA first strand; detecting miR-101-3p expression level in the HepG2 cell on an ABI 7300 real-time fluorescence quantitative PCR instrument by using SYBR Premix Ex Taq II real-time fluorescence quantitative PCR detection kit; the miR-101-3p real-time fluorescence quantitative PCR expression detection takes U6 (upstream primer of U6: 5'-GGTCGGGCAGGAAAGAGGGC-3', downstream primer of U6: 5'-GCTAATCTTCTCTGTATCGTTCC-3') as an internal reference; and calculating the relative expression quantity of miR-101-3p by adopting a 2 -△△Ct method in result analysis. The real-time fluorescent quantitative PCR detection is carried out for 5 minutes at 95 ℃ and then amplified for 40 cycles, wherein each cycle is carried out for 5 seconds at 95 ℃ and amplified for 31 seconds at 60 ℃; the reaction was then terminated at 95℃for 15 seconds, 60℃for 1 minute, 95℃for 15 seconds, and 60℃for 15 seconds.
Delta Ct (n) =ct target gene (n) -Ct reference gene (n); ΔΔct (n) = - Δct (n) - [ Δct (1); ct= -1/lg (1+Ex). Times. lgX 0 + lgN/lg (1+Ex), where N is the number of cycles of the amplification reaction, X 0 is the initial template amount, ex is the amplification efficiency, and N is the amount of amplified product when the fluorescent amplification signal reaches a threshold intensity.
3. Constructing an evaluation formula of hydrogen peroxide damage resistance of docosahexaenoic acid: r= |a-a|/1.0×100%; wherein A is: 200. the ratio of miR-101-3p/U6 in the mu M hydrogen peroxide damage sample (hydrogen peroxide damage model sample group); a is the ratio of miR-101-3p/U6 in 200 mu M hydrogen peroxide treatment plus 100 mu M DHA intervention sample (hydrogen peroxide damage plus DHA intervention sample group); the expression level of miR-101-3p/U6 in the negative control sample was set to 1.0.
The results showed that the ratio of miR-101-3p/U6 in HepG2 cells of the hydrogen peroxide damage model sample group was marked as A, which was reduced by 65% compared to the negative control group, and the ratio of miR-101-3p/U6 in HepG2 cells of the hydrogen peroxide damage plus DHA intervention sample group was marked as a, which was increased by 74% compared to the hydrogen peroxide damage model sample group (FIG. 3).
The results showed that r= |a-a|/1.0×100%, r=12%.
From the above examples, it can be seen that the docosahexaenoic acid protection can increase the ratio of miR-101-3p/U6 in the hydrogen peroxide injury HepG2, which indicates that miR-101-3p can be used as a molecular marker for evaluating the liver injury protection effect of the docosahexaenoic acid.
The invention provides miR-101-3p as a marker for evaluating the capacity of docosahexaenoic acid in resisting hydrogen peroxide to induce liver injury for the first time. In a hydrogen peroxide induced liver injury cell model, the docosahexaenoic acid obviously improves the proliferation rate of cells, improves the ratio of miR-101-3p/U6, provides a miRNA marker for evaluating the hydrogen peroxide induced liver injury resistance of the docosahexaenoic acid, and realizes the rapid and accurate evaluation of the liver injury protection capability of the docosahexaenoic acid in the early stage of liver injury. The application direction of microRNA miR-101-3p is expanded, and a basis is provided for the establishment of an evaluation method for protecting liver injury by docosahexaenoic acid.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> University of beauty set
<120> A microRNA regulated by docosahexaenoic acid and application thereof
<130> 5
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> RNA
<213> miR-101-3p
<400> 1
uacaguacug ugauaacuga a 21
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<211> 21
<212> DNA
<213> Upstream primer of miR-101-3p
<400> 2
gccgccacat gtgaggcaag g 21
<210> 3
<211> 26
<212> DNA
<213> MiR-101-3p downstream primer
<400> 3
aattgaaaaa aaaaagtgat tattat 26
<210> 4
<211> 20
<212> DNA
<213> Upstream primer of U6
<400> 4
ggtcgggcag gaaagagggc 20
<210> 5
<211> 23
<212> DNA
<213> Downstream primer of U6
<400> 5
gctaatcttc tctgtatcgt tcc 23
Claims (2)
1. The application of microRNA regulated by docosahexaenoic acid as an early molecular diagnosis marker in preparing a detection reagent for evaluating the hydrogen peroxide induction hepatic injury resistance of docosahexaenoic acid is characterized in that: the microRNA is miR-101-3p, and the nucleotide sequence of the microRNA is 5'-UACAGUACUGUGAUAACUGAA-3'; the expression level of miR-101-3p in the liver cells can be improved by the treatment of the docosahexaenoic acid, and the liver injury protection capability of the docosahexaenoic acid can be evaluated by detecting the change of the expression level of miR-101-3p in the liver cells caused by the treatment of the docosahexaenoic acid.
2. A method for evaluating the ability of docosahexaenoic acid to resist hydrogen peroxide induced liver injury, comprising: the method is not used for diagnosis or treatment of disease; the method comprises the following steps:
(1) Constructing a hydrogen peroxide induced liver injury cell model, calculating the ratio of miR-101-3p/U6 expression in the hydrogen peroxide induced liver injury cell model by using the U6 expression as an internal reference and carrying out real-time fluorescence quantitative PCR, and marking the ratio as A;
(2) The hydrogen peroxide induced liver injury cell model is treated by adopting docosahexaenoic acid, and the ratio of miR-101-3p/U6 expression quantity in the treated cell model is calculated by real-time fluorescence quantitative PCR and is marked as a;
(3) Setting the ratio of miR-101-3p/U6 in normal cells without induced injury to be 1.0, and evaluating the hepatic injury induced by the docosahexaenoic acid on hydrogen peroxide according to the following formula; the capacity of resisting hydrogen peroxide to induce liver injury R= |A-a|/1.0X100%.
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| RU2011144780A (en) * | 2011-11-07 | 2013-05-20 | Общество С Ограниченной Ответственностью "Консорциум-Пик" | PHARMACEUTICAL COMPOSITION BASED ON VEGETABLE DHA FOR TREATMENT AND PREVENTION OF LIVER DISEASES |
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| RU2011144780A (en) * | 2011-11-07 | 2013-05-20 | Общество С Ограниченной Ответственностью "Консорциум-Пик" | PHARMACEUTICAL COMPOSITION BASED ON VEGETABLE DHA FOR TREATMENT AND PREVENTION OF LIVER DISEASES |
| CN109477144A (en) * | 2015-09-29 | 2019-03-15 | 国家儿童医院研究所 | Method for detecting hepatic fibrosis-renal tubular ectasia syndrome and the responsiveness to therapy |
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