CN113913508B - Application of miR-195-3p detection reagent in preparation of product for diagnosing oxidative stress injury of liver - Google Patents
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Abstract
The invention belongs to the field of molecular diagnosis, and particularly relates to an application of a miR-195-3p detection reagent in preparation of a product for diagnosing oxidative stress injury of liver. The sequence of the miR-195-3p is shown as SEQ ID NO.1, the invention clarifies the action of the miR-195-3p in the process of inducing oxidative stress injury of the liver by Hcy by detecting the expression level of the miR-195-3p in liver tissues and liver cells, provides a theoretical basis for diagnosing the oxidative stress injury of the liver from a molecular level in the future, and has great theoretical significance and potential practical value.
Description
Technical Field
The invention belongs to the field of molecular diagnosis, and particularly relates to an application of a miR-195-3p detection reagent in preparation of a product for diagnosing oxidative stress injury of liver.
Background
Liver damage caused clinically by metabolic disease is a common cause of reduced liver function. Research shows that under normal conditions, the body can protect the liver from being damaged through an antioxidant system, and when homeostasis is disturbed to cause oxidative stress, the occurrence and development of liver damage can be promoted, so that more serious liver diseases can be caused, including drug-induced liver diseases, alcoholic liver diseases, other non-alcoholic steatohepatitis and the like. Therefore, oxidative stress is considered as one of the major mechanisms of the development of metabolic liver diseases. Oxidative stress not only causes changes in induced lipids, proteins and DNA, but more importantly, it can modulate pathways that control normal biological function, thereby causing liver damage.
Homocysteine (Hcy) is an intermediate in the normal biosynthetic metabolic processes of methionine and cysteine. Under normal physiological conditions, the reference value for the total homocysteine (tHcy) concentration in human plasma is between 5 and 10 μ M. Hcy accumulates abnormally in the body, can induce Hyperhomocysteinemia (HHcy), and causes damage to various tissues and organs. The liver is an important organ of amino acid metabolism. Research shows that HHcy is related to the occurrence and the progression of various liver-related diseases such as hepatic steatosis and nonalcoholic fatty liver, but the action mechanism of Hcy in oxidative stress-induced metabolic liver injury is still unclear at present.
microRNAs (miRNAs) are highly conserved non-coding RNA molecules of about 22 nucleotides in length that regulate gene expression primarily by degrading mRNA at the post-transcriptional level. Recently, studies have reported a number of miRNAs that are aberrantly expressed in liver disease. Wherein miR-195 is a member of microRNA 15 family. Research shows that miR-195 plays a role in various liver diseases, for example, in type II diabetes, the expression of miR-195 in the liver is up-regulated along with the rise of blood sugar, miR-195 also can play an inhibition role in liver cancer metastasis by down-regulating the expression of VEGFA and FGF2, and a miR-195 inhibitor can relieve the damage of the oxidative stress of the liver of a diabetic mouse. However, the role of miR-195 in Hcy-induced hepatic oxidative stress leading to metabolic liver injury remains unclear.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide the application of the miR-195-3p detection reagent in the preparation of products for diagnosing oxidative stress injury of the liver.
The miR-195-3p detection reagent provided by the invention is applied to the preparation of products for diagnosing oxidative stress injury of the liver, and the sequence of the miR-195-3p is shown in SEQ ID NO. 1.
Further, the reagent comprises: and (3) detecting the miR-195-3p expression level by qRT-PCR.
Furthermore, the reagent for detecting the expression level of the miR-195-3p by qRT-PCR comprises a primer for specifically amplifying miR-195-3 p.
Further, the detection sample is animal liver tissue or liver cells.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers that miR-195-3p is up-regulated in oxidative stress of liver induced by Hcy for the first time, and miR-195-3p promotes aggravation of oxidative stress of liver induced by Hcy to cause liver injury, provides a basis for diagnosis and prognosis of oxidative stress injury of liver by detecting the expression level of miR-195-3p in liver tissues and liver cells, provides a theoretical basis for diagnosing metabolic liver diseases from a molecular level in the future, and has great theoretical significance and potential practical value.
Drawings
FIG. 1 shows that the detection kits for MDA (A) and T-SOD (B) detect cbs respectively +/+ Group and cbs +/- The MDA content and the T-SOD activity in the liver tissue of the mice are formed, * P<0.05， ** P<0.01。
FIG. 2 shows that MDA (A) and T-SOD (B) detection kits detect MDA content and T-SOD activity of hepatocytes after Hcy treatment, ** P<0.01。
FIG. 3 depicts the cbs detection by qRT-PCR +/+ Group and cbs +/- The expression level of miR-195-3p in liver tissues of the mice in the group, ** P<0.01。
FIG. 4 shows that the expression level of miR-195-3p in the Hcy-treated hepatocytes is detected by qRT-PCR, ** P<0.01。
FIG. 5 shows miR-195-3p mimic (A) and inhibitor (B) transfected in hepatocytes (HL-7702), expressed by miR-195-3p detected by qRT-PCR, ** P<0.01。
FIG. 6 shows the changes of MDA content (A) and T-SOD activity (B) in hepatocytes after transfection of miR-195-3p mimic, and the changes of MDA content (C) and T-SOD activity (D) in hepatocytes after transfection of miR-195-3p inhibitor, ** P<0.01。
FIG. 7 shows cbs +/- MDA content (A) and T-SOD activity (B) in liver tissues after miR-195-3p recombinant lentivirus is injected into mouse tail vein, * P<0.05， ** P<0.01。
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
The specific technical scheme of the invention is as follows:
(1) Establishment of cbs Gene knockout (cbs) +/- ) Mouse Gao Tongxing cysteinemia model: cbs +/- Heterozygote mice were purchased from Jackson laboratories, USA, and were housed in Ningxia university of medicine laboratory animal center, and were bred with cbs +/+ The group was a normal control group.
(2) After isoflurane anesthesia, separating liver tissues of each group of mice, and respectively detecting cbs by using MDA and T-SOD detection kits +/+ Group and cbs +/- MDA content and T-SOD activity in liver tissue of mice. Culturing hepatic cell (HL-7702) in vitro, and detecting MDA content and T-SOD activity under Hcy respectively with MDA and T-SOD detection kit.
(3) qRT-PCR detected the expression of miR-195-3p in mouse liver tissue and hepatocytes (HL-7702). miR-195-3p mimics (miR-195-3 p-mimic) and inhibitors (miR-195-3 p-inhibitor) are transfected in hepatocytes (HL-7702), and the influence of the mimics on MDA content and T-SOD activity is detected.
(4)cbs +/- After miR-195-3p recombinant lentivirus (Lv-miR-195-3 p), empty vector control (Lv-GFP) and PBS with the same volume are injected into a mouse through tail vein, the change of MDA content and T-SOD activity in liver tissues is detected.
The effect of miR-195-3p in the oxidative stress injury process of the liver is further clarified by the following experiments.
1 test subject
1.1 Experimental animals
cbs +/+ And cbs +/- A mouse.
1.2 cell lines
HL-7702 cell line (human hepatocytes).
2 instruments and equipment and experimental reagent
2.1 Primary reagents
Sterile cell culture flasks, pipettes, filters (Corning, usa); fetal bovine serum, RPMI 1640 medium (Gibco, usa); streptomycin qing, trypsin digest (solibao, china); homocysteine (Sigma, usa); blood/cell/tissue gene RNA extraction reagent (Omega, usa); reverse transcription kit (Takara, japan); qRT-PCR mix (QIAGEN, germany); MDA and T-SOD detection kit (Nanjing, china); the miR-195-3p and U6 primers were synthesized by Ruibo Biotechnology, inc., guangzhou; the miR-195-3p recombinant lentivirus (Lv-miR-195-3 p) and empty vector control (Lv-GFP) are provided by Shanghai Ji Ma pharmaceutical technology, inc.
2.2 Main Instrument
Thermostated cell culture chambers (Thermo Fisher Scientific, usa); clean bench (Antai technologies, inc., suzhou, china); 5415 model D microcuvette centrifuge (Eppendorf, germany); precision electronic balances (Sartorius, germany); ice maker (AF 10 SCOTSMAN, usa); fluorescent quantitative PCR instrument (Funglyn, shanghai); general PCR machine (Bio-Rad, USA); micropipettes (Eppendorf, germany); pure water instruments (Heal Force, china); fully automatic enzyme labeling machine (Bio-TEK); centrifuge (Thermo Fisher, eppendorf, usa); vortex mixer (Dalong, china); a constant temperature metal bath (ka You Di); a negative pressure aspirator (packer), and the like.
3 method
3.1 cell culture
At 37 ℃ C, 5% CO 2 Human hepatocytes (HL-7702) were cultured in a concentration incubator with 10% fetal bovine serum and 1% streptomycin-containing RPMI-1640, and when the cell density reached 70%, the hepatocytes were interfered with Hcy at final concentrations of 0. Mu.M and 100. Mu.M, respectively, as Control group and Hcy group, respectively. After 48h of cell intervention, cells were harvested for subsequent studies.
3.2 determination of MDA content and T-SOD Activity
The MDA content and the T-SOD activity are detected by strictly operating according to the kit instructions. Collecting cell lysates of different treatment groups or accurately weighing liver tissues of mice of each group, adding 9 times of volume of physiological saline according to the weight (g) and volume (ml) of 1:9, mechanically homogenizing under the condition of ice-water bath to prepare 10% homogenate, centrifuging for 10min at 2500-3000 r/min, and measuring after quantifying BCA protein of supernatant. The cells were collected, and after adding physiological saline according to the amount of the cells, the cells were disrupted by sonication to collect protein BCA for quantification, and then the measurement was carried out. The calculation formula is as follows:
3.3 fluorescent quantitative PCR (qRT-PCR) for detecting the expression of miR-195-3p
8978 extraction of zxft 8978
Extracting RNA according to the kit instructions, adding 500. Mu.L of TRK lysate (20. Mu.L of beta-hydroxyethanol is added to every 1ml of TRK lysate before use) into liver tissues and liver cells, homogenizing by using a homogenizer, and transferring the lysis sample into a 1.5ml RNase-free centrifuge tube; standing and cracking the sample for about 5min in a super clean bench (30 min after ultraviolet irradiation disinfection), and properly prolonging the time according to the sample cracking condition to completely separate the nucleic acid-protein compound; centrifuging at 4 deg.C and 12,000 rpm for 5min to remove protein, fat, etc., and transferring the supernatant into a new 1.5ml RNase-free centrifuge tube; adding 50% volume of anhydrous ethanol/sample, oscillating and mixing uniformly by a vortex oscillator, transferring the sample into a binding mini column, centrifuging at 12,000 rpm for 1min, and discarding waste liquid; placing the column in a new collection tube of 2ml, adding 300 microliter of RNA wash buffer I, centrifuging at 12,000 rpm for 1min at high speed, and discarding the waste liquid; adding 500 mu L of RNA wash buffer I, centrifuging at 12, 000rpm for 1min, and discarding the waste liquid; adding 500 mu L of RNA wash buffer I again, centrifuging at 12,000 rpm for 1min, and discarding the waste liquid; centrifuging for 2min again, taking the adsorption column out of the collecting pipe, standing at normal temperature for 5-10 min, and removing the residual rinsing liquid; preparing a new centrifugal tube with 1.5ml and no RNase, marking, placing an air-dried adsorption column into the centrifugal tube, vertically suspending in the air, dripping 70 mu L of DEPC water into the center of an adsorption membrane, standing at room temperature for waiting for 2min, 12 at 4 ℃, and centrifuging at 000rpm for 2min to obtain RNA, wherein the RNA is stored at-80 ℃ to prevent RNA degradation.
3.3.2 reverse transcription
This step was done in an ultra clean bench illuminated by uv light, using RNase-free tips during the operation, placing 200 μ l RNase-free centrifuge tubes on ice, and then adding the following ingredients in table 1 in sequence:
TABLE 1 reverse transcription System
After the above systems are mixed evenly, the mixture is instantaneously centrifuged to carry out RT reaction, and the reaction procedure is as follows: 15min at 37 ℃, 5s at 85 ℃ and heat preservation at 4 ℃. The reacted product is preserved at-20 ℃ in a short time and at-80 ℃ for a long time, and repeated freeze thawing is avoided.
3.3.3qRT-PCR detection of miR-195-3p expression
The following systems in Table 2 were added to 200. Mu.l RNase-free centrifuge tubes, respectively, in that order:
TABLE 2qRT-PCR reaction System
After all samples are added, vortex oscillation and uniform mixing are carried out, the samples are placed into a fluorescent quantitative PCR instrument for reaction after instantaneous centrifugation, and the reaction procedure is as follows: the reaction was run for 45 cycles at 95 ℃ 2min,95 ℃ 5s,60 ℃ 10s, with U6 as the internal control. Relative amounts according to the gene of interest =2 -△△Ct As a result, the miR-195-3p primer was designed and synthesized by Ruibo Biotechnology, inc., guangzhou.
3.4miR-195-3p mimetics and inhibitor transfection
When the cell growth is fused to 70%, transferring the miR-195-3p analogue (miR-195-3 p-mic), the inhibitor (miR-195-3 p-inhibitor) and corresponding control groups, namely micic-NC and inhibitor-NC, into the hepatocyte with good growth state respectively, transfecting for 6 hours, then changing the liquid, and transfecting for 48 hours for subsequent experiments.
3.5 mouse caudal vein injection of miR-195-3p recombinant lentivirus
The virus titer was 2X 10 7 TU/mL miR-195-3p recombinant lentivirus (Lv-miR-195-3 p) and Lv-GFP virus cbs +/- The mice were sacrificed 30 days after injection of lentivirus, and liver tissues were taken and rapidly frozen in liquid nitrogen for subsequent experiments.
4. Statistical treatment
All data types are quantitative data, and the statistical results are in terms of mean ± standard deviation
The mean of two samples was tested using Student's t, the mean of multiple samples was tested using One-way ANOVA, the two-by-two comparisons between groups were tested using Student-Newman-Keuls, and the test level was α =0.05.
5 results
5.1cbs +/+ And cbs +/- MDA content and T-SOD activity in liver tissue of mouse
Detection of cbs by MDA and T-SOD detection kit respectively +/+ And cbs +/- MDA content and T-SOD activity in liver tissue of mice, the results are shown in FIG. 1: and cbs +/+ Group comparison, cbs +/- The MDA content in the liver tissue of the group mice is obviously increased (P)<0.05 The T-SOD activity is obviously reduced (P)<0.01 The difference is significant).
5.2 Effect of Hcy on MDA content and T-SOD Activity in hepatocytes
MDA content and T-SOD activity in the liver cells after Hcy treatment are detected by MDA and T-SOD detection kits, and the result is shown in figure 2: compared with the Control group, the content of MDA in the Hcy group is obviously increased (P < 0.01), the activity of T-SOD is obviously reduced (P < 0.01), and the difference is obvious.
5.3cbs +/+ And cbs +/- Expression condition of miR-195-3p in liver tissue of mouse
qRT-PCR detection of cbs +/+ And cbs +/- The expression level of miR-195-3p in mouse liver tissue is shown in figure 3: and cbs +/+ Group comparison, cbs +/- The expression level of miR-195-3P in liver tissues of mice is obviously increased (P)<0.01 The difference is significant).
5.4 Effect of Hcy on miR-195-3p in hepatocytes
The expression level of miR-195-3p in the hepatocytes after Hcy treatment is detected by qRT-PCR, and the result is shown in FIG. 4: compared with the Control group, the expression level of miR-195-3P in the Hcy group is obviously increased (P is less than 0.01), and the difference is significant.
5.5 expression of miR-195-3p after transfection of miR-195-3p mimic and inhibitor
In order to reveal the role of miR-195-3p in oxidative stress injury of liver caused by Hcy, miR-195-3p mimics (miR-195-3 p-mic) and inhibitors (miR-195-3 p-inhibitor) are transfected in liver cells, and the transfection conditions of the miR-195-3 p-mic and miR-195-3p-inhibitor after transfection are detected by qRT-PCR (quantitative reverse transcription-polymerase chain reaction), and the results are shown in FIG. 5: compared with the mimic-NC group, the expression level of miR-195-3P in the liver cells is obviously increased (P < 0.01) after miR-195-3P-mimic transfection, and the expression level of miR-195-3P-inhibitor transfection is obviously reduced (P < 0.01), which indicates that the transfection is successful and can be used for subsequent experiments.
5.6 Effect of miR-195-3p on MDA content and T-SOD activity in hepatocytes
MDA content and T-SOD activity in hepatocytes after miR-195-3 p-imic and miR-195-3p-inhibitor transfection are measured by an MDA and T-SOD detection kit, and the results are shown in figure 6: compared with a mimic-NC group or an inhibitor-NC group, after miR-195-3P-mimic transfection, the MDA content is remarkably increased, the T-SOD activity is remarkably reduced (P < 0.01), while after miR-195-3P-inhibitor transfection, the MDA content is remarkably reduced, the T-SOD activity is remarkably increased (P < 0.01), and the difference is remarkable.
5.7miR-195-3p vs cbs +/- Effect of MDA content and T-SOD Activity in liver tissue of mice
Detection of cbs by MDA and T-SOD detection kits +/- Intravenous injection of mouse tailThe MDA content and the T-SOD activity in the liver tissue after the miR-195-3p recombinant lentivirus result are shown in figure 7: compared with the Lv-GFP group, the MDA content in liver tissues of the Lv-miR-195-3P group is remarkably increased (P)<0.01 The T-SOD activity is obviously reduced (P)<0.05 The difference is significant).
Conclusion 6
Hcy can induce liver tissues and liver cells to generate oxidative stress, miR-195-3p is up-regulated in the oxidative stress of the liver induced by Hcy, and miR-195-3p can promote aggravation of the oxidative stress of the liver induced by Hcy so as to cause liver injury.
The invention feeds cbs with high methionine feed +/- The mouse is used for duplicating a hyperhomocysteinemia model, observing the MDA content and the T-SOD activity in liver tissues and liver cells, observing the action of miR-195-3p in the process of promoting oxidative stress injury of the liver by Hcy, providing a new theoretical basis for clinically preventing and treating metabolic liver diseases in future and having very important significance.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Ningxia medical university
Application of miR-195-3p detection reagent in preparation of product for diagnosing oxidative stress injury of liver
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<170> SIPOSequenceListing 1.0
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<212> RNA
<213> Intelligent (Homo sapiens)
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Claims (4)
- The application of the miR-195-3p expression level detection reagent in the preparation of products for diagnosing oxidative stress injury of the liver caused by hyperhomocysteinemia is characterized in that the sequence of the miR-195-3p is shown as SEQ ID NO. 1.
- 2. The application of the miR-195-3p expression level detection reagent in the preparation of products for diagnosing liver oxidative stress injury caused by hyperhomocysteinemia according to claim 1, wherein the reagent comprises: and (3) detecting the expression level of miR-195-3p by qRT-PCR.
- 3. The application of the miR-195-3p expression level detection reagent in the preparation of products for diagnosing liver oxidative stress injury caused by hyperhomocysteinemia according to claim 2, wherein the reagent for detecting miR-195-3p expression level through qRT-PCR comprises a primer for specifically amplifying miR-195-3 p.
- 4. The application of the miR-195-3p expression level detection reagent in the preparation of products for diagnosing liver oxidative stress injury caused by hyperhomocysteinemia according to claim 1, wherein the detection sample is animal liver tissue or liver cells.
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| 张宏红等.miR- 195- 3p 在同型半胱氨酸诱导的 肝氧化应激损伤中的调控作用研究.《中国临床药理学杂志》.2022,第第38卷卷(第第38卷期),2044-2048页. * |
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