CN116334206A - Application of miR-541in preparation of noninvasive diagnosis and prognosis evaluation kit for hepatic fibrosis/cirrhosis - Google Patents
Application of miR-541in preparation of noninvasive diagnosis and prognosis evaluation kit for hepatic fibrosis/cirrhosis Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to application of miR-541in preparation of a noninvasive diagnosis and prognosis evaluation kit for liver fibers/liver cirrhosis. The invention utilizes the serum miRNA extraction, reverse transcription and RT-PCR method to measure the expression quantity of miR-541in serum of patients with cirrhosis, and combines follow-up information to determine the correlation between the expression quantity of miR-541 and prognosis of patients with cirrhosis, and miR-541 can be used for preparing noninvasive molecular markers for judging prognosis of patients with cirrhosis, and has important guiding significance for preventing or delaying treatment such as decompensation of cirrhosis in the decompensation period.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of micro ribonucleic acid (RNA), in particular to application of miR-541in preparation of a noninvasive diagnosis and prognosis evaluation kit for liver fibers/liver cirrhosis.
Background
Liver fibrosis is an early stage of cirrhosis, and once it progresses to cirrhosis, particularly to decompensated cirrhosis, many complications such as hepatic encephalopathy, esophageal varices rupture hemorrhage, ascites complicated infection, etc. follow up, and survival rate drops sharply, which is one of the serious diseases endangering the life of patients. It is mainly characterized by the continuous activation of hepatic stellate cells (Hepatic stellate cells, HSCs) and excessive deposition of extracellular matrix in the liver under the long-term stimulation of various acute and chronic injury factors such as viruses (HBV/HCV, toxins, medicines, alcohol, oxidative stress factors and the like), and HSCs activation is a key link of hepatic fibrosis. The liver fibrosis mechanism is not clear at present, and especially key targets, cells and molecules are to be further studied and confirmed. Various signaling pathways, molecules, can inhibit or maintain the development of liver fibrosis by inhibiting or maintaining HSCs activation and proliferation.
Early identification and intervention of liver fibrosis is expected to reverse fibrosis, blocking its progression to irreversible cirrhosis or liver cancer. The current diagnosis method mainly comprises the imaging examination of liver-penetrating pathology detection, ultrasound, nuclear magnetic resonance and the like, blood markers, biochemical models, liver hardness value detection and the like, and the liver-penetrating pathology detection is still a gold standard for diagnosing liver fibrosis, but the liver-penetrating pathology detection is large in wound, is difficult to accept by a plurality of patients, has high imaging examination cost of nuclear magnetic resonance and the like, and is prohibitively expensive for a plurality of patients; with the popularization and progress of noninvasive diagnosis technologies in recent years, more and more researches focus on related noninvasive diagnosis means such as serum markers and biochemical models, wherein micrornas (miRNAs) exist stably in serum, and are favored by researchers, and can be used as good noninvasive diagnosis biomarkers, treatment targets and prognostic factors. At present, the research on noninvasive diagnosis and prognosis analysis of miRNAs in liver cirrhosis is small in quantity, small in sample size and limited to a certain index, and the miRNAs are not deeply researched, and many lack of related molecular mechanism evidence, and the clinical prediction requirements are not satisfied.
The related noninvasive diagnostic markers for predicting the liver fibrosis/cirrhosis disease and prognosis are urgently needed in the field, and the research has important significance for clinically treating the liver fibrosis/cirrhosis and preventing liver cancer.
microRNA 541 (microRNA 541, miR-541) is a non-coding RNA (UGGUGGGCACAGAAUCUGGACU, SEQ ID NO: 1) of 22 nucleotides in length, is located on human chromosome 14, and is one of the miRNA cluster members of the DLK1/DIO3 imprinted gene region. We have found that when studying hepatocyte nuclear factor 4α, it can transcriptionally regulate miR-379-656 cluster of DLK1/DIO3 imprinting gene region, and miR-541 is also in the cluster. We have previously suggested that miR-541 can inhibit liver cancer, is associated with prognosis of liver cancer, and can inhibit Sorafenib resistance by inhibiting autophagy (Xu WP, et al miR-541potentiates the response of human hepatocellular carcinoma to sorafenib treatment by inhibiting autophagy.Gut.2020;69 (7): 1309-1321). miR-541 can influence cell differentiation, regulate and control lung and kidney fibrosis and reduce expression in damaged tissues besides influencing tumors such as liver cancer, cholangiocellular carcinoma and the like. Liver fibrosis is a repair reaction of liver to injury factors, and the molecular mechanism is not completely clear, and effective therapeutic targets and prognosis markers are limited.
However, no report has been made at present on whether miR-541 can be used as a noninvasive diagnosis marker for liver cirrhosis or not as to the expression change of miR-541in serum of patients with liver cirrhosis
Disclosure of Invention
The invention aims to provide a novel application of miR-541, in particular to an application in preparing a hepatic fibrosis/cirrhosis noninvasive diagnostic marker and a prognosis evaluation kit.
Through extensive and intensive research, the inventor discovers that the expression level of miR-541in serum of a patient suffering from liver cirrhosis can be judged by adopting a serum miRNA extraction method, a reverse transcription method and an RT-PCR method for detecting the expression level of miR-541in serum of the patient suffering from liver cirrhosis, and the conditions of decompensation complications such as ascites and the like of the patient suffering from liver cirrhosis in a decompensation period and the survival condition of the patient suffering from liver cirrhosis can be judged for the first time. Based on the correlation between the expression quantity of miR-541 and liver cirrhosis, the miR-541 is used as a molecular marker to detect the expression quantity of the miR-541, so that prognosis judgment of a patient suffering from liver cirrhosis can be guided.
In a first aspect of the invention, the application of miR-541 as a diagnostic marker in preparation of a liver fibrosis/cirrhosis prognosis evaluation kit is provided.
Furthermore, the prognosis evaluation kit comprises a reagent for detecting the expression level of miR-541in serum of a patient suffering from liver cirrhosis by using a miRNA reverse transcription and RT-PCR method.
Further, the prognosis evaluation kit is a noninvasive prognosis evaluation kit.
Furthermore, the application refers to that miR-541 is used as a molecular marker, and the expression quantity of miR-541in serum of a patient suffering from liver cirrhosis is analyzed by utilizing serum miRNA extraction, reverse transcription and RT-PCR.
Furthermore, in the application, the high and low expression standard of miR-541 is defined by the median (1.07 x 10E-7) of miR-541 expression scores in 84 cases of liver cirrhosis serum; when the expression is higher than the median miR-541, the expression is lower than the median miR-541.
The finding of the correlation between miR-541 and liver cirrhosis provides a brand-new way for predicting the severity of liver cirrhosis, occurrence of complications and survival or death of patients, has an important role in judging prognosis of patients with liver cirrhosis, and has important guiding significance for preventing or delaying treatment of decompensation and the like of liver cirrhosis in a decompensation period. When the expression level of miR-541 is lower than the median, complications such as ascites, hepatic encephalopathy and the like are likely to occur in the liver cirrhosis in the compensatory stage, and the patient suffering from the liver cirrhosis is likely to die.
In a second aspect, the invention provides application of a reagent for detecting the expression level of serum miR-541in preparation of a liver fibrosis/cirrhosis prognosis evaluation kit.
Furthermore, the reagent for detecting the expression level of the serum miR-541 is a reagent for detecting the expression level of the miR-541in serum of a patient suffering from liver cirrhosis by using a miRNA reverse transcription and RT-PCR method.
In a third aspect, the invention provides a noninvasive prognosis evaluation kit for liver fibrosis/cirrhosis, which comprises a reagent for detecting the expression level of miR-541in serum of a patient suffering from cirrhosis by using a miRNA reverse transcription and RT-PCR method.
The invention has the advantages that:
the invention utilizes the serum miRNA extraction, reverse transcription and RT-PCR method to measure the expression quantity of miR-541in serum of patients with cirrhosis, and combines follow-up information to determine the correlation between the expression quantity of miR-541 and prognosis of patients with cirrhosis, and miR-541 can be used for preparing noninvasive molecular markers for judging prognosis of patients with cirrhosis, and has important guiding significance for preventing or delaying treatment such as decompensation of cirrhosis in the decompensation period.
Drawings
FIG. 1 shows that miR-541 has reduced expression in activated hepatic stellate cells and liver cirrhosis tissue. Wherein A is the expression of miR-541 after LX-2 cells are treated by TGF-beta with different concentration gradients, B is the expression of miR-541 at different time points after LX-2 cells are treated by 5ng/ml TGF-beta, and C is the expression of miR-541in liver cirrhosis tissues and normal liver tissues.
FIG. 2 shows that miR-541 inhibits proliferation of hepatic stellate cells in vitro. Wherein, A is that the over-expression miR-541 inhibits the proliferation of LX-2, and B is that the down-regulation miR-541 promotes the proliferation of LX-2.
FIG. 3 shows that miR-541 inhibits hepatic stellate cell activation in vitro. Wherein, A is the expression of the mRNA of the markers Acta2 and Col1a1 of the liver fibrosis, B is the expression of the protein of the markers Acta2 and Col1a1 of the liver fibrosis, C is the expression of the mRNA of the markers Acta2 and Col1a1 of the liver fibrosis promoted by the miR-541, and D is the expression of the protein of the markers Acta2 and Col1a1 promoted by the miR-541.
FIG. 4 shows that miR-541 is expressed in serum of patients with liver cirrhosis and is related to the severity of liver cirrhosis. Wherein A is the expression of miR-541in serum of normal people, patients in liver cirrhosis compensating period and liver cirrhosis decompensating period, B is the correlation between the level of miR-541in serum of patients in liver cirrhosis and MELD score, and C is the comparison of the expression of miR-541in serum of patients in liver cirrhosis with Child-Pugh in different grades.
Fig. 5 is a graph comparing complications of the patients with compensated liver cirrhosis grouped according to the expression level of serum miR-541. Wherein A is the comparison of concurrent ascites between high and low expression groups of serum miR-541, and B is the comparison of concurrent hepatic encephalopathy between high and low expression groups of serum miR-541.
Fig. 6 is a graph of the survival of complications-free patients with cirrhosis grouped according to serum miR-541 expression level.
Fig. 7 is a graph of overall survival for the grouping of cirrhosis patients based on serum miR-541 expression levels.
Detailed Description
The following examples are provided to illustrate the present invention in detail, and the following examples are provided to illustrate the detailed embodiments and the specific operation procedures based on the technical scheme of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1:
LX-2 was stimulated with different concentrations of TGF- β (0 ng/ml, 2.5ng/ml, 5ng/ml, 10 ng/ml), corresponding cells were collected and RNA was extracted after 48h stimulation, miR-541 expression was detected using RT-PCR, which indicated that miR-541 expression was significantly reduced at 5ng/ml TGF- β (FIG. 3), and further that 5ng/ml TGF- β was used to stimulate LX-2, and miR-541 expression was detected at different time points (0 h, 12h, 24h, 48 h) and was found to be significantly reduced after 24h, 48h (FIG. 1). Suggesting that miR-541 expression is reduced in activated hepatic stellate cells.
Example 2:
randomly selecting 20 liver fibrosis/liver cirrhosis patients, and 20 liver tissue specimens for liver discard (liver tissue specimens are all from Ruijin Hospital), grinding the tissues, extracting RNA by Trizol method, detecting miR-541 expression by RT-PCR, and displaying miR-541 expression in liver cirrhosis tissue to be reduced (figure 1).
The specific method for detecting the expression quantity of miR-541in cells and tissues comprises the following steps:
1. the collected cell or tissue samples were subjected to total RNA extraction by Trizol method and quantified. The specific operation is as follows:
(1) Grinding the tissue or cultured cells, adding 1ml Trizol; repeatedly blowing for several times until the mixture is uniform and has no sticky feeling, fully lysing tissues or cells, and transferring the tissues or cells into a 1.5ml Ep tube;
(2) Adding 200 μl (1/5 of Trizol volume) of chloroform into each Ep tube, shaking thoroughly, mixing, and standing at room temperature for about 5 min;
(3) In advance precooling to 12000g in a centrifuge at 4 ℃ for 15 minutes;
(4) The solution can be in three layers, the upper transparent liquid is carefully transferred into a new clean 1.5ml Ep tube, 500 μl of isopropanol (equal volume to the upper transparent liquid) is added, the Ep tube is inverted up and down for 6-8 times to mix uniformly, and the solution is precipitated for about 30 minutes at-80 ℃;
(5) Centrifuge 12000g at 4deg.C for 10 min, at which time white precipitate was visible at the bottom of the tube;
(6) The supernatant was aspirated, and 1ml of 85% ethanol (DEPC water) was added to the Ep tube to gently pop up the pellet;
(7) Centrifuging 7500g of the mixture for 5 minutes at 4 ℃, discarding the supernatant again, rinsing once again, or directly inverting the Ep tube at room temperature and airing;
(8) When the white precipitate is semitransparent, adding a proper amount of DEPC water to dissolve RNA;
(9) The RNA concentration and 260nm/280nm values were measured and used directly or RNA was stored in a-80℃refrigerator for use.
2. Reverse transcription by tail-adding method, the configuration system and the conditions are as follows:
reaction conditions: 37 ℃ 60min,85 ℃ 5min, 4-
3、RT-PCR
Utilizing SYBR Premix Ex Taq kit and Step-one real-time fluorescence quantitative PCR instrument, adopting U6 as internal reference, and analyzing the expression quantity of serum miRNA
PCR reaction system:
reaction conditions for PCR: the calculation analysis was performed by the delta Ct method at 94℃for 30s,94℃for 10s, and 60℃for 30s for 40 cycles (two-step method).
Example 3:
the miR-541 analogue (miR-541 mic) synthesized by a chemical method is used for transfecting activated human hepatic stellate cells (LX-2), up-regulating the expression of miR-541in the cells or transfecting 2 methoxy-modified miR-541 complementary strand (miR-541 inhibitor) to inhibit the activity of miR-541in the cells, detecting the number of living cells by using a CCK8 kit (cell-counting kit 8), drawing a growth curve, and observing the change of proliferation capacity. The results show that up-regulating miR-541 inhibits the proliferation capacity of LX-2, and down-regulating miR-541 promotes the proliferation capacity of LX-2 (FIG. 2). miR-541 is suggested to inhibit proliferation of activated hepatic stellate cells.
Example 4:
miR-541mimic or miR-541inhibitor transfects activated LX-2, up-regulates or down-regulates miR-541 expression, cultures cells for 48 hours, extracts RNA by a Trizol method, carries out reverse transcription to obtain cDNA, and defines the mRNA expression of Acta2 and Col1a1 by RT-PCR; western Blot detects expression of the protein of alpha-SMA and Col1a 1. The results suggest that up-regulation of miR-541 inhibited the expression of Acta2 and Col1a1, and down-regulation of miR-541 promoted the expression of Acta2 and Col1a1 (FIG. 3). The results show that miR-541 can inhibit activation of hepatic stellate cells.
Example 5:
randomly selecting 84 serum samples of patients with liver cirrhosis (the serum is from Shanghai long-term hospital, and the liver cirrhosis is confirmed by 2 clinicians) and 50 normal human serum samples, extracting RNA by adopting a serum extraction miRNA kit, and detecting the expression quantity of miR-541in the serum of the patients with liver cirrhosis by using an RT-PCR method, wherein the method comprises the following steps:
1. total miRNA in Serum was extracted using a miraasy Serum/Plasma Advanced Kit procedure as follows.
Reverse transcription of miRNA, and the method is similar to that of tissue miRNA.
RT-PCR, the method is similar to that of tissue, and Ce-miR-39 is adopted as an internal reference.
The miRNA extraction comprises the following specific steps:
(1) Taking out serum specimen from-80 deg.c refrigerator, thawing and centrifuging.
(2) 200ul of serum was transferred to a fresh 1.5ml EP tube.
(3) 60ul Buffer RPL was added, shaken for at least 5 seconds, mixed well, and then allowed to stand at room temperature for 3 minutes.
(4) 3.5ul of mi Reasy Serum/Plasma Spike-In Control was interposed and mixed well.
(5) Adding 20ul Buffer RPP, shaking for at least 20 seconds, mixing, and standing at room temperature for 3 minutes.
(6) Centrifuge at 12000g for 3 min at room temperature.
(7) The supernatant was transferred to a fresh 1.5ml ep tube, added with an equal volume of isopropanol, and mixed well.
(8) Adding RNeasy UCP MinElute column into the uniformly mixed liquid, centrifuging for 15s, not less than 8000g, and discarding the lower liquid.
(9) 700ul Buffer RWT was added to UCP column, centrifuged for 15 seconds, not less than 8000g, and the supernatant was discarded.
(10) Adding 500ul Buffer RPE into UCP column, centrifuging for 15s, not less than 8000g, and discarding the supernatant.
(11) Adding 500ul of 80% ethanol into UCP column, centrifuging for 2 min to not less than 8000g, and discarding the supernatant.
(12) UCP column is placed in a new 1.5ml EP tube, after airing, 20ul of double distilled water without RNase is added, incubated for 1 min at room temperature, and RNA is eluted by 12000g centrifugation for 1 min, and is directly used for the next step or stored at-80 ℃.
Comparing the levels of miR-541in serum of liver cirrhosis and normal people, the result indicates that miR-541 expression in serum of liver cirrhosis decompensated patients is lower than that of normal people, but no statistical significance exists between the two groups, and expression in liver cirrhosis decompensated patients is obviously lower than that of normal people, and the result is statistically significant, and miR-541in serum of liver cirrhosis decompensated patients is obviously lower than that in decompensated patients (figure 4). As suggested by the Child-pugh classification, miR-541 levels were significantly lower in phase C than in phase B, both lower than in phase A (FIG. 4). And miR-541 is inversely correlated with MELD score (figure 4). Suggesting that the expression of miR-541in liver cirrhosis serum is related to liver cirrhosis severity.
Comparing the difference of complications such as ascites and hepatic encephalopathy of patients in the compensatory period, the result indicates that the miR-541 high-expression group is not easy to cause ascites and hepatic encephalopathy related complications, and otherwise, the related complications are more easy to cause (figure 5).
Total survival (fig. 6) and no complications survival (fig. 7) were plotted. The result shows that the survival time and the total survival time of the patients with high expression of miR-541in liver cirrhosis are obviously longer than those with low expression.
From the test results, the severity of the patients suffering from liver cirrhosis, the occurrence of complications and the survival or death of the patients can be predicted by detecting the expression level of miR-541in liver cirrhosis serum by adopting an RT-PCR method. When miR-541 is lower than the median, decompensation-related complications easily occur in the liver cirrhosis in the compensation period, and the liver cirrhosis patients easily die. Obviously, miR-541 has correlation with the severity of liver cirrhosis, so that detection of the expression quantity of miR-541 serving as a molecular marker can predict liver cirrhosis decompensation and other events and judge prognosis. Accordingly, it will be apparent to those skilled in the art that serum miRNA extraction kits, RT-PCR, etc. can be used to prepare a preparation or kit for determining prognosis of cirrhosis.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (8)
- Application of miR-541 as a diagnostic marker in preparation of liver fibrosis/cirrhosis prognosis evaluation kit.
- 2. The application of miR-541 as a diagnostic marker in preparing a hepatic fibrosis/cirrhosis prognosis evaluation kit, which is characterized in that the kit comprises a reagent for detecting the expression level of miR-541in serum of a patient suffering from cirrhosis by using a miRNA reverse transcription and RT-PCR method.
- 3. The use of miR-541 as a diagnostic marker in the preparation of a liver fibrosis/cirrhosis prognosis evaluation kit according to claim 1, wherein the kit is a noninvasive prognosis evaluation kit.
- 4. The application of miR-541 as a diagnostic marker in preparing a liver fibrosis/liver cirrhosis prognosis evaluation kit, which is characterized in that miR-541 is used as a molecular marker, and the expression level of miR-541in serum of a liver cirrhosis patient is analyzed by serum miRNA extraction, reverse transcription and RT-PCR.
- 5. The use of miR-541 as a diagnostic marker in the preparation of a kit for prognosis evaluation of liver fibrosis/cirrhosis of claim 4, wherein, in the use, when the expression level of miR-541in serum of a patient suffering from cirrhosis is less than 1.07 x 10e-7, complications such as ascites, hepatic encephalopathy, etc., are likely to occur in cirrhosis in the compensatory stage, and the patient suffering from cirrhosis is likely to die.
- 6. Application of a reagent for detecting serum miR-541 expression quantity in preparation of a hepatic fibrosis/cirrhosis prognosis evaluation kit.
- 7. The application of the reagent for detecting the expression level of the serum miR-541in preparing a liver fibrosis/liver cirrhosis prognosis evaluation kit, which is characterized in that the reagent for detecting the expression level of the serum miR-541 is a reagent for detecting the expression level of the miR-541in serum of a patient suffering from liver cirrhosis by using a miRNA reverse transcription and RT-PCR method.
- 8. The noninvasive prognosis evaluation kit for liver fibrosis/cirrhosis is characterized by comprising a reagent for detecting the expression level of miR-541in serum of a patient suffering from cirrhosis by using a miRNA reverse transcription and RT-PCR method.
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