CN110548041A - Application of LNA-anti-miR-150 in preparation of medicine for preventing or treating renal fibrosis - Google Patents

Abstract

The invention relates to application of a miR-150 inhibitor in kidney fibrosis prevention and treatment, and belongs to the field of biological medicines. The inhibitor can inhibit miR-150 signal pathways, and relieve kidney fibrosis caused by glomerular injury and tubulointerstitial injury by up-regulating SOCS1 gene. The invention also discloses a mature structural formula of miR-150 and a structure of an inhibitor, and a method, an effect and a mechanism for treating renal fibrosis by applying the miR-150 inhibitor.

Description

Application of LNA-anti-miR-150 in preparation of medicine for preventing or treating renal fibrosis

Technical Field

The invention belongs to the field of pharmacology, and particularly relates to application of LNA-anti-miR-150 in preparation of a medicine for preventing or treating renal fibrosis.

Background

microRNAs (miRs) are short, single-stranded, non-coding RNAs composed of about 22 nucleotides that regulate the translation of proteins that ultimately interfere with the execution of biological functions in 50% of the human genome. Recent studies have demonstrated that aberrant expression of miRs is involved in the mechanism of development of renal fibrosis due to various renal diseases. First inventors discovered and verified a positive correlation of miR-150 with chronic activity index of renal pathology in 25 Lupus Nephritis (LN) patients using microarray chip analysis (microRNA microscopy) technology in duplicate kidney biopsy wax blocks during us work with quantitative polymerase chain reaction (qPCR). Meanwhile, the miR-150 level of the kidney tissue is found to be obviously and positively correlated with the transforming growth factor beta 1(transforming growth factor-beta 1, TGF-beta 1), fibronectin (fibronectin, FN), collagen I (COLLAGEN I, COL1) and collagen III (COLLAGEN III, COL3) in the kidney, and negatively correlated with the anti-fibrosis protein-cytokine signal transduction inhibitor 1 (SOCS 1). And further research on a cellular level shows that miR-150 increases the synthesis of profibrotic protein by down-regulating the target protein SOCS1 to cause kidney fibrosis.

Fibrosis of the kidney can be caused by both glomerulosclerosis due to glomerular injury and interstitial fibrosis of the kidney due to tubular injury, and is a common pathological feature of Chronic Kidney Disease (CKD). The incidence rate of CKD in China is 10.8% (2012, Lancet 379, 815-125822) and the incidence rate of CKD in the United states is 14% (2017, Lancet 389: 1238-1252). CKD eventually progresses to end-stage renal disease (ESRD) due to kidney fibrosis, and dialysis or kidney transplantation will be required for life maintenance, which brings huge economic burden to society and families. LN accounts for the first place of secondary glomerular diseases discovered in renal biopsy in China, and although new medicines for treating systemic lupus erythematosus are emerging continuously in the last two decades, the LN incidence rate is always high (50-70%), and is particularly higher in Asian population. Focal Segmental Glomerulosclerosis (FSGS) is the most common primary glomerular disease that causes ESRD. Chronic interstitial nephritis (CITN) patients due to the use of various nephrotoxic drugs are also not uncommon in clinical work. The onset of the disease of CITN is hidden, no obvious clinical expression exists, and a specific clinical diagnosis marker is lacked, so that the early detection cannot be realized, and the kidney fibrosis cannot be timely and effectively treated and developed into another important cause of ESRD. Therefore, effective medicines are searched for inhibiting renal fibrosis, so that the occurrence of ESRD is delayed and controlled, the proportion of patients needing renal replacement therapy is reduced, the life quality of the patients is improved, the medical burden of the patients and the society is reduced, and great economic benefits are brought to families of the patients and the whole society.

The compound related to the miR-150 inhibitor can inhibit the expression of the kidney miR-150, and effectively relieves the kidney fibrosis. The present invention satisfies this need. The invention applies the nucleic acid preparation for preventing and treating the kidney fibrosis for the first time.

Disclosure of Invention

Aiming at the clinical background and the hazard of the renal fibrosis and no medicine for effectively radically treating or relieving the renal fibrosis pathological changes exists clinically at present, the invention provides the application of LNA-anti-miR-150 in preparing the medicine for preventing or treating the renal fibrosis. The application aims to solve the technical problems of treating renal fibrosis lesions and the like clinically.

The invention provides a mature sequence SEQ ID NO.1 of miR-150: UCUCCCAACCCUUGUACCAGUG, wherein the sequence of the miR-150 inhibitor LNA-anti-miR-150 is SEQ ID NO. 2: TACAAGGGTTGGGAG are provided.

Further, the miR-150 inhibitor LNA-anti-miR-150 is applied to preparation of a medicine for preventing or treating renal fibrosis.

Further, the miR-150 inhibitor LNA-anti-miR-150 uses an LNA ^TM structure to inhibit miR-150 effect stably.

Further, the LNA ^TM is a novel nucleic acid analog comprising a 2 '-oxo-4' carbon methylene linkage that limits the flexibility of the ribofuranose ring, locking its structure into a rigid bicyclic mode, thus increasing hybridization efficiency and superior stability.

The invention provides the biological safety of LNA-anti-miR-150 as a novel nucleic acid medicament in a mouse body. The invention provides a therapeutic effect of relieving kidney fibrosis in a mouse model LNA-anti-miR-150 for spontaneous lupus and lupus nephritis. The invention provides a therapeutic effect of LNA-anti-miR-150 in a mouse model of doxorubicin-induced focal segmental glomerulosclerosis for relieving renal fibrosis. The invention provides a therapeutic effect of a mouse model LNA-anti-miR-150 for relieving renal fibrosis caused by renal interstitial injury induced by a large amount of folic acid. The invention provides a mechanism for controlling the expression of SOCS1 by inhibiting the expression of kidney miR-150 through LNA-anti-miR-150, and finally preventing and treating kidney fibrosis.

In conclusion, we believe that miR-150 plays an important role in the development and progression of renal fibrosis. LNA-anti-miR-150 can effectively relieve and treat kidney fibrosis caused by glomerular injury and tubulointerstitial injury, and provides a basis for clinically developing a novel medicament for treating the kidney fibrosis.

Compared with the prior art, the invention has the technical progress that: the invention has the innovation points and the beneficial effects that the miR-150 inhibitor LNA-anti-miR-150 can be proved to be capable of effectively relieving renal fibrosis caused by glomerular injury and tubulointerstitial injury, and improving and protecting the kidney. Compared with other existing medicines, LNA-anti-miR-150 can hinder the onset and progress of renal fibrosis from multiple aspects, has guaranteed safety, and has an obvious inhibition effect on the onset of renal fibrosis, so that the renal function is protected, and a theoretical basis is provided for clinically preparing LNA-anti-miR-150 related prevention and treatment of renal fibrosis.

Drawings

FIG. 1 is a graph showing biosafety evaluation of LNA-anti-miR-150 in a lupus nephritis mouse model in example 2, wherein A is a graph showing changes in body weight of LNA-anti-miR-150 before and after 8 weeks of treatment, B is a graph showing serum creatinine levels of mice in the LNA-anti-miR-150 treatment group and the placebo treatment group, C is a graph showing serum urea nitrogen levels of mice in the LNA-anti-miR-150 treatment group and the placebo treatment group, D is a graph showing serum glutamic-pyruvic transaminase levels of mice in the LNA-anti-miR-150 treatment group and the placebo treatment group, and E is a graph showing serum glutamic pyruvic transaminase levels of mice in the LNA-anti-miR-150 treatment group and the placebo treatment group.

FIG. 2 is a graph showing the therapeutic effect of LNA-anti-miR-150 in example 3 on kidney fibrosis in lupus nephritis mice, in which A is a graph showing the level of mouse serum anti-double-stranded DNA antibody, B is a graph showing the level of mouse urine albumin/urine creatinine, C is a graph showing the staining of mouse kidney tissue iodonic acid Schiff, and D is a graph showing the staining of mouse kidney tissue collagen fibers.

FIG. 3 is a graph showing the therapeutic effect of LNA-anti-miR-150 of example 4 on kidney fibrosis in a mouse model of Adriamycin-induced focal segmental glomerulosclerosis, wherein A is a graph of urine albumin/urine creatinine levels, B is a graph of serum albumin levels, C is a graph of total serum cholesterol levels, D is a graph of serum urea nitrogen levels, and E is a graph of mouse kidney tissue iodosnowfield and collagen fiber staining.

FIG. 4 is a graph showing the therapeutic effect of LNA-anti-miR-150 in example 5 on renal interstitial fibrosis in a mouse model of large-dose folate-induced renal interstitial fibrosis.

FIG. 5 is a graph of LNA-anti-miR1-50 inhibiting the expression of kidney miR-150 and increasing the expression of cytokine signal transduction inhibitory factor 1 in the mouse model of lupus nephritis in example 6, wherein A is a graph of miR-150 level in mouse kidney tissue, B is a graph of messenger RNA cytokine signal transduction inhibitory factor 1 expression level in mouse kidney tissue, C is a graph of detecting the expression level of cytokine signal transduction inhibitory factor 1 protein in mouse kidney tissue by immunoblotting, and D is a graph of detecting the expression level of cytokine signal transduction inhibitory factor 1 protein in mouse kidney tissue by immunofluorescence.

FIG. 6 is a graph of the expression of LNA-anti-miR1-50 in the doxorubicin-induced focal segment glomerulosclerosis mouse model to inhibit kidney miR-150 and increase cytokine signaling inhibitory factor 1 in example 7, wherein A is a graph of miR-150 level in mouse kidney tissue, B is a graph of messenger RNA cytokine signaling inhibitory factor 1 expression level in mouse kidney tissue, C is a graph of the expression level of cytokine signaling inhibitory factor 1 protein in mouse kidney tissue detected by immunoblotting, and D is a graph of the expression level of cytokine signaling inhibitory factor 1 protein in mouse kidney tissue detected by immunofluorescence.

FIG. 7 is a graph of LNA-anti-miR1-50 inhibiting the expression of kidney miR-150 and increasing the expression of cytokine signaling inhibitory factor 1 in a mouse model of large-dose folate-induced renal interstitial fibrosis in example 8, wherein A is a graph of miR-150 level in mouse kidney tissue, B is a graph of messenger RNA cytokine signaling inhibitory factor 1 expression level in mouse kidney tissue, C is a graph of the expression level of cytokine signaling inhibitory factor 1 protein in mouse kidney tissue detected by immunoWestern blotting, and D is a graph of the expression level of cytokine signaling inhibitory factor 1 protein in mouse kidney tissue detected by immunofluorescence.

Detailed Description

Example 1 materials and methods

(1) reagent consumable

1) Experimental animals:

Spontaneous lupus nephritis mice (purchased from Jackson Lab), C57BL/6 (donated to the Pistatic laboratory);

BABL/c mice (purchased from Beijing Wittingle science and technology);

CD1 mouse (purchased from Beijing Wittiaxle science and technology).

2) medicine preparation:

miR-150 inhibitor with placebo (Exiqon);

Folic acid (Sigma, usa);

Doxorubicin (japan and light).

3) Reagent:

(2) Mouse kidney disease model and Experimental groups

1) Spontaneous lupus nephritis

Wild type normal control group, C57BL/6 mouse + placebo group

C57BL/6 mouse + LNA-anti-miR-150

Lupus nephritis mice: lupus nephritis mouse + placebo group

Lupus nephritis mouse + LNA-anti-miR-150

end point of experiment: LNA-anti-miR-1502mg/kg was injected subcutaneously twice a week for 8 weeks.

2) Focal segmental glomerulosclerosis

normal control group: no treatment is carried out;

Group of adriamycin: the disposable adriamycin is injected into vein at 11 mg/kg;

Doxorubicin + placebo control group: doxorubicin + placebo;

Doxorubicin + LNA-anti-miR-150 treatment group: doxorubicin + LNA-anti-miR-150;

end point of experiment: LNA-anti-miR-1502mg/kg was injected subcutaneously twice a week for 6 weeks.

3) Folate-induced renal interstitial fibrosis

Normal control group: no treatment is carried out;

Folic acid group: carrying out intraperitoneal injection of 250mg/kg of folic acid for one time;

Folate + placebo control group: folic acid + placebo;

Folate + LNA-anti-miR-150 treatment group: folic acid + LNA-anti-miR-150;

End point of experiment: LNA-anti-miR-1502mg/kg was injected subcutaneously twice a week for 4 weeks.

(3) Retention and biochemical index detection of blood and urine specimen

and collecting random urine sample on the same day as the experimental end point, centrifuging at 3000 rpm for 5 minutes, taking the supernatant, and freezing and storing in a deep freezing refrigerator of-80 ℃ for later use. Anaesthetizing at the end of experiment, collecting arterial blood by abdominal aorta puncture, standing overnight at 4 ℃, centrifuging for 5 minutes at 5000 r/min, taking supernatant, and freezing and storing the supernatant in a deep freezing refrigerator at-80 ℃ for later use continuously for 2 times.

Double-stranded DNA immunoglobulin G enzyme-linked immunosorbent assay kit (Alpha Diagnostic International) was used. Double-stranded DNA was detected in serum. Serum urea nitrogen, creatinine, albumin, cholesterol, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and other biochemical indexes are detected by an autoanalyzer (Hitachi 917). Urine albumin and creatinine were detected with Albuwell M elisa kit and a matched creatinine assay kit (Exocell corporation) and urine albumin/urine creatinine was calculated.

(4) Retention of Kidney tissue and Paraffin section preparation

And (4) anaesthetizing the end point of the experiment, irrigating the kidney by using 4-degree PBS after arterial blood is collected by abdominal aorta puncture, and taking out the double kidneys after residual blood in the kidneys is completely removed. 1/2 the upper left kidney is fixed in 10% formalin to make paraffin block for iodine acid snow, collagen fiber and immunofluorescence staining.

Preparing a paraffin section: fixing: fixing the kidney tissue blocks with 4% paraformaldehyde for 48 hours; and (3) dehydrating: sequentially immersing the fixed kidney tissue blocks in 70% ethanol for 12 hours, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, absolute ethanol I for 30 minutes and absolute ethanol II for 20 minutes; and (3) transparency: placing the dehydrated kidney tissue blocks into dimethylbenzene for 8 minutes in sequence; wax dipping, embedding and slicing: immersing in paraffin I for 1 hour and paraffin II for 1 hour, embedding in paraffin, and slicing with a slicer to obtain slices with a thickness of 3 μm.

(5) iodine acid Schiff dyeing

xylene I5 minutes, xylene II 5 minutes, absolute ethyl alcohol 1 minute, 95% ethyl alcohol 1 minute, 85% ethyl alcohol 1 minute, distilled water 30 seconds, wipe dry the moisture around the tissue, circle the tissue outline with immunohistochemical pen, drip periodic acid on the tissue, incubate 10 minutes at room temperature and back to go, drip the snow agent on the tissue, incubate 10 minutes at 37 ℃ and back to go, sodium metaphosphate differentiates 30 seconds/times, 2 times, tap water washes 10 minutes, hematoxylin dyes 10 minutes, distilled water slightly washes, 75% hydrochloric acid alcohol differentiates 1 minute, tap water washes 3 minutes, 85% ethyl alcohol 1 minute, 95% ethyl alcohol 1 minute, absolute ethyl alcohol I1 minute, absolute ethyl alcohol II 1 minute, xylene I5 minutes, xylene II 5 minutes, neutral glue seal and dry; and taking pictures under a direct microscope.

(6) Collagen fiber dyeing

Xylene I minute, xylene II 5 minute, absolute ethyl alcohol 1 minute, 95% ethyl alcohol 1 minute, 85% ethyl alcohol 1 minute, distilled water washing 30 seconds, collagen fiber composite dye solution 20 minutes, 0.2% acetic acid washing 3 minutes, repeating for 3 times, 5% phosphotungstic acid 9 minutes, 0.2% acetic acid washing for 3 minutes, repeating for 3 times, toluidine blue 4 minutes. 1 minute of 85% ethanol, 1 minute of 95% ethanol, 1 minute of absolute ethanol I, 1 minute of absolute ethanol II, 5 minutes of xylene I and 5 minutes of xylene II, and sealing and airing neutral glue; and taking pictures under a direct microscope.

(7) immunofluorescence staining

Paraffin sections were placed in an oven at 56 ℃ for 2 hours to prevent tissue loss. The sections were removed from the oven and placed in 100% xylene and slowly shaken to remove paraffin, left for 10 minutes and repeated 2 times. The slices were taken out and put into absolute ethanol for 5 minutes, repeated for 2 times, and then xylene was removed. Then, the elution is carried out according to a sequential gradient, and the elution is sequentially put into 95 percent, 85 percent and 75 percent of ethanol for 5 minutes respectively, and finally the washing is carried out for 2 times by double distilled water for 5 minutes each time. Preparing an antigen repairing solution, and performing antigen repairing in the repairing solution prepared from 4.5ml of citric acid solution, 20.5ml of citric acid sodium salt solution and 225ml of double distilled water. Firstly heating the repairing liquid to boiling by a microwave oven, stopping immediately after boiling, then inserting the slices, adjusting the microwave oven to high fire, heating for 5 seconds, waiting for 20 seconds, repeating the steps for 20 minutes, and then cooling for 1 hour at room temperature. Removing peroxidase: incubate with 3% hydrogen peroxide in deionized water for 10 min to inactivate endogenous peroxidase, shake wash for 3 min in phosphate buffer, repeat 3 times. And (3) sealing: sections were non-specific antigen blocked with 1.5% sheep serum and left at 37 ℃ for 30 minutes to reduce non-specific background staining. The diluted primary antibody was added and refrigerated overnight at 4 ℃ in a wet box. Phosphate buffer was washed with shaking at room temperature for 3 minutes 3 times. And (3) keeping out of the sun, dropwise adding fluorescent secondary antibody (diluted by 1: 200), incubating at 37 ℃ for 60 minutes in the sun, and washing by a phosphate buffer at room temperature for 3 minutes each time in a shaking table. 4' 6-diamidino-2-phenylindole (1 mg/ml) was added dropwise to the mixture at room temperature for 10 minutes in the absence of light. Protected from light, washed with phosphate buffer at room temperature for 3 minutes each time. And (5) sealing the chip by using the anti-quenching sealing agent. And taking pictures under a direct microscope.

(8) Real-time quantitative fluorescent PCR

And (4) anaesthetizing the experimental end point, irrigating the kidney by using 4-degree PBS after arterial blood is collected by abdominal aorta puncture, and taking out the double kidneys after residual blood in the kidney is completely removed. 1/2 the upper right kidney was stored at-80 degrees for total RNA extraction from the kidneys.

and (3) RNA extraction: cutting kidney tissue, placing in a 1.5ml centrifuge tube without RNase, adding 1000 μ l Trizol and a frozen steel ball, placing in a tissue homogenizer, and homogenizing for 3 minutes with shaking at a frequency of 25 Hz; sucking the homogenate by using a pipette gun, transferring the homogenate into a new 1.5ml RNA-free enzyme centrifuge tube, adding 300 mu l chloroform, repeatedly oscillating for 15 seconds, and standing for 10 minutes at room temperature; placing the centrifuge tube after standing in the previous step into a low-temperature high-speed centrifuge, centrifuging for 15 minutes at 4 ℃ at 12000 rpm; sucking out the upper layer liquid without touching the middle layer white membrane by using a pipette gun, transferring the upper layer liquid into a new 1.5ml RNase-free centrifuge tube, adding isopropanol with the same volume, repeatedly reversing and uniformly mixing, and standing for 10 minutes at 4 ℃; placing the centrifuge tube after standing in the previous step into a low-temperature high-speed centrifuge, centrifuging for 15 minutes at 4 ℃ at 12000 rpm; pouring the upper layer liquid, so that white lumps at the bottom of the centrifuge tube are separated out, adding 800ml of 75% ethanol prepared by DEPC water, rotating at 12000 r/min, centrifuging for 5min, and repeating for 2 times; carefully sucking out the ethanol by a pipette gun, and air-drying the ethanol at room temperature for 1 hour; to each sample, DEPC water was added in an amount of 15. mu.l to dissolve RNA, and the RNA concentration was measured by Nanodrop, and the sample concentration was adjusted to 50 ng/. mu.l in a lump.

reverse transcription:

c, detecting miR-150: mu.l of RNase-free octal tube is added with 10 mu.l of RNA with the concentration of 50 ng/mu.l and a certain amount of reverse transcription kit reagent, the total reaction solvent is prepared into 20 mu.l, and the 20 mu.l of reverse transcription kit reagent is placed in PCR for reverse transcription at 37 ℃ for 1 hour and 85 ℃ for 5 seconds.

Reaction system:

② messenger RNA detection: adding 10 mul RNA with the concentration of 50 ng/mul and a certain amount of RT kit reagent into 200 mul RNA-free enzyme octa-plex tube, preparing 20 mul total reaction solvent, placing in PCR for reverse transcription, and temporarily storing at 37 ℃ for 15 minutes-85 ℃ for 15 seconds-4 ℃ to real-time quantitative fluorescent PCR.

Reaction system:

Real-time quantitative fluorescent PCR:

after reverse transcription into cDNA, real-time quantitative PCR reaction is carried out in a 384-well plate, and the reaction system is as follows:

Reaction conditions

The fluorescent dye SYBR Green PCR kit was used for real-time quantitative PCR analysis. Real-time fluorescence detection was detected using an ABI Q6 Sequence Detector.

(9) Western blot (Western-blot)

tissue protein extraction:

Cutting the residual frozen kidney tissue by using a sterile blade to obtain about 40mg, placing the cut residual frozen kidney tissue into a 2ml RNase-free centrifuge tube, adding 1000 mu l of protein lysate into the centrifuge tube according to the weight of 1g of the tissue, adding frozen steel balls, placing the centrifuge tube into a tissue homogenizer, and performing vibration homogenization at the frequency of 25 Hz for 3 minutes. The lysate is sucked out and transferred into a new 1.5ml centrifuge tube and placed on ice; the sonicator was set to 50 Hz for 10 seconds 3 times per sample. Transferring to a low-temperature high-speed centrifuge for centrifugation at 4 ℃ and 10000 r/min for 10 min. The supernatant was aspirated and placed in a new 1.5ml centrifuge tube, and the ice box was transferred to a deep freezer at-80 ℃ for storage.

Protein concentration determination by BCA method:

Preparing 2mg/ml BSA standard solution, adding the BSA standard solution into a 96-well plate according to the volume of 0,2,4,8,16 and 20 mul, repeating twice for each standard sample, adding 1 mul of protein samples to be detected into the 96-well plate respectively, and repeating twice for each sample. Preparing a BCA working solution according to the volume ratio of the BCA reagent A to the B which is 50:1, adding the BCA working solution into a standard substance and a sample to be detected, placing the sample at 37 ℃ for 20-30 minutes at 100ul per hole, measuring the absorbance value of the sample at 562nm in an enzyme labeling instrument, drawing a standard curve, and calculating the protein concentration of the sample.

Western blot：

after the protein concentration of the extracted protein is measured by a BCA method, the loading buffer is added according to the volume ratio after the protein concentration of 30-50 mug protein per hole is calculated, and the protein is denatured by heating at 95 ℃ for 5min after being fully mixed. The proteins were subjected to SDS-PAGE gel electrophoresis and Western blotting. Preparing a dry glass plate: washing with deionized water, spraying 75% ethanol, and drying at room temperature by ventilation; preparing a 15ml centrifuge tube to prepare 7.5% or 10% separation gel according to the formula of the solution, gently mixing the separation gel uniformly, and quickly injecting the separation gel into a gap between glass plates which are arranged in advance by using a 1ml gun head until the distance is about 2cm from the top. Slowly adding a proper amount of deionized water into the top layer by using a rubber head dropper to completely cover the liquid level so as to prevent the influence of air on gel polymerization, and standing at room temperature for about 30 minutes until the separation gel is polymerized; after 30 minutes, the separation gel is polymerized, the top water is poured out, 4% of concentrated gel is prepared according to the formula and is injected on the separation gel until the concentrated gel overflows, so that no gas exists between the separation gel and the separation gel. A comb was inserted into the concentrated gel, and left at room temperature for about 30 minutes, taking care to avoid air bubbles. After the gel was polymerized satisfactorily, the comb was removed and the gel was placed in the Bio-Med electrophoresis chamber. Adding TRIS GLYCIE buffer solution into the electrophoresis tank, and checking whether the buffer solution leaks from the inside and outside of the gel; sequentially adding a uniform mixing solution of sample protein and a loading buffer solution into a specific comb hole, and adding a protein pre-dyeing Marker; electrophoresis was carried out at room temperature, the voltage for gel concentration was selected to be 80V, and after about 30 minutes, the voltage was increased to 100V until the sample completely reached the gel for separation, and electrophoresis was continued until the macroscopic dye reached the bottom of the gel. After SDS-PAGE electrophoresis is finished, taking down the gel, cutting off the upper concentrated gel, and soaking the gel in a Transfer buffer; taking a 0.45-micrometer PVDF membrane with the size consistent with that of the separation gel, soaking and activating in methanol for 10s, then placing in a Transfer buffer, and taking out the PVDF membrane by using tweezers; film transfer: soaking the film Transfer clip, the sponge pad and the filter paper in a Transfer buffer for wetting, and placing the components in the following sequence: the sponge pad, the filter paper, the gel, the PVDF membrane, the filter paper and the sponge pad are packaged into a sandwich type, and air bubbles between the gel and the PVDF membrane are removed; clamping each layer by a film rotating clamp, putting the film rotating clamp into an electrophoresis tank, connecting one side of a PVDF film with an anode and the other side of a glue film with a cathode, and rotating the film at 100V for 1.5 hours; after the film is turned, taking out the PVDF film by using a pair of tweezers, and cutting a corner at the upper right corner of the film to make a mark; washing the membrane with clear water and TBST in sequence on a shaking bed at room temperature for 5 minutes respectively; placing the PVDF membrane in 3% BSA blocking solution, and incubating for 1 hour in a shaking table at room temperature; i, incubation of an antibody: recovering the confining liquid, washing the membrane for 3 times by TBST (TBST), adding a proper amount of I-type anti-dilution liquid into a plate for completely immersing the PVDF membrane, and incubating overnight in shaking table oscillation at 4 ℃; taking out the PVDF membrane, washing the PVDF membrane for 3 times by using TBST solution, wherein each time lasts for 10 minutes; II, anti-incubation: placing the PVDF membrane in a plate, adding a proper amount of II-resistant diluent, immersing the PVDF membrane, and oscillating by a shaking table at room temperature for 1 hour for incubation; taking out the PVDF membrane, washing the PVDF membrane for 3 times by TBST, and washing the PVDF membrane for 10 minutes each time; placing the PVDF film in ECL luminous liquid, placing the ECL luminous liquid in a chemical imaging system, and exposing; and analyzing and calculating the ratio of the grey value of the protein to the grey value of the internal reference protein by using Image J software, and taking the ratio as the relative expression quantity result of the target protein.

(10) Statistical analysis

And all data statistical analysis adopts Graphpad Prism 5 software, the expression form of the data is mean +/-standard deviation, two groups of data are compared by adopting t test, multiple groups of data are compared by adopting single-factor or multi-factor variance analysis, and the statistical difference is considered when p is less than 0.05.

example 2 evaluation of the biological safety of LNA-anti-miR-150 in a mouse model of spontaneous lupus nephritis

Lupus nephritis mice are divided into LN placebo treatment group and LN + miR-150 inhibitor treatment group (LNA-anti-miR-150), and C57BL/6 (wild type) mice are adopted as normal control group. LNA-anti-miR-1502mg/Kg is injected subcutaneously 2 times a week for 8 weeks. Before treatment, blood was collected 8 weeks after treatment and the body weight was weighed. The serum is used for detecting creatinine, urea nitrogen, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase. FIG. 1 is a biosafety assessment of LNA-anti-miR-150 in a lupus nephritis mouse model, wherein A is the body weight change before and after 8 weeks of LNA-anti-miR-150 treatment, and the body weight of the mouse is not changed before and after LNA-anti-miR-150 treatment. The body weight of mice in the LNA-anti-miR-150 treatment group and the placebo treatment group is not obviously changed. B is the serum creatinine level of the mice of the treatment group applying LNA-anti-miR-150 and the treatment group applying placebo, and the serum creatinine level of the mice of the treatment group applying LNA-anti-miR-150 and the treatment group applying placebo is not different. C is the serum urea nitrogen level of mice in the LNA-anti-miR-150 treatment group and the placebo treatment group, and the serum urea nitrogen level of the mice in the LNA-anti-miR-150 treatment group and the placebo treatment group is not different. D is the serum glutamic-oxaloacetic transaminase level of mice in the LNA-anti-miR-150 treatment group and the placebo treatment group, and the serum glutamic-oxaloacetic transaminase level of the mice in the LNA-anti-miR-150 treatment group and the placebo treatment group is not different. E is the serum glutamic pyruvic transaminase level of the mice in the LNA-anti-miR-150 treatment group and the placebo treatment group, and the serum glutamic pyruvic transaminase level of the mice in the LNA-anti-miR-150 treatment group and the placebo treatment group is not different. As can be seen from the figure, in lupus nephritis mice in LNA-anti-miR-150 injection, 2mg/kg, weekly 2 times, subcutaneous injection, total 8 weeks, mouse creatinine, urea nitrogen, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, weight did not change.

example 3 therapeutic Effect of LNA-anti-miR-150 on Kidney fibrosis in Lupus nephritis mice

Lupus nephritis mice are divided into a placebo treatment group and a miR-150 inhibitor treatment group (LNA-anti-miR1-50), and C57BL/6 (wild type) mice are used as a normal control group. The miR-150 inhibitor is injected subcutaneously at the concentration of 2mg/Kg 2 times per week for 8 weeks. Blood, urine and kidney tissues were collected 8 weeks after treatment. Titer of anti-dsDNA antibody, urine was used to determine the ratio of urine protein to creatinine. A portion of the kidney tissue was used for iodosnowflake staining and collagen fiber staining on paraffin sections.

FIG. 2 is a graph showing the therapeutic effect of LNA-anti-miR-150 on kidney fibrosis in lupus nephritis mice, wherein A is the serum anti-dsRNA antibody level of the mice in the LNA-anti-miR-150 treatment group and the placebo treatment group, and the serum anti-dsRNA antibody of the mice in the LNA-anti-miR-150 treatment group is significantly reduced compared with the placebo treatment group in the lupus nephritis mice. And B is the urinary albumin/urinary creatinine level of the mice using the LNA-anti-miR-150 treatment group and the placebo treatment group, and the urinary albumin/urinary creatinine of the mice is obviously reduced in the lupus nephritis mice using the LNA-anti-miR-150 treatment group and the placebo treatment group. C, performing iodoacid snow-flake staining on kidney tissues of mice applying the LNA-anti-miR-150 treatment group and the placebo treatment group, and obviously improving the kidney pathology of the mice in the lupus nephritis mice by applying the LNA-anti-miR-150 treatment group and the placebo treatment group. And D, staining the collagen fibers of the kidney tissues of the mice applying the LNA-anti-miR-150 treatment group and the placebo treatment group, and obviously improving the kidney pathology of the mice applying the LNA-anti-miR-150 treatment group and the placebo treatment group in lupus nephritis mice. As can be seen from the figure: LNA-anti-miR-150 reduces the serum double-stranded DNA level and the urinary protein level of the LN mouse, and pathologically and obviously relieves the renal tissue fibrosis.

Example 4 therapeutic Effect of LNA-anti-miR-150 on Kidney fibrosis in Adriamycin-induced focal segmental glomerulosclerosis mouse model

The BABL/c mice were divided into a normal control group, an adriamycin-injected group, an adriamycin + placebo-injected group, and an adriamycin + LNA-anti-miR 1-50-injected group. LNA-anti-miR1-50(2mg/Kg) was injected subcutaneously 2 times a week for 6 weeks. Blood, urine and kidney tissues were collected 6 weeks after treatment. The serum is used for detecting albumin, cholesterol and urea nitrogen, and the urine is used for detecting the ratio of urine albumin to creatinine. A portion of the kidney tissue was used for paraffin sectioning for iodoacid snowflake staining and collagen fiber staining.

Fig. 3 is a graph of the therapeutic effect of LNA-anti-miR-150 on kidney fibrosis in a doxorubicin-induced focal segmental glomerulosclerosis mouse model, where a is the increase in urinary albumin/urinary creatinine levels in mice after 6 weeks of intravenous doxorubicin administration for a time period that is less than the decrease in urinary albumin/urinary creatinine levels in focal segmental glomerulosclerosis mice treated with LNA-anti-miR-150 in the placebo-treated group. And B, after 6 weeks of intravenous doxorubicin injection, the serum albumin level of the mice is reduced, and the serum albumin level of the focal segment glomerulosclerosis mice treated by the LNA-anti-miR-150 is higher than that of the mice treated by the placebo. C is that the serum total cholesterol level of the mice is increased after 6 weeks of intravenous adriamycin once, and the serum total cholesterol level of the focal segment glomerulosclerosis mice is reduced after the mice are treated by LNA-anti-miR-150 compared with the mice treated by the placebo. D is that after 6 weeks of one-time intravenous adriamycin injection, the serum urea nitrogen level of the mice is increased, and the serum urea nitrogen level of the focal segment glomerulosclerosis mice is reduced by using LNA-anti-miR-150 treatment compared with that of the mice treated by using placebo. And E is the staining of mouse kidney tissue iodoacid snow and collagen fibers, and the kidney pathology of the focal segmental glomerulosclerosis mouse is obviously improved after the treatment by using LNA-anti-miR-150. As can be seen from the figure: LNA-anti-miR-150 can reduce albuminuria of doxorubicin-induced focal segmental glomerulosclerosis mice, increase serum albumin levels and reduce cholesterol and urea nitrogen levels. Pathologically, markedly relieved glomerulosclerosis.

Example 5 therapeutic Effect of LNA-anti-miR-150 on Kidney fibrosis in a mouse model of Large dose folate-induced Kidney interstitial fibrosis

The CD1 mice were divided into normal control group, folic acid injection group, folic acid + placebo injection group, folic acid + LNA-anti-miR1-50 injection group, LNA-anti-miR1-50(2mg/Kg) subcutaneous injection, 2 times per week, total injection for 4 weeks. Renal tissue was harvested 4 weeks after treatment. Part of kidney tissue was used for iodine acid snowflake staining and collagen fiber staining of paraffin sections.

FIG. 4 is a graph showing the treatment effect of LNA-anti-miR-150 on renal fibrosis in a mouse model of renal interstitial fibrosis induced by a large dose of folic acid, the mouse renal tissue is stained with iodoxysnow and collagen fibers, and the renal pathology of the mouse with renal interstitial fibrosis treated by using LNA-anti-miR-150 is obviously improved. As can be seen from the figure: LNA-anti-miR-150 can slow the fibrosis of the renal tubular interstitium.

Example 6 LNA-anti-miR1-50 in Lupus nephritis mouse model inhibits the expression of kidney miR-150 and increases the expression of cytokine signal transduction inhibitory factor 1

Lupus nephritis mice are divided into LN placebo treatment group and LN + miR-150 inhibitor treatment group (LNA-anti-miR-150), and C57BL/6 (wild type) mice are adopted as normal control group. LNA-anti-miR-1502mg/Kg is injected subcutaneously 2 times a week for 8 weeks. Renal tissue was harvested 8 weeks after treatment. Extracting RNA in the kidney tissue, detecting miR-150 in the kidney tissue and increasing the expression of the cytokine signal transduction inhibitory factor 1 by real-time quantitative fluorescence PCR, extracting protein, carrying out Western Blot detection on the cytokine signal transduction inhibitory factor 1 protein, and simultaneously carrying out immunofluorescence staining on the kidney tissue for increasing the cytokine signal transduction inhibitory factor 1.

FIG. 5 is an expression diagram of LNA-anti-miR1-50 inhibiting kidney miR-150 expression and increasing cytokine signal transduction inhibitory factor 1 in a lupus nephritis mouse model in a mouse model, wherein A is miR-150 level in mouse kidney tissues, miR-150 expression in lupus nephritis mouse kidney tissues is increased, and miR-150 in mouse kidney tissues is obviously reduced by applying an LNA-anti-miR-150 treatment group compared with a placebo treatment group. B is the expression level of the messenger RNA cell factor signal transduction inhibitor 1 in the kidney tissue of the mouse, the expression of the messenger RNA cell factor signal transduction inhibitor 1 in the kidney tissue of the lupus nephritis mouse is reduced, and the level of the messenger RNA cell factor signal transduction inhibitor 1 in the kidney tissue of the mouse is increased by applying the LNA-anti-miR-150 treatment group compared with applying the placebo treatment group. C, detecting the expression level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse by an immunoblotting method, reducing the expression of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the lupus nephritis mouse, and increasing the level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse by applying the LNA-anti-miR-150 treatment group compared with the placebo treatment group. D is the expression level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse detected by an immunofluorescence method, the expression of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the lupus nephritis mouse is reduced, and the level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse is increased by applying the LNA-anti-miR-150 treatment group compared with applying the placebo treatment group. As can be seen from the figure: LNA-anti-miR1-50 inhibits the expression of kidney miR-150 in a lupus nephritis mouse model, and increases the expression of a cytokine signal transduction inhibitory factor 1.

Example 7 LNA-anti-miR1-50 inhibits the expression of kidney miR-150 and increases the expression of cytokine signaling inhibitory factor 1 in Adriamycin-induced focal segmental glomerulosclerosis mouse model

The BABL/c mice were divided into a normal control group, an adriamycin-injected group, an adriamycin + placebo group, and an adriamycin + LNA-anti-miR-150 group. LNA-anti-miR-1502mg/Kg is injected subcutaneously 2 times a week for 6 weeks. Kidney tissue was harvested 6 weeks after treatment. Extracting RNA in the kidney tissue, detecting miR-150 in the kidney tissue and increasing the expression of the cytokine signal transduction inhibitory factor 1 by real-time quantitative fluorescence PCR, extracting protein, carrying out Western Blot detection on the cytokine signal transduction inhibitory factor 1 protein, and simultaneously carrying out immunofluorescence staining on the kidney tissue for increasing the cytokine signal transduction inhibitory factor 1.

FIG. 6 is an expression diagram of LNA-anti-miR1-50 inhibiting kidney miR-150 expression and increasing cytokine signal transduction inhibitory factor 1 in an doxorubicin-induced focal segmental glomerulosclerosis mouse model, wherein A is miR-150 level in mouse kidney tissue, miR-150 expression in focal segmental glomerulosclerosis mouse kidney tissue is increased, and miR-150 in mouse kidney tissue is significantly reduced by applying an LNA-anti-miR-150 treatment group compared with a placebo treatment group. B is the expression level of the messenger RNA cell factor signal transduction inhibitor 1 in the kidney tissue of the mouse, the expression of the messenger RNA cell factor signal transduction inhibitor 1 in the kidney tissue of the mouse with focal segmental glomerulosclerosis is reduced, and the level of the messenger RNA cell factor signal transduction inhibitor 1 in the kidney tissue of the mouse is increased by using the LNA-anti-miR-150 treatment group compared with the placebo treatment group. C, detecting the expression level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse by an immunoblotting method, reducing the expression of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the focal-segment-type glomerulosclerosis mouse, and increasing the level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse by applying the LNA-anti-miR-150 treatment group compared with applying the placebo treatment group. D is the expression level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse detected by an immunofluorescence method, the expression of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the focal segmental glomerulosclerosis mouse is reduced, and the level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse is increased by applying the LNA-anti-miR-150 treatment group compared with the placebo treatment group. As can be seen from the figure: LNA-anti-miR1-50 inhibits the expression of kidney miR-150 in a focal segment glomerulosclerosis mouse model, and increases the expression of a cytokine signal transduction inhibitor 1.

Example 8 LNA-anti-miR1-50 inhibits the expression of kidney miR-150 and increases the expression of cytokine signaling inhibitory factor 1 in a mouse model of renal interstitial fibrosis induced by large dose of folic acid

the CD1 mice were divided into normal control group, folic acid injection group, folic acid + placebo injection group, folic acid + LNA-anti-miR1-50 injection group, LNA-anti-miR1-50(2mg/Kg) subcutaneous injection, 2 times per week, total injection for 4 weeks. Renal tissue was harvested 4 weeks after treatment. Extracting RNA in the kidney tissue, detecting miR-150 in the kidney tissue and increasing the expression of the cytokine signal transduction inhibitory factor 1 by real-time quantitative fluorescence PCR, extracting protein, carrying out WesternBlot detection on the cytokine signal transduction inhibitory factor 1 protein, and simultaneously carrying out immunofluorescence staining on the kidney tissue for increasing the cytokine signal transduction inhibitory factor 1.

FIG. 7 is a graph showing that LNA-anti-miR1-50 inhibits the expression of kidney miR-150 and increases the expression of cytokine signal transduction inhibitory factor 1 in a mouse model of renal interstitial fibrosis induced by large dose of folic acid, wherein A is the miR-150 level in the kidney tissue of a mouse, the expression of miR-150 in the kidney tissue of the renal interstitial fibrosis mouse is increased, and miR-150 in the kidney tissue of the mouse is significantly reduced by using the LNA-anti-miR-150 treatment group compared with the placebo treatment group. B is the expression level of the messenger RNA cytokine signal transduction inhibitor 1 in the kidney tissue of the mouse, the expression of the messenger RNA cytokine signal transduction inhibitor 1 in the kidney tissue of the mouse with the renal interstitial fibrosis is reduced, and the level of the messenger RNA cytokine signal transduction inhibitor 1 in the kidney tissue of the mouse is increased by using the LNA-anti-miR-150 treatment group compared with the placebo treatment group. C, detecting the expression level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse by an immunoblotting method, reducing the expression of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse with renal interstitial fibrosis, and increasing the level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse by applying the LNA-anti-miR-150 treatment group compared with the placebo treatment group. D is that the expression level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse is detected by an immunofluorescence method, the expression of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse with renal interstitial fibrosis is reduced, and the level of the cytokine signal transduction inhibitory factor 1 protein in the kidney tissue of the mouse is increased by applying the LNA-anti-miR-150 treatment group compared with the placebo treatment group. It can be seen from the figure that: LNA-anti-miR1-50 inhibits the expression of kidney miR-150 in a folate-induced renal interstitial fibrosis mouse model, and increases the expression of cytokine signal transduction inhibitory factor 1.

Sequence listing

<110> Shengjing Hospital affiliated to Chinese medical university

Application of <120> LNA-anti-miR-150 in preparation of medicine for preventing or treating renal fibrosis

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> RNA

<213> house mouse(Mus musculus) 、human（Homo sapiens）

<400> 1

ucucccaacc cuuguaccag ug 22

<210> 2

<211> 15

<212> RNA

<213> Artificial sequence (Artifical)

<400> 2

tacaagggtt gggag 15

Claims (4)

1. Use of a miR-150 inhibitor LNA-anti-miR-150 in preparation of a medicament for preventing or treating renal fibrosis;

   The sequence of the LNA-anti-miR-150 is SEQ ID NO. 2: TACAAGGGTTGGGAG are provided.
2. 2. The use according to claim 1, wherein the mature sequence of miR-150 is SEQ ID No. 1: UCUCCCAACCCUUGUACCAGUG are provided.
3. 3. The use according to claims 1-2, characterized in that the miR-150 inhibitor LNA-anti-miR-150 employs the LNA ^TM structure to inhibit miR-150 with stable results.
4. 4. the use according to claim 3, wherein LNA ^TM is a novel nucleic acid analogue comprising a 2 '-oxo-4' carbon methylene linkage that limits the flexibility of the ribofuranose ring, locks it into a rigid bicyclic mode, increases hybridization efficiency and has superior stability.