CN114533876A

CN114533876A - Application of SRP14 gene in treating renal insufficiency or renal injury

Info

Publication number: CN114533876A
Application number: CN202210136704.6A
Authority: CN
Inventors: 李怡
Original assignee: Sichuan Provincial Peoples Hospital
Current assignee: Sichuan Provincial Peoples Hospital
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2022-05-27
Anticipated expiration: 2040-03-30
Also published as: CN111388674B; CN111388674A; CN114533876B

Abstract

The invention discloses an application of SRP14 gene in treating renal insufficiency or renal injury, relating to the technical field of gene therapy. The invention discloses application of an inhibitor for inhibiting gene expression in preparing a medicament for treating renal insufficiency or renal injury diseases, and researches of the invention find that the inhibition of the expression of SRP14 gene can treat the renal insufficiency or the renal injury diseases for the first time.

Description

Application of SRP14 gene in treating renal insufficiency or renal injury

Divisional application

The divisional application is based on Chinese patent application with application number of 2020102376062, application date of 3-30.2020, and invention name of application of RPS7 and SRP14 gene in treating renal insufficiency or renal injury.

Technical Field

The invention relates to the technical field of gene therapy, in particular to application of SRP14 gene in treating renal insufficiency or renal injury.

Background

Renal insufficiency or injury (AKI) is highly likely to progress to Chronic Kidney Diseases (CKD) and End Stage Renal Diseases (ESRD), and renal ischemia reperfusion injury (kidney IRI) is an important cause of AKI and one of the major factors affecting early functional recovery and long-term survival of transplanted kidneys after renal transplantation.

Acute Kidney Injury (AKI) is very likely to progress to Chronic Kidney Diseases (CKD), leading to chronic renal insufficiency and end-stage renal disease (ESRD), and in recent years, the incidence and death rate of the disease are increasing, causing huge damage to families and society. Despite the high morbidity and mortality of AKI patients, AKI is not a single disease, the pathogenesis of which is complex and the specific pathogenesis of which is unknown, and the lack of early diagnosis biomarkers and effective intervention targets, which causes difficulties in the prevention and treatment of the disease. Therefore, the research on the pathogenesis of AKI and the search of possible biomarkers and intervention targets of AKI are of great clinical significance. At present, the research on the pathogenesis of AKI focuses on the effect stage after injury, but the research on the initiation mechanism is less, so that the research on the initiation mechanism of the pathogenesis of AKI is deeply discussed, and the search for new early biomarkers and therapeutic intervention targets is a problem to be solved urgently for the research on AKI, and is a precondition and key for delaying the generation and development of CKD by early treatment of AKI.

AKI is complex in pathogenesis and is characterized primarily by tubular epithelial cell injury and death. Tubular epithelial cells are highly susceptible to apoptosis, and damage to this site can lead to kidney failure. Oxidative stress, nephrotoxic-induced injury of tubular epithelial cells also predominates in the early stages of AKI, mainly by apoptosis. Therefore, the pathophysiological mechanism of renal tubular epithelial cell apoptosis has been the focus of AKI research. Apoptosis of tubular epithelial cells is closely associated with tubular epithelial cell injury, loss of renal function in mice and renal tissue damage in the early stages of AKI development. Factors secreted into the circulation by injured kidneys in AKI further induce apoptosis and inflammation in heart, lung, liver and brain, further leading to high morbidity and mortality of AKI, so that inhibition of tubular epithelial apoptosis is a key link for preventing and treating AKI. Despite the significant advances in the study of tubular epithelial apoptosis in AKI, and the combination therapy directed to multiple cell death pathways that can provide the greatest benefit in treating AKI, no truly effective specific method for preventing tubular epithelial apoptosis has been found. Therefore, continuously exploring the new mechanism of the apoptosis pathway in the early stage of AKI is a big problem facing researchers of kidney diseases all over the world at present, and is one of the technical bottlenecks of the apoptosis pathway related to the clinical use of novel compounds.

Disclosure of Invention

The invention aims to provide application of SRP14 gene in treating renal insufficiency or renal injury.

The invention is realized in the following way:

in a first aspect, the embodiments of the present invention provide the use of an inhibitor for inhibiting the expression of a gene selected from the group consisting of SRP14 gene in the manufacture of a medicament for the treatment of renal insufficiency or renal injury.

The research of the invention firstly discovers that the inhibition of the expression of the SRP14 gene can improve the survival rate of the kidney ischemia reperfusion injury model cell, which indicates that the inhibition of the expression of the SRP14 gene can treat renal insufficiency or kidney injury diseases, and correspondingly, the inhibitor for inhibiting the expression of the SRP14 gene can be prepared into the medicine for treating the renal insufficiency or the kidney injury diseases.

In alternative embodiments, the inhibitor inhibits expression of the gene at the DNA level, RNA level, or protein level.

Based on the present disclosure, one skilled in the art can easily think of inhibiting the expression of SRP14 gene in various ways, including but not limited to inhibiting the expression at RNA level and protein level, and can also inhibit the expression of gene by other similar technical means, such as directly changing the coding sequence of SRP14 gene or its regulatory sequence by gene editing technology, so that the gene cannot be transcribed into RNA, i.e., inhibiting the expression of gene at DNA molecule level. Based on this, it is easy for those skilled in the art to achieve that the expression of SRP14 gene is inhibited in any way, and it is within the scope of the present invention.

In alternative embodiments, the inhibitor inhibits expression of the gene at the RNA level.

In alternative embodiments, the inhibitor is a siRNA, shRNA or microRNA.

The types of inhibitors that inhibit gene expression at the RNA level include, but are not limited to, siRNA, shRNA, and microRNA, and those skilled in the art will readily recognize that other similar inhibitors can be used to inhibit expression of SRP14 gene at the RNA level, and therefore it is within the scope of the present invention to employ other similar inhibitors to inhibit expression of SRP14 gene at the RNA level.

In alternative embodiments, the siRNA, shRNA or microRNA may be commercially available. For example, sirnas directed against SRP14 gene were purchased from Thermo fisher, such as: SRP14siRNA (# AM16708, Assay ID #12804, RefSeq: NM-001309434.1, target: site 356 of exon 5, Thermo fisher, USA); the targeted exon sequences are as follows: gtga gctccaagga agtgaataag tttcagatg, respectively;

although the above-mentioned siRNA has a good effect of inhibiting the expression of SRP14 gene, it should be noted that the skilled in the art can easily think that other sequences of siRNA are used to inhibit the expression of SRP14 gene, and it is within the scope of the present invention to use any sequence of siRNA to inhibit the expression of SRP14 gene.

In an alternative embodiment, the renal insufficiency or renal injury disease is caused or caused by a renal ischemia reperfusion injury.

In a second aspect, the embodiments of the present invention provide a medicament for treating renal insufficiency or a renal injury disease, which comprises an inhibitor for inhibiting the expression of a gene selected from the group consisting of SRP14 genes.

The invention provides a medicine for treating renal insufficiency or renal injury diseases, which improves the survival rate of renal damaged cells by inhibiting the expression of SRP14 genes, and further has the effect of treating the renal insufficiency or the renal injury diseases.

In alternative embodiments, the inhibitor is an iRNA, shRNA or microRNA.

In alternative embodiments, the renal insufficiency or renal injury diseases include, but are not limited to, acute renal injury (AKI), chronic renal disease (CKD), and end-stage renal disease (ESRD), and other related diseases caused or resulting from renal ischemia-reperfusion injury are also within the scope of the present invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing that reoxygenation 24 hours (I/R) after 4 hours of hypoxia increased the protein expression levels of RPS7 and SRP14 in tubular epithelial cells, while RPS7 siRNA and SRP14siRNA significantly decreased the protein expression levels of RPS7 and SRP14 in tubular epithelial cells after knocking down the protein expression of RPS7 and SRP14, respectively;

FIG. 2 shows that in the normal negative control siRNA (negative siRNA) group, the survival rate of cells after hypoxia reoxygenation is obviously reduced, the survival rate of HK2 human tubular epithelial cells (IRI in the figure) after hypoxia reoxygenation can be obviously increased after SRP14siRNA or RPS7 siRNA knockdown gene expression, Ctrl represents normal oxygen condition; IRI means hypoxia for 4h and reoxygenation for 24 h;

FIG. 3 shows the result of PI/Annexin V staining detected by flow cytometry, wherein the cell injury rate in the Normal control (Normal control) group is 13.23%, the cell injury rate in the SRP14 knock-down (SKD) group is 11.94%, and the cell injury rate in the RPS7 knock-down (RKD) group is 24.38%; the cell damage rate after 24 hours of reoxygenation (IRI) after 4 hours of hypoxia is significantly increased to 43.54 percent, while the cell damage rate of IRI group after SRP14 knockdown (SKD) is reduced to 35.97 percent, the cell damage rate of IRI group after RPS7 knockdown (SKD) is reduced to 33.06 percent, and Ctrl represents normal oxygen condition; IRI means hypoxia for 4h and reoxygenation for 24 h.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The expression of SRP14 or RPS7 in HK2 human tubular epithelial cells was knocked down by SRP14siRNA and RPS7 siRNA, respectively.

1 transfection of SRP14siRNA

SRP14siRNA gene silencing was diluted to a concentration of 5nmol/ml according to the instructions (available from Thermo fisher, Inc. # AM16708, Assay ID #12804, RefSeq: NM-001309434.1).

1) 40pmol "SRP 14 siRNA" was dissolved in 100. mu.l Opti-DMEM-F12 serum free and double antibody free medium in 1.5ml EP tube and gently mixed.

2) Mu.l Lipo2000 was dissolved in 1.5ml EP tube containing 100. mu.l Opti-DMEM-F12 serum-free and double antibody-free medium, mixed well and left for 5 minutes at room temperature.

3) Adding the solution in the step 1) into the solution in the step 2), uniformly mixing and standing for 15 minutes.

4) As fast as 13 minutes, the medium in the 6-well plate was removed, the cells were washed once with Opti-DMEM-F12 serum-free and double antibody-free medium, 200. mu.l of the above-mentioned mixed mixture was added to the corresponding well of the 6-well plate, and 1800. mu.l each of Opti-DMEM-F12 double antibody-free medium was added to each well.

5) After 5 hours the liquid was changed to a double-antibody free complete medium.

2 transfection of RPS7 siRNA

RPS7 siRNA gene silencing was diluted to a concentration of 5nmol/ml according to the instructions (available from Thermo fisher, Inc. # AM16708, Assay ID #142206, RefSeq: NM-001011.3).

1) 75pmol of "RPS 7 siRNA" was dissolved in 100. mu.l Opti-DMEM-F12 serum-free and double antibody-free medium in 1.5ml of EP tube and gently mixed.

2. HK2 human tubular epithelial cell hypoxia/reoxygenation injury (IRI) model (I4h/R24h) was established in vitro using the MIC-101 hypoxia modular incubator system of Biluosnerg (Bilupus) Inc., USA.

3. Preparation of protein samples

Precooling the mortar by using liquid nitrogen, taking out the frozen tissue of a refrigerator with the temperature of minus 80 ℃ to the mortar, adding the liquid nitrogen while grinding, and keeping the temperature low. The milled tissue powder was placed in a 1.5ml EP tube. To 1mg of tissue, 40. mu.l of protein lysate (RIPA, Byunnan biosystems, P0013) and phosphatase inhibitor PMSF (Byunnan biosystems, P1048) were added (RIPA: PMSF: 100:1, ready for use).

After removing the medium from the 6-well plate in the incubator, the plate was washed 3 times with PBS. Mu.l of RIPA lysate (RIPA: PMSF 100:1, ready for use) was added to each well. The cells were then scraped off with a cell scraper and collected in a 1.5ml EP tube.

② the cell ultrasonic crusher carries out ultrasonic treatment for 10-15 times on ice, and is placed on ice for cracking for 30 min.

③ 4 ℃, 13000rmp for 10 min.

Fourthly, taking the supernatant into a new 1.5ml EP tube, and measuring the protein concentration by using a BCA method (BCA kit, Biyunyan, P0009):

diluting a protein standard solution: diluting 10 μ l protein standard with 90 μ l PBS (phosphate buffer) to obtain 0.5mg/ml protein standard;

0.5mg/ml protein standard was added to a 96-well plate in an amount of 0, 1, 2, 4, 8, 12, 16, 20. mu.l. Make up volume to 20 μ l with PBS per well and mix well; adding 19 mul PBS into each sample hole, adding 1 mul protein sample and mixing evenly;

add 200. mu.l BCA working solution to all wells;

detecting the absorbance of the standard substance and the sample at the wavelength of 595nm by using an enzyme-labeling instrument; drawing a standard curve according to the absorbance of the standard substance, and further calculating the protein concentration of the sample;

based on the protein concentration measured by BCA, the concentration of each group of samples was made the same by adding the corresponding volume of RIPA.

Fifthly, according to the protein liquid: loading buffer is 4: 1 Add 5x loading buffer (Bilun. day, P0015).

Sixthly, the protein is denatured by metal bath at 95 ℃ for 5 min. Storing at 80 deg.C

5Western blot

Preparation of PAGE glue:

the separation gel and the stacking gel were prepared according to SDS-PAGE preparation 10% and 12.5% kit instructions (SDS-PAGE kit, Biyun day, P0052A). The product content comprises 250ml of separation gel buffer solution (2X), 250ml of separation gel solution (2X), 80ml of color concentrated gel buffer solution (2X), 80ml of concentrated gel solution (2X) and 8ml of improved Ammonium Persulfate Solution (APS), wherein prepared 1.5mm gel is adopted in the experiment, and a gel-making glass plate is cleaned and naturally air-dried before gel making.

When preparing the separating gel (lower layer gel), 4.0ml of separating gel buffer solution and separating gel solution are taken firstly, 80 mul of APS is added, and the separating gel buffer solution and the separating gel solution are fully and uniformly mixed; injecting the solution into a mounted 1.5mm glue-making glass plate, and then adding absolute ethyl alcohol to cover the separation glue; and after the separation gel is solidified, removing the upper layer of absolute ethyl alcohol, and sucking the redundant absolute ethyl alcohol by using a single-channel liquid transfer device.

When preparing concentrated gel (upper layer gel), 1.0ml of each of the concentrated gel buffer solution and the concentrated gel solution is taken, and then 20 mul of APS is added to be fully and uniformly mixed. The solution is injected into a glue-making glass plate, and 1.5mm comb teeth with 10 holes or 15 holes are inserted, and bubbles are prevented from being generated when the comb teeth are inserted. After the concentrated gel is solidified, the gel can be immediately subjected to sample electrophoresis or stored in a refrigerator at 4 ℃.

(ii) electrophoresis: the prepared gel is taken out and installed on an electrophoresis frame, and the anode and the cathode are noticed. Placing the electrophoresis frame in an electrophoresis tank, and pouring electrophoresis liquid into the electrophoresis tank, wherein the height of the electrophoresis liquid is higher than that of the aluminum wire. The comb is pulled out vertically upwards, pre-run for 10min at 100V, check whether leakage exists, and then marker and protein sample are added. The gel was concentrated at 80V, the gel was separated at 120V, and bromophenol blue stopped at 1cm above the bottom of the glass plate.

③ transferring the film: and soaking the clamp, the filter paper and the sponge for membrane transfer into the transfer liquid half an hour before membrane transfer. And (3) taking out the glass plate after electrophoresis, slightly poking the glass plate, cutting off the concentrated gel and the bromophenol blue, and cutting the separation gel with the corresponding size according to the sample loading number. The remaining gum was gently poured onto the filter paper. Cutting a PVDF membrane slightly larger than the glue, slightly covering the PVDF membrane on the glue after activating the PVDF membrane by methanol, discharging bubbles between the filter paper and the glue, assembling the PVDF membrane in a membrane rotating clamp according to the sequence of a negative electrode, a sponge, 3 layers of filter paper, the glue, the membrane, 3 layers of filter paper, the sponge and a positive electrode, installing the clamp in a membrane rotating groove, filling pre-cooled 1 Xelectrotransfer liquid in the membrane rotating groove, and paying attention to the positive electrode and the negative electrode. The voltage of 100V is converted into the film. The target protein is less than 100KD and is transferred for 60min, and the target protein is more than 100KD, and the film transfer time is determined according to the molecular weight of 1KD/1 min.

Sealing: after the film transfer, the PVDF film was taken out. After marking the positive and negative and date, putting the mixture into 5% of experimental skimmed milk and sealing the mixture for 2 hours at room temperature.

Primary anti-incubation: after blocking was completed, the membrane was removed from skim milk, washed 3 times with 1 × TBST for 5 minutes, and then antibody was formulated using 1 × TBST at dilution ratio β -actin (1:1000, ZENBIO, #340042), RPS7(1:1000, Abcam, # ab230862), and SRP14(1:1000, Abcam, # ab66896), and the antibody incubation chamber was incubated overnight on a shaker in a refrigerator at 4 ℃.

Sixthly, incubation with a secondary antibody: the following day, membranes were removed from the antibody incubation cassette and washed 3 times with 1 × TBST for 5min each. HRP-labeled secondary antibodies (ZENBIO, anti-rabbit 1: 5000, # 511203; anti-mouse 1: 10000, #701051) were prepared at a dilution ratio with 1 XTBST and incubated at room temperature for 2 h.

Color development exposure: after the secondary antibody incubation, the membranes were removed from the antibody incubation cassette and washed 3 times with 1 × TBST for 5min each. An Electrochemiluminescence (ECL) reagent (Millipore) was prepared at a ratio of 1:1, and an exposure solution was dropped on the surface of the film, followed by automatic exposure imaging using an Image Quant LAS 4000mini system.

The results are shown in FIG. 1: reoxygenation 24 hours (I/R) after 4 hours of hypoxia increased the protein expression levels of RPS7 and SRP14 in tubular epithelial cells, while RPS7 siRNA and SRP14siRNA significantly reduced the protein expression levels of RPS7 and SRP14 in tubular epithelial cells after knocking down the protein expression of RPS7 and SRP14, respectively.

Example 2:

the cell treatment conditions for the preliminary modeling were the same as in example 1. Cell viability assay Using the Kit of Cell Counting Kit-8(CCK8, cat # CK04) from east China, CCK8

The formazan can be reduced into water-soluble orange yellow formazan by dehydrogenase in cells, the amount of generated formazan is in direct proportion to the number of cells, and the number of living cells can be indirectly measured.

The results are shown in FIG. 2: in the Normal negative control siRNA group (Silencer)^TMIn Negative Control No.1siRNA (# AM 4611), which has no significant similarity with mouse, rat or human gene sequence, i.e. has no interference effect, Thermo fisher, USA), the survival rate of cells after hypoxia reoxygenation is obviously reduced (ctrl in the figure), and SRP14siRNA or RPS7 siRNA can be used for knocking down gene expressionThe survival rate of HK2 human renal tubular epithelial cells (IRI in the figure) after hypoxia reoxygenation is increased.

Example 3

The cell treatment conditions for the preliminary modeling were the same as in example 1.

Detection of PI/Annexin V staining Using flow cytometry

1) Cell collection: directly collecting adherent HK2 cells into a 5ml centrifuge tube after trypsinization, wherein the number of the cells in each sample is (1-5) multiplied by 10⁶Centrifuging at 2500r/min for 5min, and discarding the culture solution.

2) Washed 2 times with pre-cooled PBS and centrifuged at 2500r/min for 5 min.

3) Resuspending the cells with 100ul Binding Buffer, setting blank control, PI single staining, annexin V single staining, double staining of experimental group, and incubating for 10-15 min at room temperature in dark place.

4) Flow cytometry analysis results

5) And (5) judging a result: apoptotic cells are resistant to all dyes used for cell viability identification, such as PI, whereas necrotic cells are not. The DNA of the cells with damaged cell membranes can be stained by PI to generate red fluorescence, and the cells with intact cell membranes can not generate the red fluorescence. Thus, PI does not stain without a red fluorescent signal at the early stage of apoptosis. Normal living cells are similar. Displaying living cells in a lower left quadrant of a scatter diagram of the bivariate flow cytometer, wherein the living cells are FITC-/PI-; the upper right quadrant is a non-viable cell, i.e., a necrotic cell, FITC +/PI +; while the lower right quadrant is apoptotic cells, FITC +/PI-. Cell injury Rate results were the sum of the upper right quadrant and the lower right quadrant (SKD: SRP14siRNA transfection; RKD: RPS7 siRNA transfection)

The results in FIG. 3 show that the cell damage rate in the Normal control (Normal control) group was 13.23%, the cell damage rate in the SRP14 knock-down (SKD) only group was 11.94%, and the cell damage rate in the RPS7 knock-down (RKD) only group was 24.38%; the cell damage rate after 24 hours of reoxygenation (IRI) after 4 hours of hypoxia is remarkably increased to 43.54 percent, while the cell damage rate of IRI group after SRP14 knockdown (SKD) is reduced to 35.97 percent, and the cell damage rate of IRI group after RPS7 knockdown (RKD) is reduced to 33.06 percent. The results fully indicate that the RPS7 and/or SRP14 gene is used as a target to inhibit the expression of the gene, and the gene can be used for treating renal insufficiency or renal injury diseases caused by or caused by renal ischemia-reperfusion injury, such as acute renal injury (AKI), chronic renal disease (CKD) or end-stage renal disease (ESRD) and the like. The invention provides a new idea for treating renal insufficiency or renal injury diseases caused or caused by renal ischemia-reperfusion injury.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of an inhibitor for inhibiting gene expression in the manufacture of a medicament for the treatment of renal insufficiency or renal injury, wherein the gene is selected from the group consisting of SRP14 gene; the inhibitor inhibits the expression of the gene at the DNA level, RNA level, or protein level;

when the inhibitor inhibits the expression of the gene on the RNA level, the inhibitor is siRNA, shRNA or microRNA; when the inhibitor is siRNA, the siRNA inhibiting SRP14 gene is AM16708 siRNA, the reference sequence: NM-001309434.1; the renal insufficiency or renal injury disease is caused or caused by renal ischemia reperfusion injury.

2. A medicament for treating renal insufficiency or renal injury, which comprises an inhibitor for inhibiting the expression of a gene selected from the group consisting of SRP14 gene; the renal insufficiency or renal injury disease is caused or caused by renal ischemia reperfusion injury; the inhibitor is used for inhibiting the expression of the gene on the level of RNA, the inhibitor is siRNA, shRNA or microRNA, when the inhibitor is siRNA, the siRNA for inhibiting the SRP14 gene is AM16708 siRNA, and the reference sequence: NM _ 001309434.1.

3. The medicament according to claim 2, wherein the renal insufficiency or renal injury disease is acute renal injury, chronic renal disease or end-stage renal disease.