CN114533876B - Application of SRP14 gene in treating renal insufficiency or renal injury - Google Patents
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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 the application of an inhibitor for inhibiting gene expression in the preparation of a medicament for treating renal insufficiency or renal injury diseases, and the research of the invention discovers for the first time that the inhibition of the expression of SRP14 gene can treat the renal insufficiency or the renal injury diseases, and the invention provides a new idea and strategy for treating the renal insufficiency or the renal injury diseases.
Description
Divisional application
The divisional application is based on the application number of 2020102376062, the application date of 3/30/2020, and the invention name of the application of the RPS7 and SRP14 genes in treating renal insufficiency or renal injury.
Technical Field
The invention relates to the technical field of gene therapy, in particular to application of an 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 likely to progress to Chronic Kidney Diseases (CKD), leading to chronic renal insufficiency and End Stage Renal Disease (ESRD), and the incidence and fatality rate of the disease have been increasing in recent years, causing great 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 the injured kidney in the AKI can further induce apoptosis and inflammation in the heart, lung, liver and brain, further leading to high morbidity and mortality of the AKI, so that the inhibition of tubular epithelial apoptosis is a key link for preventing and treating the AKI. Despite the significant progress made in the study of tubular epithelial apoptosis in AKI, and the combination therapy directed to multiple cell death pathways could provide the greatest benefit in treating AKI, no truly effective specific method for preventing tubular epithelial apoptosis has yet 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 an SRP14 gene in treating renal insufficiency or renal injury.
The invention is realized by the following steps:
in a first aspect, the embodiments of the present invention provide the use of an inhibitor that inhibits the expression of a gene selected from the group consisting of SRP14 genes in the manufacture of a medicament for the treatment of renal insufficiency or renal injury.
The research of the invention discovers for the first time that the survival rate of cells of a kidney ischemia reperfusion injury model can be improved by inhibiting the expression of the SRP14 gene, which shows that the inhibition of the expression of the SRP14 gene can treat renal insufficiency or renal injury diseases, and correspondingly, the inhibitor for inhibiting the expression of the SRP14 gene can be prepared into a medicament for treating the renal insufficiency or the renal 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 against SRP14 gene were purchased from Thermo fisher, such as: SRP14siRNA (# AM16708, assay ID #12804, refSeq; the targeted exon sequences are as follows: gtga gctccaagga agtgaataag tttcagatg;
although the above-mentioned siRNA has a good effect of inhibiting expression of SRP14 gene, it should be noted that those skilled in the art can easily think that other sequences of siRNA are used to inhibit expression of SRP14 gene, and it is within the scope of the present invention to use any sequence of siRNA to inhibit 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 renal injury diseases.
In alternative embodiments, the inhibitor inhibits expression of the gene at the DNA level, RNA level, or protein level.
In alternative embodiments, the inhibitor is an iRNA, shRNA or microRNA.
In an alternative embodiment, the renal insufficiency or renal injury disease is caused or caused by a renal ischemia reperfusion injury.
In alternative embodiments, the renal insufficiency or kidney injury diseases include, but are not limited to, acute Kidney Injury (AKI), chronic Kidney 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 required 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 those skilled in the art can also obtain other related drawings based on 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 the 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 is used for knocking down gene expression, and Ctrl represents normal oxygen condition; IRI means hypoxia for 4h and reoxygenation for 24h;
FIG. 3 shows the result of PI/Annexin V staining detected by flow cytometry, wherein the cell damage rate in the Normal control (Normal control) group is 13.23%, the cell damage rate in the simple SRP14 knock-down (SKD) group is 11.94%, and the cell damage rate in the simple RPS7 knock-down (RKD) group is 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, 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 24h.
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
SRP14siRNA and RPS7 siRNA were used to knock down the expression of SRP14 or RPS7 in HK2 human tubular epithelial cells, respectively.
1 transfection of SRP14siRNA
SRP14siRNA gene silencing was diluted to a concentration of 5nmol/ml according to the instructions (purchased from Thermo fisher, inc. # AM16708, assay ID #12804, refSeq.
1) 40pmol of "SRP14 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) 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 at room temperature for 5 minutes.
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 serum-free and 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 5nmol/ml according to the instructions (purchased from Thermo fisher, # AM16708, assay ID #142206, refSeq.
1) 75pmol of RPS7 siRNA "was dissolved in 1.5ml of EP tube containing 100. Mu.l of Opti-DMEM-F12 serum-free and double antibody-free medium 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 at room temperature for 5 minutes.
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 serum-free and 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. An HK2 human renal tubular epithelial cell hypoxia/reoxygenation injury (IRI) model (I4 h/R24 h) was established in vitro using the MIC-101 hypoxia modular incubator system of biruex roburg (Bilupus) corporation, usa.
3. Preparation of protein samples
(1) Precooling the tissue in a mortar by using liquid nitrogen, taking out the frozen tissue of a refrigerator with the temperature of-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. Per 1mg of tissue, 40 μ l of protein lysate (RIPA, pecan organisms, P0013), phosphatase inhibitor PMSF (pecan organisms, P1048) were added (RIPA: PMSF =100, 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, ready for use). The cells were then scraped off with a cell scraper and collected in a 1.5ml EP tube.
(2) The cell ultrasonic disruptor is ultrasonically treated for 10-15 times on ice and then is placed on ice for cracking for 30min.
(3) Centrifuge at 13000rmp for 10min at 4 ℃.
(4) The supernatant was taken in a new 1.5ml EP tube and the protein concentration was measured by BCA method (BCA kit, cloudy day, P0009):
diluting 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 according to 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.
(5) According to the protein solution: loading buffer =4:1 Add 5x loading buffer (Biyun day, P0015).
(6) The protein was denatured by a metal bath at 95 ℃ for 5min. Storing at 80 deg.C
5Western blot
(1) Preparation of PAGE gel:
the separation gel and the concentrated gel were prepared according to SDS-PAGE preparation 10% and 12.5% kit instructions (SDS-PAGE kit, biyunyan, 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 concentrated gel buffer solution and concentrated gel solution are respectively taken, 20 mul of APS is added, and the concentrated gel buffer solution and the concentrated gel solution are 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 ℃.
(2) Electrophoresis: and taking out the prepared glue, installing the glue on an electrophoresis rack, and paying attention to the positive and negative electrodes. Placing the electrophoresis rack in an electrophoresis tank, and pouring electrophoresis liquid into the electrophoresis tank, wherein the height of the electrophoresis tank 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) Film transferring: and soaking the clamp, the filter paper and the sponge for membrane transfer into the transfer liquid half an hour before membrane transfer. And taking out the glass plate after electrophoresis, slightly pulling the glass plate open, cutting off the concentrated gel and bromophenol blue, and cutting the separation gel with corresponding size according to the number of samples. 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.
(4) And (3) sealing: after the film transfer, the PVDF film was taken out. After marking the positive and negative dates, putting the mixture into 5% of test skim milk and sealing the mixture for 2 hours at room temperature.
(5) Primary antibody incubation: after blocking was completed, the membrane was removed from the skim milk, washed 3 times with 1 × TBST for 5 minutes, and then antibodies were formulated using 1 × TBST at dilution ratios β -actin (1.
(6) And (3) secondary antibody incubation: 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 (zengio, anti-rabbit 1, #511203; anti-mouse 1 10000, # 701051) were formulated at a dilution ratio with 1 × TBST and incubated at room temperature for 2h.
(7) Developing and exposing: after the secondary antibody incubation, the membranes were removed from the antibody incubation cassette and washed 3 times with 1 × TBST for 5min each. Electrochemiluminescence (ECL) reagent (Millipore) was prepared as 1:1, and the exposure solution was dropped onto the membrane surface and imaged by automatic exposure using 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 survival rate was measured using the Japanese Dong Keren Cell Counting Kit-8 (CCK 8, cat # CK 04) Kit, the main component of 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) TM In Negative Control No.1siRNA (# AM 4611), which has no significant similarity with mouse, rat or human gene sequences, 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 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 is used for knocking down gene expression.
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: adherent HK2 cells are directly collected into a 5ml centrifuge tube after being digested by pancreatin, and the number of the cells of each sample is (1-5) multiplied by 10 6 Centrifuging at 2500r/min for 5min, and discarding the culture solution.
2) Washed 2 times with pre-cooled PBS and centrifuged at 2500r/min for 5min.
3) Resuspending the cells with 100ul Binding Buffer, setting blank control, PI single staining, annexin V single staining, experimental group double staining, and incubating for 10-15 min at room temperature in dark.
4) Flow cytometry analysis results
5) And (4) judging a result: apoptotic cells are resistant to all dyes used for the identification of cell activity, 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 the living cells in the left lower quadrant of a scatter diagram of the bivariate flow cytometer, wherein the 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-. The cellular injury 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 significantly 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 targeted to inhibit the expression thereof, and can treat renal insufficiency or renal injury diseases caused or caused by renal ischemia-reperfusion injury, such as acute renal injury (AKI), chronic renal disease (CKD) or end-stage renal disease (ESRD). 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)
1. Use of an inhibitor that inhibits gene expression for the manufacture of a medicament for increasing survival of HK2 human tubular epithelial cells after hypoxia reoxygenation, wherein the gene is selected from the group consisting of SRP14 genes; the inhibitor is to inhibit the expression of the gene at the RNA level;
the inhibitor is used for inhibiting the expression of the gene on the RNA level, and the inhibitor is siRNA; the siRNA inhibiting SRP14 gene is AM16708 siRNA, and the reference sequence: NM _001309434.1; target: exon 5 at position 356; the targeted exon sequences are as follows: gtga gctccaagga agtgaataag tttcagatg.
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| Inhibition of HMGB1 protects the retina from ischemia-reperfusion, as well as reduces insulin resistance proteins;Liu et al.;《PLOS ONE》;20170523;第12卷(第5期);全文 * |
| Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration;Hesketh et al.;《Journal of Visualized Experiments》;20140630;全文 * |
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| CN114533876A (en) | 2022-05-27 |
| CN111388674B (en) | 2022-05-03 |
| CN111388674A (en) | 2020-07-10 |
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