CN117925827A - Application of KDM5B in auxiliary diagnosis of renal fibrosis - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a biomarker for auxiliary diagnosis of renal fibrosis and application thereof. The invention firstly provides a biomarker for kidney fibrosis auxiliary diagnosis, namely KDM5B gene or coded protein thereof. Up-regulation of KDM5B expression in fibrosed human tubular epithelial cells and up-regulation of KDM5B expression levels in mouse fibrosed kidney tissue; thus, KDM5B may be used as an auxiliary diagnostic marker for renal fibrosis to aid in the diagnosis of renal fibrosis. The invention also provides a kit for auxiliary diagnosis of renal fibrosis, which contains a detection reagent of KDM5B gene or protein coded by the same. According to the invention, the ROC curve AUC of the KDM5B diagnosis for distinguishing the kidney fibrosis mice from the normal control mice can reach 0.882, so that the auxiliary diagnosis of the kidney fibrosis by the KDM5B has higher diagnosis value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of KDM5B in auxiliary diagnosis of renal fibrosis.
Background
Renal fibrosis is a chronic and progressive pathophysiological process of damage to cells inherent to the kidneys caused by a variety of causative factors, independent of underlying etiology. Renal fibrosis is mainly characterized by abnormal deposition of extracellular matrix (ECM), a key pathological stage from normal to Chronic Kidney Disease (CKD) to end-stage kidney disease (ESRD), and is considered to be the best histological predictor of reduced kidney function, as well as one of the major factors affecting prognosis of renal transplant patients. Kidney fibrosis affects half of the population of adults over 70 years and 10% of the world, bringing a tremendous economic burden to society, however no targeted drug is currently available to treat kidney fibrosis. Therefore, the pathogenesis of the renal fibers and a new target point are deeply explored, and the research and development of new drugs by using the target point have important research significance and clinical value.
Renal fibrosis is a complex pathophysiological process in which multiple factors and multiple signaling pathways are involved, and its pathogenesis has not been completely elucidated so far. Studies show that the epigenetic histone modification has a remarkable regulation and control effect on regulating TGF beta 1-induced renal fibrosis and ECM gene expression and downstream fibrosis promotion genes. Histone lysine demethylase 5B (KDM 5B) specifically removes dimethyl and trimethyl (H3K 4me2/me 3) of lysine (K4) at position 4 of histone 3 (H3), thereby inhibiting expression of the target gene. Abnormal expression of KDM5B is closely related to the occurrence and development of various diseases. Evidence suggests that KDM5B is a key regulator of the H3K 4-methylated group in early mouse embryo preimplantation stage development. KDM5B is also overexpressed, amplified, or mutated in many cancer types. In cancer cells, KDM5B regulates expression of oncogenes and tumor suppressors by mediating H3K4 methylation levels. In addition, KDM5B was found to bind to the promoter that activates transcription factor 3 (Atf 3) and inhibit expression of ATF3 by demethylating the activated H3K4me2/3 modification, thereby promoting heart failure. However, as a key molecule for epigenetic histone modification, the role of KDM5B in the progression of renal fibrosis has not been reported. Because histone modification is closely related to pathogenesis of renal fibrosis, the key epigenetic regulation and control effect of KDM5B in the pathogenesis of abnormal renal fibrosis is clarified, and the KDM5B is expected to become an important candidate target for intervention of renal fibrosis.
Disclosure of Invention
In view of the problems and deficiencies in the prior art, it is an object of the present invention to provide the use of KDM5B in the assisted diagnosis of renal fibrosis.
In order to achieve the aim of the invention, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a biomarker for auxiliary diagnosis of renal fibrosis, wherein the biomarker is KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragment of KDM5B protein.
The expression of KDM5B in human renal tubular epithelial cells (HK-2) which are subjected to fibrosis change induced by TGF beta 1 is detected by Western Blot, and the expression level of KDM5B in the HK-2 cells which are subjected to fibrosis change induced by TGF beta 1 is obviously higher than that of normal HK-2 cells, and the difference has statistical significance; in addition, detection of KDM5B by Western Blot and immunohistochemical staining showed significantly higher levels of expression in kidney tissue of UUO (unilateral ureteral obstruction) mice fibrosis than sham operated mice, and the differences were statistically significant.
In a second aspect, the invention provides the use of a detection reagent for a KDM5B gene or KDM5B mRNA or KDM5B protein or a peptide fragment of KDM5B protein in the preparation of a product for the assisted diagnosis of renal fibrosis.
According to the above application, preferably, the product detects the expression level of the KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragment of KDM5B protein in the sample by RT-PCR, real-time quantitative PCR, in situ hybridization, northern Blot, western Blot, chip, high throughput sequencing platform, immunohistochemistry or enzyme linked immunosorbent.
According to the above application, preferably, the product contains an antibody that specifically binds to a KDM5B protein or a peptide fragment of a KDM5B protein, or contains a KDM5B mRNA primer that specifically amplifies a KDM5B gene or KDM5B mRNA, or contains a probe that specifically detects a KDM5B gene or KDM5B mRNA.
According to the above application, preferably, the antibody is a monoclonal antibody, a polyclonal antibody or a single domain antibody.
According to the above application, preferably, the test sample of the product is a cell, tissue or serum.
According to the above application, preferably, the product is a chip, a preparation or a kit.
According to the above-mentioned application, preferably, the kidney fibrosis is kidney fibrosis caused by various kidney diseases.
In a third aspect, the invention provides a kit for auxiliary diagnosis of renal fibrosis, wherein the kit contains a detection reagent of KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragments of KDM5B protein.
According to the above kit, preferably, the detection reagent is a reagent for detecting the expression level of KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragment of KDM5B protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, northern Blot, western Blot, chip, high throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent.
According to the above kit, preferably, the detection reagent is an antibody that specifically binds to a KDM5B protein or a peptide fragment of a KDM5B protein, or a KDM5B mRNA primer that specifically amplifies a KDM5B gene or a KDM5B mRNA, or a probe that specifically detects a KDM5B gene or a KDM5B mRNA.
According to the above kit, preferably, the test sample is a cell, a tissue or serum.
According to the above kit, preferably, the kidney fibrosis is kidney fibrosis caused by various kidney diseases.
Compared with the prior art, the invention has the following positive and beneficial effects:
(1) According to the invention, the expression of KDM5B in fibrotic human renal tubular epithelial cells is up-regulated for the first time, and the expression level of KDM5B in the kidney tissue of the mouse fibrosis is up-regulated, so that KDM5B can be used as an auxiliary diagnostic index of kidney fibrosis to assist in diagnosing the kidney fibrosis. Moreover, the ROC curve AUC of the KDM5B diagnosis for distinguishing the kidney fibrosis mice from the normal control mice can reach 0.882, which shows that the KDM5B has higher diagnosis value for auxiliary diagnosis of the kidney fibrosis.
(2) The invention adopts KDM5B specific small interfering RNA (si-KDM 5B) and a specific inhibitor TK-129 thereof to inhibit KDM5B expression or activity, and can obviously lighten and alleviate kidney fibrosis, so that the KDM5B can be used as a drug, a drug target or a target gene in gene therapy, is applied to prevention, alleviation or/and treatment of kidney fibrosis, can provide a new strategy for preventing and treating the kidney fibrosis, and simultaneously provides a new direction for further researching the cause of the kidney fibrosis and the corresponding prevention and treatment strategy.
Drawings
FIG. 1 is a graph showing Western Blot detection of levels of FN1, α -SMA and KDM5B protein expression in TGF- β1-induced human tubular epithelial cells HK-2; a is a representative Western Blot banding pattern; B-D is the result of Western Blot quantitative analysis; GAPDH was used as an internal control, n=3, < p <0.01;
FIG. 2 is a graph showing the results of Masson staining for detection of UFO and Sham mouse kidney tissue fibrosis; a is a representative Masson-stained picture, scale is 100 μm; b is the statistical result of kidney tissue fibrosis area of UFO group mice and Sham mice; n=6, p <0.001;
FIG. 3 is a graph showing the results of Western Blot detection of the expression of the fibrosis markers FN1 and alpha-SMA protein in kidney tissue of UFO and Sham mice; a is a representative Western Blot banding pattern; b is a Western Blot quantitative analysis result; GAPDH as an internal control, n=3, < p <0.01, < p <0.001;
FIG. 4 is a graph showing the results of immunohistochemical staining and Western Blot detection of KDM5B expression in UFO-induced mouse fibrotic kidney tissue; a is a representative immunohistochemical staining pattern; b is the result of quantitative analysis of immunohistochemical staining, and the scale is 20 μm; c is a representative Western Blot banding pattern; d is the result of Western Blot quantitative analysis, using GAPDH as an internal control. n=3, p <0.01, p <0.001;
FIG. 5 is a graph showing the results of Masson staining for detection of UFO and Sham mouse kidney tissue fibrosis; a is a representative picture of Masson staining; b is the quantitative analysis result of Masson staining, scale is 100 μm, n=12, ×p <0.001;
FIG. 6 is a graph showing the results of immunohistochemical staining for detection of KDM5B expression in kidney tissue of UFO and Sham mice; a is a representative picture of immunohistochemical staining; b is the result of immunohistochemical staining quantitative analysis, scale 50 μm, n=12, ×p <0.001;
FIG. 7 is a graph of ROC of KDM5B diagnosis distinguishing UFO mice from Sham mice;
FIG. 8 is a graph showing the result of Western Blot detection of knockdown of KDM5B in HK-2; a is a strip picture of a representative Western Blot, and B is a quantitative analysis result of the Western Blot; GAPDH as an internal control, n=3, < p <0.001;
FIG. 9 is a graph showing the effect of Western Blot detection of KDM5B knockdown on the levels of FN1 and alpha-SMA protein expression in TGF-beta 1-induced HK-2; a is a representative Western Blot band; b is the Western Blot quantitative analysis result of KDM5B protein; c is the result of Western Blot quantitative analysis of FN1 protein; d is the Western Blot quantitative analysis result of the alpha-SMA protein; GAPDH as an internal control, n=3, < p <0.01, < p <0.001;
FIG. 10 is a graph showing the results of the detection of the viability of HK-2 cells in CCK-8 assay at concentrations of 2.5. Mu.M, 5. Mu.M and 10. Mu.M TK-129 in solution for 24 h;
FIG. 11 is a graph of the effect of Western Blot detection of TK-129 inhibition of KDM5B activity on the levels of FN1 and alpha-SMA protein expression in TGF-beta 1-induced HK-2; a is a representative Western Blot band; b is the Western Blot quantitative analysis result of FN1 protein; c is the result of Western Blot quantitative analysis of alpha-SMA protein; GAPDH as an internal control, n=3, p <0.05, < p <0.01, < p <0.001;
FIG. 12 is a graph showing the results of RT-PCR and Western Blot detection of KDM5B overexpression in HK-2; a is a schematic diagram of a structure map of a KDM5B over-expression plasmid; b is RT-PCR detection KDM5B mRNA expression; c is a representative Western Blot band; d is the Western Blot quantitative analysis result of KDM5B protein; GAPDH as an internal control, n=3, < p <0.01, < p <0.001;
FIG. 13 is a graph of the effect of KDM5B overexpression on FN1 and α -SMA protein expression levels in HK-2; a is a representative Western Blot band; b is the Western Blot quantitative analysis result of FN1 protein; c is the result of Western Blot quantitative analysis of alpha-SMA protein; GAPDH as an internal control, n=3, < p <0.05, < p <0.01;
FIG. 14 is a graph showing the results of Western Blot detection of KDM5B expression in kidney tissue of a kidney tubular epithelial cell-specific KDM5B knockout mouse; a is a schematic diagram of a structural map of KDM5B knockout adeno-associated virus AAV2/9-KDM 5B-shRNA; b is a kidney in-situ injection schematic diagram of the mice; c is a representative Western Blot band; d is the Western Blot quantitative analysis result of KDM5B protein, with GAPDH as an internal control, n=3, p <0.001;
FIG. 15 is a graph showing the effect of Masson staining on detection of kidney tubule epithelial cell specific KDM5B knockout on UFO-induced kidney fibrosis in mice; a is a representative Masson-stained picture, scale is 100 μm; b is the statistical result of kidney tissue fibrosis area of each experiment grouping mouse; n=3, p <0.001;
FIG. 16 is a graph showing the effect of immunofluorescent staining to detect the effects of a tubular epithelial cell-specific KDM5B knockout on FN1 and α -SMA expression in UFO-induced kidney tissue of mice; a is a representative FN1 immunofluorescence staining picture, and the scale is 100 μm; b is the result of the immunofluorescence staining expression statistics of FN1 protein in kidney tissues of mice in each experimental group; c is a representative alpha-SMA immunofluorescence staining picture with a scale of 100 μm; d is the result of immunofluorescence staining expression statistics of the alpha-SMA protein in kidney tissues of mice in each experimental group; n=3, p <0.05, p <0.01, p <0.001.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
The following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, components, and/or combinations thereof.
The experimental methods in the following examples, in which specific conditions are not specified, are all conventional in the art or according to the conditions suggested by the manufacturer; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: expression level study of KDM5B in TGF-beta 1-induced human tubular epithelial cells (HK-2)
TGF-beta 1 was used to detect the level of KDM5B expression in HK-2 cells and normal HK-2 cells using Western Blot.
1. Experimental cells and culture method:
Human tubular epithelial cells (HK-2) were from the Shanghai ATCC cell bank. The old medium in the flask was discarded, 2mL of sterile PBS was added, gently shaken, discarded, and 2mL of 0.25% pancreatin pre-heated at 37℃was added for digestion for 2-3min. The dishes were gently shaken to shed the cells and digestion was stopped by adding 2mL fresh DMEM/F12 complete medium (10% FBS+1% diabody). The cells were collected, centrifuged at 1000rpm for 5min at room temperature, the supernatant was discarded, the cells were resuspended in fresh complete medium, added to a petri dish, and placed in a 5% CO 2 incubator for continued culture.
2. The experimental method comprises the following steps:
HK-2 cells were collected and, after counting with a cell counting plate, plated in 6-well plates with 1X 10 5 cells per well. The cell groupings were: control group and tgfβ1 group. The next day, after the cell morphology is completely restored, TGF-beta 1 solution (the solvent is citric acid, the concentration of TGF-beta 1 solution is 10ug/mL, the dilution is needed for 1000 times, the final action concentration is 10 ng/mL) is added into TGF-beta 1 group, the Control group is added with the same dose of Control solvent (citric acid), and the cells are collected after 24 hours of action. Protein expression changes of Control group, TGF beta 1 group fibrosis markers, fibracton (FN 1), and alpha-SMA, and KDM5B were detected separately using Western Blot.
3. Experimental results:
Western Blot detection of TGF-beta 1 induced changes in the expression of the fibrosis markers FN1 and alpha-SMA and KDM5B protein in human tubular epithelial cells HK-2, the results are shown in FIG. 1.
As can be seen from FIG. 1, the expression of the fibrosis markers FN1 and alpha-SMA protein was significantly increased in the TGF-beta 1 treated group HK-2 compared to the Control group, and the differences were statistically significant, indicating that TGF-beta 1 induced a fibrotic change in the HK-2 cells. Moreover, KDM5B expression was significantly elevated in fibrotic HK-2, and the differences were statistically significant.
Example 2: expression level study of KDM5B in mouse fibrotic kidney tissue
The expression level of KDM5B in kidney tissue of kidney-fibrotic mice was detected using Western Blot.
1. Experimental animal selection and feeding conditions:
SPF-grade Wild (WT) male C57BL/6 mice of 8 weeks old were purchased from Liaoning long biotechnology Co., ltd, were kept in an SPF environment with a temperature of 18-23℃and a humidity of 40% -60% at 12 hours light/12 hours darkness, and were given normal feeding to ordinary mice.
2. The experimental method comprises the following steps:
after one week of adaptive feeding, the mice were randomly divided into Sham operation (Sham) groups and Unilateral Ureteral Obstruction (UUO) groups, each group of 6 animals.
The UUO model construction method comprises the following steps: under isoflurane gas anesthesia, fixing the lateral position of the mouse on an operating table, sterilizing the skin by adopting alcohol, making a left abdominal incision, exposing and freeing a left ureter by about 1cm, ligating 1/3 position and 1/3 position on the ureter by using a 6-0 surgical line, cutting the ureter between two ligature points, and finally suturing the abdominal cavity and the skin layer by layer. Whereas Sham group mice opened only the abdominal cavity and free left ureter, no ligation treatment was performed.
At 7 days after the model, killing each group of mice, collecting kidney tissues, fixing half of the kidney tissues with 4% paraformaldehyde for 48 hours, embedding paraffin, cutting into 4-mu M thick slices, performing Masson dyeing to detect the fibrosis condition of the kidney tissues of UFO mice, and evaluating whether the construction of the UFO-induced kidney fibrosis model of the mice is successful; the remaining kidney tissues are frozen by liquid nitrogen and then stored at the temperature of minus 80 ℃, and the subsequent Western Blot is adopted to detect the expression condition of fibrosis markers FN1 and alpha-SMA in UUO mouse kidney tissues; meanwhile, immunohistochemical staining and Western Blot experiments are adopted to detect the expression change of KDM5B in kidney tissues of UFO mice fibrosis.
3. Experimental results
(1) Masson staining:
The Masson staining results are shown in fig. 2. As can be seen from fig. 2, compared with Sham mice, UUO mice have obvious collagen deposition in kidney tissue, and the statistical fibrosis area is significantly increased, which indicates that the construction of the UUO mice kidney fibrosis model is successful.
(2) Western Blot detection of the expression of the fibrosis markers FN1 and α -SMA in kidney tissue of UFO and Sham mice:
the results of Western Blot detection of the fibrosis markers FN1 and alpha-SMA in kidney tissue of UFO mice are shown in FIG. 3. As can be seen from FIG. 3, the expression of the fibrosis markers FN1 and alpha-SMA protein in kidney tissue of UUO mice is significantly increased compared with that of Sham group mice, and the difference has statistical significance, further indicating that the construction of the kidney fibrosis model of UO mice is successful.
(3) Immunohistochemical staining and Western Blot detection of KDM5B expression in kidney tissue of UUO and Sham mice fibrosis:
The results of KDM5B immunohistochemical staining and Western Blot detection in UFO and Sham mouse fibrotic kidney tissue are shown in FIG. 4. As can be seen from fig. 4, KDM5B expressed significantly higher in kidney tissue of UUO mice than Sham mice, and the differences were statistically significant.
Example 3: value assessment of KDM5B for diagnosis of renal fibrosis
1. Experimental animal selection and feeding conditions:
SPF grade 7 week old C57 BL/6 male mice, weighing 16-18g, purchased from Liaoning long biotechnology Co., ltd, were kept in SPF environment with temperature controlled at 18-23℃and humidity maintained at 40% -60% under 12 hours light/12 hours darkness, and were given normal drinking water and ordinary rat food feeding.
2. The experimental method comprises the following steps:
After one week of adaptive feeding, the mice were randomly divided into Sham operation (Sham) groups and Unilateral Ureteral Obstruction (UUO) groups of 12 animals each. The UUO model construction method is the same as that of embodiment 2, and will not be described in detail here.
7 Days after the model, each group of mice was sacrificed, kidney tissue was harvested, paraffin embedded after fixation with 4% paraformaldehyde for 48 hours, cut into 4- μm thick sections, and UUO-induced kidney tissue fibrosis was detected using Masson staining. The expression change of KDM5B in kidney tissues of UFO mice and Sham mice is detected by adopting immunohistochemical staining, the experimental result is subjected to statistical analysis, an ROC curve is drawn, AUC values are counted, and the value of KDM5B for diagnosing renal fibrosis is evaluated.
3. Experimental results:
The Masson staining results are shown in fig. 5. As can be seen from fig. 5, the UUO mice showed significant fibrosis in kidney tissue compared to Sham mice, indicating that UUO-induced mouse kidney fibrosis model construction was successful.
Immunohistochemical staining results of UUO mice fibrillated kidney tissue KDM5B are shown in fig. 6. As can be seen from fig. 6, KDM5B expressed significantly higher in UUO mice fibrotic kidney tissue than Sham mice, and the differences were statistically significant.
Statistical analysis was performed on the immunohistochemical staining results of KDM5B, and a ROC curve was drawn for the diagnosis of KDM5B to distinguish UFO group mice from Sham mice, and the results are shown in FIG. 7.
As can be seen from fig. 7, the AUC of ROC curve for differentiating UUO group mice from Sham mice using the diagnosis of KDM5B reached 0.882, which is effective for differentiating kidney fibrosis mice from normal control mice. Therefore, the KDM5B can be used for auxiliary diagnosis of renal fibrosis and has higher diagnostic value.
Example 4: effect of in vitro KDM5B knockdown or Activity inhibition on TGF-beta 1-induced fibrosis of human tubular epithelial cells (HK-2)
1. HK-2 cell KDM5B knockdown:
(1) Experimental cell and culture method
Human tubular epithelial cells (HK-2) were from the Shanghai ATCC cell bank. The old medium in the flask was discarded, gently washed with 2mL of sterile PBS, discarded, and digested with 2mL of 0.25% pancreatin pre-warmed at 37℃for 2-3min. The dishes were gently shaken to shed the cells and digestion was stopped by adding 2mL fresh DMEM/F12 complete medium (10% FBS+1% diabody). The cells were collected, centrifuged at 1000rpm for 5min at room temperature, the supernatant was discarded, the cells were resuspended in fresh complete medium, added to a petri dish, and placed in a 5% CO 2 incubator for continued culture.
(2) Experimental method
The small interfering RNAs with the specificity of KDM5B (si-KDM 5B-1, si-KDM5B-2, si-KDM 5B-3) and the control siRNA (si-Ctrol) are synthesized by Ji Ma gene design, and the nucleotide sequences are respectively as follows:
si-KDM5B-1forward 5’-GGACUUAUUUCAGCUUAAUTT-3’，
si-KDM5B-1reverse 5’-AUUAAGCUGAAAUAAGUCCTT-3’；
si-KDM5B-2forward 5’-GACCUGGAGAGAGCUUUAATT-3’，
si-KDM5B-2reverse 5’-UUAAAGCUCUCUCCAGGUCTT-3’；
si-KDM5B-3forward 5’-GCCCAAGAGUCGAUCUAAATT-3’，
si-KDM5B-3reverse 5’-UUUAGAUCGACUCUUGGGCTT-3’；
si-Ctrol forward 5’-UUCUCCGAACGUGUCACGUTT-3’，
si-Ctrol reverse 5’-ACGUGACACGUUCGGAGAATT-3’。
human tubular epithelial cells HK-2 were cultured in DMEM/F12 (10% FBS,100U/mL penicillin, 100. Mu.g/mL streptomycin) medium at 37℃in a 5% carbon dioxide incubator, and when the cell density reached 80%, pancreatin was digested, fresh medium was resuspended, counted, plated in 6-well plates, and the number of cells per well was 1X 10 5, and placed in the cell incubator for overnight culture. The next day, the small interfering RNAs specific for KDM5B (si-KDM 5B-1, si-KDM5B-2, si-KDM 5B-3) and control RNA (si-Ctrol) were transfected with Lipofectamine RNAiMAX Transfection Reagent, respectively, and after 48 hours of action, cells were collected and the knockdown of KDM5B in HK-2 cells was detected using western blot.
(3) Experimental results
The experimental results of the Western Blot detection of the KDM5B knockdown are shown in FIG. 8.
As can be seen from FIG. 8, the expression of the protein of the SIKDM 5B-1, the SIKDM 5B-2 and the SIKDM 5B-3 treatment group KDM5B was significantly reduced compared with that of the SI-Ctrol, which indicates that the knockdown of the protein of the KDM5B in the HK-2 was successful and that the sequence knockdown effect of the SIKDM 5B-1 was best, and the subsequent experiments were carried out by using the sequence.
2. Effect of KDM5B knockdown on tgfβ1 induction of FN1 and α -SMA protein expression in HK-2 by Western Blot detection:
(1) Experimental cell and culture method
Human tubular epithelial cells (HK-2) were from the Shanghai ATCC cell bank. The old medium in the flask was discarded, gently washed with 2mL of sterile PBS, discarded, and digested with 2mL of 0.25% pancreatin pre-warmed at 37℃for 2-3min. The dish was gently shaken to detach the cells, and 2mL of fresh DMEM/F12 complete medium (10% FBS,100U/mL penicillin, 100. Mu.g/mL streptomycin) was added to terminate the digestion. The cells were collected, centrifuged at 1000rpm for 5min at room temperature, the supernatant was discarded, the cells were resuspended in fresh complete medium, added to a petri dish, and placed in a 5% CO 2 incubator for continued culture.
(2) Experimental method
HK-2 cells were cultured in vitro, digested with pancreatin, resuspended in fresh DMEM/F12 complete medium and plated in 6-well plates with 1X 10 5 cells per well. The experimental group is as follows: control group (si-Ctrol), KDM5B knockdown group (si-KDM 5B), TGFβ1 treatment group (si-Ctrol +TGFβ1), KDM5B knockdown+TGFβ1 treatment group (si-KDM 5B+TGFβ1). The next day, using Lipofectamine RNAiMAX Transfection Reagent to separately transfect si-KDM5B and si-Ctrol, after 24 hours adding TGF beta 1 (solvent is citric acid, TGF beta 1 solution concentration is 10ug/mL, needed to dilute 1000 times, final effect concentration is 10 ng/mL), and further incubation for 24 hours. The cells were collected and assayed for total protein concentration using RIPA lysis and BCA protein quantification kit, and Western Blot was used to detect the effect of KDM5B knockdown on TGF-beta 1-induced protein expression of KDM5B, FN1 and α -SMA in HK-2.
(3) Experimental results:
The experimental results of the Western Blot detection of KDM5B knockdown on TGF-beta 1 induced FN1 and alpha-SMA protein expression in HK-2 are shown in FIG. 9.
As can be seen from fig. 9, the tgfβ1 treated group showed significantly elevated levels of KDM5B and fibrosis marker proteins FN1 and α -SMA protein expression compared to the control group, indicating that KDM5B was up-regulated in tgfβ1-induced fibrosis HK-2. Meanwhile, KDM5B knockdown significantly reduced tgfβ1-induced FN1 and α -SMA expression compared to tgfβ1-treated groups. The results show that in vitro knock-down of KDM5B can significantly improve the TGF-beta 1 induced fibrosis response of HK-2 cells.
3. Western Blot detection of the effect of inhibition of KDM5B Activity on TGF-beta 1-induced expression of FN1 and alpha-SMA protein in HK-2:
(1) Experimental cell and culture method
Human tubular epithelial cells (HK-2) were from the Shanghai ATCC cell bank. The old medium in the flask was discarded, gently washed with 2mL of sterile PBS, discarded, and digested with 2mL of 0.25% pancreatin pre-warmed at 37℃for 2-3min. The dish was gently shaken to detach the cells, and 2mL of fresh DMEM/F12 complete medium (10% FBS,100U/mL penicillin, 100. Mu.g/mL streptomycin) was added to terminate the digestion. The cells were collected, centrifuged at 1000rpm for 5min at room temperature, the supernatant was discarded, the cells were resuspended in fresh complete medium, added to a petri dish, and placed in a 5% CO 2 incubator for continued culture.
(2) Concentration of KDM5B Activity inhibitor TK-129 ((R) -1-Cyclopropyl-3- (1- (3-isopropyl-1H-pyracl-5-carboyl) -pyrrolidin-yl) urea) screening
Culturing HK-2 cells in vitro, digesting with pancreatin, re-suspending, and spreading in 6-well plate with 1×10 5 cells per well; a series of KDM5B specific small molecule inhibitor TK-129 solutions (TK-129 solution concentration of 2.5. Mu.M, 5. Mu.M, 10. Mu.M; TK-129 solution solvent DMSO) were added to 96-well plates and allowed to react for 24 hours, and the cell viability was measured using a CCK-8 assay and the effect concentration of TK-129 was screened. The results are shown in FIG. 10.
As can be seen from FIG. 10, there was no statistical difference in the survival rate of HK-2 cells after 24 hours of the 2.5. Mu.M, 5. Mu.M, 10. Mu.M TK-129 solution compared to the Control (Control), indicating that none of 2.5. Mu.M, 5. Mu.M, 10. Mu.M TK-129 showed cytotoxicity, and subsequent experiments were performed using 5. Mu.M and 10. Mu.M, respectively.
(3) Effect of KDM5B Activity inhibitor TK-129 on TGF-beta 1-induced FN1 and alpha-SMA protein expression in HK-2
HK-2 cells were cultured in vitro, digested with pancreatin, resuspended and plated in 6-well plates with 1X 10 5 cells per well. The experimental setup groups were: control (Ctrl), TK-129 treated (TK-1295. Mu. M, TK-12910. Mu.M), TGF-beta.1 treated (TGF-beta.1), TGF-beta.1+TK-129 dosed (TGF-beta.1+TK-1295. Mu. M, TGF. Beta.1+TK-12910. Mu.M). The next day, control reagent (DMSO) and 5. Mu.M and 10. Mu.M TK-129 solution (TK-129 solution in DMSO) were added to HK-2 cells, respectively, and after two hours of incubation, TGF-beta 1 (TGF-beta 1 solution in citric acid at a concentration of 10ug/mL, diluted 1000-fold, and final concentration of 10 ng/mL) was added and incubation was continued for 24 hours. Cells were collected and assayed for total protein concentration using RIPA lysis and BCA protein quantification kit, and Western Blot was used to detect the effect of KDM5B activity inhibitor TK-129 on TGF-beta 1-induced expression of the fibrosis markers FN1 and alpha-SMA protein in HK-2. The experimental results are shown in FIG. 11.
As can be seen from fig. 11, the tgfβ1 treated group had significantly increased expression levels of the fibrosis marker proteins FN1 and α -SMA protein compared to the control group; whereas high doses of TK-129 (10. Mu.M) significantly reduced TGF-beta.1-induced FN1 and alpha-SMA expression compared to the TGF-beta.1 treated group. The above results indicate that inhibiting KDM5B activity in vitro with TK-129 significantly reduces the TGF-beta 1 induced fibrosis response of HK-2.
Example 5: effect of in vitro KDM5B overexpression on human tubular epithelial cells (HK-2)
1. Experimental cell and culture method
Human tubular epithelial cells (HK-2) were from the Shanghai ATCC cell bank. The old medium in the flask was discarded, gently washed with 2mL of sterile PBS, discarded, and digested with 2mL of 0.25% pancreatin pre-warmed at 37℃for 2-3min. The dish was gently shaken to detach the cells, and 2mL of fresh DMEM/F12 complete medium (10% FBS,100U/mL penicillin, 100. Mu.g/mL streptomycin) was added to terminate the digestion. The cells were collected, centrifuged at 1000rpm for 5min at room temperature, the supernatant was discarded, the cells were resuspended in fresh complete medium, added to a petri dish, and placed in a 5% CO 2 incubator for continued culture.
2. Experimental method
The KDM5B over-expression plasmid (pc-KDM 5B) and the control plasmid (pc-DNA 3.1) were synthesized by hantazicar biosignature, and the structural map of pc-KDM5B is shown in FIG. 12A. HK-2 cells were plated in 6-well plates with a cell count of 1X 10 5 per well. The following day, plasmid transfection was performed using Lipofectamine TM transfection reagent 3000. After 48 hours of action, cells were collected and the expression of KDM5B in HK-2 cells was detected separately using RT-PCR and Western Blot. The effect of KDM5B overexpression on the expression of the fibrosis markers FN1 and alpha-SMA protein in HK-2 was further examined using Western Blot.
3. Experimental results
(1) RT-PCR and Western Blot detection of KDM5B overexpression effect in HK-2
The results of RT-PCR and Western Blot detection of KDM5B overexpression in HK-2 are shown in FIG. 12. As can be seen from FIG. 12, both the levels of KDM5B mRNA and protein were significantly increased in the Ad-KDM5B group compared to the Ad-Control group, indicating successful over-expression of KDM5B in HK-2.
(2) Western Blot detection of the Effect of KDM5B overexpression on FN1 and alpha-SMA protein expression levels in HK-2
The results of Western Blot detection of the effect of KDM5B overexpression on FN1 and alpha-SMA protein expression levels in HK-2 are shown in FIG. 13. As can be seen from FIG. 13, the fibrosis markers FN1 and alpha-SMA protein expression were significantly elevated in the HK-2 group of pc-KDM5B compared to the control group of pc-cDNA3.1, indicating that KDM5B overexpression induced a HK-2 fibrosis response.
Example 6: effect of tubular epithelial cell-specific KDM5B knockout on UUO model mouse kidney fibrosis
1. Experimental animal selecting and raising conditions
SPF grade 8 week old C57 BL/6 male mice, weighing 16-18g, purchased from Liaoning long biotechnology Co., ltd, were kept in SPF environment with temperature controlled at 18-23℃and humidity maintained at 40% -60% under 12 hours light/12 hours darkness, and were given normal feeding to ordinary mice.
2. Experimental method
KDM5B knockout adeno-associated virus (AAV 2/9-KDM5B-shRNA, structure map of AAV2/9-KDM5B-shRNA is shown as A in FIG. 14) was constructed by hantaa using a kidney tubule epithelial cell specific promoter Ksp-cadherin, and no-load adeno-associated virus control (AAV 2/9-Ctrol). The nucleotide sequence of KDM5B shRNA is: 5'-TTCGCTTGTGATGTCGATAAA-3' the control nucleotide sequence is: 5'-TTCTCCGAACGTGTCACGT-3'. The experimental mice were randomly divided into AAV9-Ctrol Sham, AAV9-KDM5B-SHRNA SHAM, AAV9-Ctrol UUO, and AAV9-KDM5B-shRNA UUO groups. In situ renal injection was performed at a volume of 60. Mu.L, with an AAV9-KDM5B-shRNA viral titer of 1.3X10 12 v.g/mL and an AAV9-Ctrol viral titer of 1.4X10 12 v.g/mL. The specific operation is as follows: under isoflurane gas anesthesia, mice were fixed on an operating table in a horizontal position, after skin was sterilized, left side abdominal incision was made, left side kidney was exposed, kidneys were carefully fixed with fingers, and virus was injected into six parts of the upper and lower stages of the kidneys and the outer peripheral edge, respectively, using a microinjector (measuring range 100 μl). After injection, the kidneys are reset, muscles and skin are sutured in sequence, and after the mice are fully awakened, the mice are returned to the squirrel cage for normal feeding.
After 3 weeks, the UUO or Sham model was run on each group of mice, see example 2 for specific procedures. After 1 week after the UUO model, mice were sacrificed, left kidney tissue was taken, half of kidney tissue was paraffin-embedded after 48 hours fixation with 4% paraformaldehyde, and kidney paraffin tissue was prepared into 4 μm thick sections for later use using a microtome. The other half kidney tissue is frozen by liquid nitrogen for 5 minutes and then is preserved at-80 ℃ for standby. Taking kidney tissues, and detecting the expression condition of KDM5B by using western blot. The effect of tubular epithelial cell-specific KDM5B knockout on mouse kidney fibrosis was further examined using Masson staining. Immunofluorescence staining was used to detect the effect of tubular epithelial cell-specific KDM5B knockout on the expression of fibrosis markers FN1 and α -SMA in UUO-induced mouse kidney tissue.
3. Experimental results
(1) Detection of expression of KDM5B in kidney tissue of a tubular epithelial cell-specific KDM5B knockout mouse by Western Blot:
The results of Western Blot detection of KDM5B expression in kidney tissue of kidney tubule epithelial cell specific KDM5B knockout mice are shown in FIG. 14. As can be seen from FIG. 14, KDM5B was significantly elevated in kidney tissue from AA2/9-Ctrol UUO mice compared to AA2/9-Ctrol Sham mice. Compared with an AA2/9-Ctrol UUO mouse, the expression of KDM5B in kidney tissues of the AA2/9-KDM5B-shRNA UO mouse is obviously reduced, which indicates that the construction of the kidney tubule epithelial cell specific KDM5B knockout mouse is successful.
(2) Effect of tubular epithelial cell specific KDM5B knockout on UUO-induced mouse kidney fibrosis was examined using Masson staining:
the results of the Masson staining test are shown in FIG. 15. As shown in fig. 15, compared with the AAV9-Ctrol Sham group, the kidney tissue of the AAV9-KDM5B-SHRNA SHAM group is not significantly changed, which indicates that the kidney epithelial cell specific KDM5B knockout has no effect on the kidney of the mouse in the basal state, whereas the kidney tubular of the mouse of the AAV9-Ctrol UUO model group is dilated, the kidney fibrosis area is significantly increased, which indicates that the kidney fibrosis model is successfully constructed; compared with an AAV9-Ctrol UUO model group, the expansion degree of a mouse kidney tubule and the area of kidney fibrosis of the UFO group of AAV9-KDM5B-shRNA are reduced, which shows that the specific KDM5B knockout of the epithelial cells of the kidney tubule can obviously reduce the kidney fibrosis reaction of the mouse induced by the UFO.
(3) Immunofluorescence staining to detect effects of tubular epithelial cell specific KDM5B knockout on expression of fibrosis markers FN1 and α -SMA in UUO-induced mouse kidney tissue:
The results of immunofluorescent staining to examine the effect of tubular epithelial cell-specific KDM5B knockout on FN1 and α -SMA expression in UUO-induced mouse kidney tissue are shown in fig. 16.
As can be seen from fig. 16, the expression of fibrosis markers FN1 and α -SMA in the kidney tissue of mice in the AAV9-Ctrol UUO model group was also significantly increased compared to the AAV9-Ctrol Sham model group, while FN1 and α -SMA in the kidney tissue of mice in the AAV9-KDM5B-shRNA UUO model group was significantly decreased compared to the AAV9-Ctrol UUO model group, further indicating that the kidney fibrosis of UUO-induced mice was significantly improved by the specific knockout of KDM5B from renal tubular epithelial cells.
Taken together, in vitro KDM5B knockdown or inhibition of activity significantly reduces TGF-beta 1-induced fibrosis response in human tubular epithelial cells HK-2. The in vivo specific knockout of KDM5B in renal tubular epithelial cells can also significantly improve the renal fibrosis response of mice.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
- Application of detection reagent of KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragment of KDM5B protein in preparing kidney fibrosis auxiliary diagnosis product.
- 2. The use according to claim 1, wherein the product is used for detecting the expression level of the KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragments of KDM5B protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, northern Blot, western Blot, chip, high throughput sequencing platform, immunohistochemistry or enzyme linked immunosorbent.
- 3. The use according to claim 2, wherein the product comprises an antibody that specifically binds to a KDM5B protein or a peptide fragment of a KDM5B protein, or comprises a KDM5B mRNA primer that specifically amplifies a KDM5B gene or a KDM5B mRNA, or comprises a probe that specifically detects a KDM5B gene or a KDM5B mRNA.
- 4. The use according to claim 3, wherein the antibody is a monoclonal antibody, a polyclonal antibody or a single domain antibody.
- 5. The use according to any one of claims 1 to 4, wherein the test sample of the product is a cell, tissue or serum.
- 6. The use according to claim 5, wherein the product is a chip, a formulation or a kit.
- 7. A kit for auxiliary diagnosis of renal fibrosis, which is characterized by comprising a detection reagent of KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragment of KDM5B protein.
- 8. The kit of claim 7, wherein the detection reagent is a reagent for detecting the expression level of KDM5B gene or KDM5B mRNA or KDM5B protein or peptide fragment of KDM5B protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, northern Blot, western Blot, chip, high throughput sequencing platform, immunohistochemistry or enzyme linked immunosorbent.
- 9. The kit according to claim 7, wherein the detection reagent is an antibody that specifically binds to a KDM5B protein or a peptide fragment of a KDM5B protein, or a KDM5B mRNA primer that specifically amplifies a KDM5B gene or a KDM5B mRNA, or a probe that specifically detects a KDM5B gene or a KDM5B mRNA.
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| CN202410282823.1A CN117925827A (en) | 2024-03-13 | 2024-03-13 | Application of KDM5B in auxiliary diagnosis of renal fibrosis |
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| CN202410282823.1A CN117925827A (en) | 2024-03-13 | 2024-03-13 | Application of KDM5B in auxiliary diagnosis of renal fibrosis |
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