Application of MYO1C related reagent and kit

Abstract

The invention relates to the technical field of biology, in particular to application of a reagent related to MYO1C and a kit. The invention finds that MYO1C mRNA is obviously differentially expressed in uEVs of a T2DN patient, and verifies that the expression of MYO1C in T2DN is increased, so that the invention provides application of a reagent for detecting the expression quantity of MYO1C in preparation of a diabetic nephropathy detection product.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to applications of MYO1C-related reagents and a kit. Background Art

[0002] T2DN is one of the major microvascular complications of T2DM, characterized by progressive renal damage and decreased renal function. The renal molecular pathology of diabetic nephropathy (DN) is multifaceted, involving various biochemical pathways and genetic factors. In recent years, with the development of molecular biology, especially the improvement of molecular pathology, the mechanism of the occurrence and development of DN has gradually become clear. As a complex metabolic disease, its development involves the interaction and regulation of multiple biomolecules. Under the high glucose state of DN, the disorder of glomerular hemodynamics causes glomerular hyperfiltration. At the same time, the production of angiotensin II in the kidney constricts the efferent arterioles, leading to increased glomerular pressure and stimulating the expression of proinflammatory and profibrotic mediators; while multiple intracellular signaling pathways can lead to the continuous release of proinflammatory mediators, profibrotic factors and immune cell recruitment, which promotes renal damage through inflammation and fibrosis. Recent studies have emphasized the key role of podocyte dysfunction in the early stages of DN and its contribution to renal function damage. The early signs of DN, such as progressive proteinuria, are closely related to podocyte damage and GBM destruction, making DN a disease with "podocyte disease" as a prominent feature. Therefore, in order to inhibit the development of DN, it is necessary to clarify the relationship between DN and biomolecules. Summary of the invention

[0003] In order to solve the above problems, the present invention provides applications of MYO1C-related reagents and a kit.

[0004] The use of a reagent for detecting the expression of MYO1C in the preparation of a diabetic nephropathy detection product. The NCBI gene number of MYO1C is 4641.

[0005] Preferably, the reagent for detecting the expression level of MYO1C includes a reagent for detecting the expression level of MYO1C in a sample using sequencing technology, probe hybridization technology, gene chip technology or fluorescent quantitative PCR technology.

[0006] Preferably, the reagent for detecting the expression level of MYO1C is the primer sequence shown in SEQ ID NO.1-2.

[0007] A diagnostic kit comprising the reagent.

[0008] Use of the reagent for inhibiting the expression of MYO1C in the preparation of a drug for treating diabetic nephropathy.

[0009] Preferably, the agent for inhibiting MYO1C expression includes MYO1C-siRNA-1, MYO1C-siRNA-2 or MYO1C-siRNA-3; The sense strand of MYO1C-siRNA-1 is 5′-CCCAUUAUGAGCCAGUGCUUU-3′, and the antisense strand is 5′-AAAGCACUGGCUCAUAAUGGG-3′; The sense strand of MYO1C-siRNA-2 is 5′-GCAGAGGAUUGAUUACGCCAA-3′, and the antisense strand is 5′-UUGGCGUAAUCAAUCCUCUGC-3′; The sense strand of MYO1C-siRNA-3 is 5′-UGUAGCUCAAAGAAUCCCAUU-3′, and the antisense strand is 5′-AAUGGGAUUCUUUGAGCUACA-3′.

[0010] Preferably, the agent for inhibiting MYO1C expression further comprises an adenovirus vector.

[0011] Preferably, the agent for inhibiting MYO1C expression is a p38 inhibitor.

[0012] Preferably, the p38 inhibitor is SB 203580.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention finds MYO1C mRNA It was significantly differentially expressed in uEVs of T2DN patients, and it was verified that MYO1C expression was increased in T2DN. Therefore, the use of reagents for detecting MYO1C expression in the preparation of diabetic nephropathy detection products was proposed.

[0014] Based on the discovery that MYO1C expression is increased in T2DN, the present invention further studies and finds that knockdown Myo1c It can reduce renal inflammatory response and pathological damage in diabetic nephropathy mice. AAV podocyte-specific knockdown of MYO1C can reduce renal damage in diabetic nephropathy mice. Administration of p38 inhibitors can alleviate the damage of MYO1C overexpression to normal podocytes. Therefore, the use of reagents that inhibit MYO1C expression in the preparation of drugs for the treatment of diabetic nephropathy is proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1For CCK-8 detection of the effects of high glucose and high fat on HPC cell activity, HPC were treated with BSA (containing 28.8 mM mannitol, CON group) or different concentrations of PA (30 mM glucose, HG+PA group) for different time periods, “*” indicates comparison with the CON group at 48 h, “#” indicates comparison between the two designated groups, nsP ≥ 0.05, **P < 0.01, ##P < 0.01, data from n = 3 independent experiments.

[0016] Figure 2 Characterization and identification of HPC-derived EVs. A is a representative TEM image of uEVs (bar = 100 nm on the right); B is a histogram of NanoFCM particle size analysis of uEVs; B is a fluorescence scatter plot of CD9 analyzed by NanoFCM of uEVs; C is the identification of EVs markers by Western blot.

[0017] Figure 3 RT-qPCR expression verification of differential RNA in podocyte-derived EVs, A is the expression level of hsa-miR-21-5p; B is the expression level of hsa-miR-22-3p; C is the expression level of hsa-miR-378a-3p, A, B, and C are all relative quantifications with miR-125b-5p as reference; D is the expression level of MYO1C mRNA; E is the expression level of SP100 mRNA, D and E are all relative quantifications with GAPDH as reference, data are from n=3 independent experiments, and the inter-group comparison was performed using the one-way ANOVA with Tukey's multiple comparison test.

[0018] Figure 4 RT-qPCR expression verification of differential RNA in podocytes, A is the expression level of hsa-miR-21-5p; B is the expression level of hsa-miR-22-3p; C is the expression level of hsa-miR-378a-3p, A, B, and C are all relative quantifications with miR-125b-5p as reference; D is the expression level of MYO1C mRNA; E is the expression level of SP100 mRNA, D and E are all relative quantifications with GAPDH as reference, data are from n=3 independent experiments, and the inter-group comparison was performed using the one-way ANOVA with Tukey's multiple comparison test.

[0019] Figure 5Figure 2 shows the normal light microscopy bright field image of human podocytes induced by high glucose and high fat stimulation, with MYO1C overexpressed. Bar = 50 μm. B shows the expression of podocyte marker proteins NPSH2, SYNPO, WT-1, inflammatory factor protein TNF-α, apoptosis-related protein cleave-Caspased3 and MYO1C in HPCs detected by Western blotting, with GAPDH as the internal reference. NG: normal culture group, HM: hypertonic control group, HGHF: high glucose and high fat group. C shows the transcriptional expression levels of MYO1C, SYNPO, NPHS2 and TNF-α mRNA detected by RT-qPCR, with GAPDH as the reference. -ΔΔCT Relative quantification, data are from n = 4 independent experiments.

[0020] Figure 6 Immunofluorescence experiment to detect the expression of SYNPO and MYO1C in HPC cells under different treatment groups. Immunofluorescence staining was used to detect the expression level and co-localization of SYNPO (red) and MYO1C (green), bar = 20 μm.

[0021] Figure 7 Figure 2 shows the general condition and serum biochemical test of mice in each group. A shows the weight change of mice in db / m and db / db+HFD groups tested at 8, 10, 14 and 18 weeks of age, respectively; B shows the blood glucose change of mice in db / m and db / db+HFD groups tested at 8, 10, 14 and 18 weeks of age, respectively; C shows the change of urine microalbumin to urine creatinine ratio (UACR) detected by collecting urine of mice in db / m and db / db+HFD groups at 8, 10, 14 and 18 weeks of age; D shows the change of kidney weight to body weight ratio (KW / BW) detected by collecting kidney tissues after mice were killed at 18 weeks of age; EH shows the change of urea nitrogen (E), serum creatinine (F), triglycerides (G) and total cholesterol (H) detected by collecting serum after mice were killed at 18 weeks of age; n=8 in each group, data are expressed as mean ± standard error, and Mann-Whitney U test, ns, P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

[0022] Figure 8The renal tissue staining pathology, histological scoring and electron microscopic analysis of mice, A is the PAS pathological staining of renal tissue of each group of mice; B is the MASSON pathological staining of small kidney tissue of each group, bar=20μm; C is the number of podocytes and foot process morphology observed under transmission electron microscopy, bar=2μm; D is the average glomerular tuft area of ​​each group of mice; E. The percentage of PAS-positive area of ​​renal tissue to the area of ​​glomerular tuft; F. The average glomerular basement membrane (GBM) thickness of each group of mice under electron microscopy; G. The length of GBM was measured and the number of foot processes of GBM per μm was calculated to evaluate the degree of foot process disappearance, n=5-6 in each group, and the one-way ANOVA with Dunnett's multiple comparison test was used for inter-group comparison.

[0023] Fig. 9 Figure 6 ELISA detection of inflammatory factors in mouse renal tissue homogenate. A. Mice were killed at 18 weeks of age, and renal tissues were collected. The macroscopic size of kidneys of db / m and db / db+HFD mice was compared; BF. Mice were killed at 18 weeks of age, and renal tissues were collected. ELISA was used to detect the expression levels of inflammatory factors MCP-1 (B), TNF-α (C), IL-1β (D), IL-18 (E) and IL-6 (F) in tissue homogenates of the two groups of mice. Data are expressed as mean ± standard error. Mann-Whitney U test was used for comparison among the groups, ns, P≥0.05, *P<0.05, **P<0.01, ***P<0.001.

[0024] Fig.10 Immunohistochemistry and Western blot were used to detect the expression of MYO1C in the kidneys of diabetic nephropathy mice. A is the immunohistochemical staining of MYO1C in renal tissue. Glomerulus is the glomerular area, bar=10μm; Tubulointerstitium is the tubule and its interstitial area, bar=20μm; B is the immunohistochemical semi-quantitative analysis of MYO1C, "*" indicates that the staining of the glomerular area of ​​the db / db+HFD group mice is compared with the glomerular area of ​​the db / m control group, "#" indicates that the staining of the tubular and interstitial area of ​​the db / db+HFD group mice is compared with the tubular and interstitial area of ​​the db / m control group, and the Mann-Whitney U test was used for comparison between the groups, *P<0.05, **P<0.01, ##P<0.01; C is Western The protein expression of MYO1C was analyzed by immunoblotting, with GAPDH as the internal reference; D is the semi-quantitative analysis result of MYO1C protein band, the data are expressed as mean ± standard error, and the inter-group comparison was performed by one-way ANOVA with Dunnett's multiple comparison test, n=6-8 per group.

[0025] Fig.11MYO1C is overexpressed in the kidneys of diabetic mice, accompanied by podocyte damage and increased p-p38. A is the Western blot analysis of MYO1C, WT-1, NPHS2, Desmin, p38, and p-p38 protein expression, with GAPDH as the internal reference; B is the semi-quantitative analysis of protein bands; C is the RT-qPCR experiment to detect the expression levels of Myo1c, Tnf-α, and Nphs2 mRNA, with Gapdh as the reference to calculate 2 -ΔΔCT Relative quantification; data are expressed as mean ± standard error, and the groups were compared using the Mann-Whitney U test; ns, P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; n = 6-8 per group.

[0026] Fig.12 To detect the expression of MYO1C in mouse kidney tissue by immunofluorescence, the expression level of MYO1C (green) was detected by immunofluorescence staining of paraffin sections of mouse kidney tissue, and the cell nuclei were labeled with DAPI (blue), bar=20μm.

[0027] Fig.13 Figure 2 shows the expression of MYO1C in renal tissue of patients with diabetic nephropathy. A shows, from left to right, podocyte changes observed under electron microscope (TEM), PAS pathological staining of renal tissue paraffin (PAS), immunofluorescence staining of frozen sections of renal tissue for MYO1C (green), immunofluorescence nucleus DAPI (blue), immunofluorescence merge (Merge), and immunohistochemical staining of renal tissue paraffin for MYO1C (IHC); bar=2μ in TEM, bar=20μm in other images; B shows the ratio of integrated optical density (IOD) of semi-quantitative MYO1C positive staining by immunohistochemistry to staining area; C shows the expression level of MYO1C mRNA detected by RT-qPCR, calculated with GAPDH as reference. -ΔΔCT Relative quantification; data are expressed as mean ± standard error, and the inter-group comparison was performed using the Mann-Whitney U test, n=6-8.

[0028] Fig.14 It shows that transfection of siRNA can knock down the expression level of MYO1C in podocytes. A is the RT-qPCR experiment to detect the expression level of each group MYO1C mRNA The expression level of GAPDH A was used as a reference to calculate the relative quantification of 2-ΔΔCT; data were expressed as mean ± standard error, and the groups were compared using one-way ANOVA with Dunnett's multiple comparison test; B was detected by Western blot to detect the protein expression of MYO1C in HPCs of each group, with GAPDH as the internal reference, n = 4 independent experiments.

[0029] Fig.15 It shows that knocking down MYO1C by transfection with siRNA can protect podocytes from injury. A is the expression of related proteins in HPCs of each group detected by Western blot, with GAPDH as the internal reference. B is the semi-quantitative analysis result of protein bands. The data are expressed as mean ± standard error. "*" indicates comparison with NG+siControl group, "#" indicates comparison with HG+siControl group. Mann-Whitney U Test; ns, P ≥ 0.05, *P < 0.05, ** P< 0.01, #P<0.05, ## P< 0.01; C is the expression level of MYO1C (green) detected by cell immunofluorescence staining, the cell nucleus was marked with DAPI (blue), and the cytoskeleton (red) was detected by F-actin staining, bar=20μm, n=3 independent experiments.

[0030] Fig.16 It shows that AAV knockdown of MYO1C can alleviate renal function damage and proteinuria levels in DN mice. A is a schematic diagram of intervention using AAV knockdown of MYO1C: 10-week-old db / db Mice were randomly divided into groups and injected once via tail vein with MYO1C knockdown AAV containing podocyte-specific promoter or empty control. The body weight, blood glucose and UACR of mice were measured every two weeks. The 8th week after injection was taken as the observation endpoint. The mice were euthanized and serum and kidney tissue samples were collected. The body weight (B), blood glucose (C) and UACR (D) levels of mice in each group were measured at 0, 2, 4, 6 and 8 weeks after AAV intervention (n=10). At the observation endpoint of the 8th week after AAV intervention, the kidney weight to body weight ratio (E), serum BUN (F) and Scr (G) levels of mice in each group were measured. Data are expressed as mean ± SD. The inter-group comparison was performed using one-way ANOVA with Dunnett's multiple comparison test; *** P< 0.001.

[0031] Fig.17 The expression of MYO1C and other target proteins in each group of mice are shown. A is the immunohistochemical staining of MYO1C in the kidney tissue of each group of mice, and B is the semi-quantitative immunohistochemical analysis of MYO1C. "*" indicates AAV- Myo1c Glomerular region staining of mice in group db / db The glomerular areas of the two groups were compared, and the inter-group comparisons were performed using one-way ANOVA with Dunnett's multiple comparison test, *** P<0.001; C is the expression of MYO1C and other proteins analyzed by Western blot, with GAPDH as the internal reference; D is the semi-quantitative analysis result of protein bands, data are expressed as mean ± standard error, and the inter-group comparison was performed using one-way ANOVA with Dunnett's multiple comparison test, bar=20μm; n=6-10 per group.

[0032] Fig.18 Figure 4: ELISA detection of inflammatory factors in renal tissue homogenates of mice in each group. AE means that mice were killed at the end point of observation and renal tissues were collected. ELISA was used to detect the expression levels of inflammatory factors MCP-1 (A), TNF-α (B), IL-1β (C), IL-6 (D) and IL-18 (E) in tissue homogenates of the three groups of mice. The data are expressed as mean ± standard error. The inter-group comparison was performed using one-way ANOVA with Dunnett's multiple comparison test. n=10 per group.

[0033] Fig.19 The results showed that AAV knockdown of MYO1C could alleviate renal pathology and podocyte injury in DN mice. A is the PAS pathological staining of renal tissue of each group of mice (bar=20μm) and the number of podocytes and foot process morphology observed under TEM microscope (bar=2μm); B is the average glomerular tuft area of ​​each group of mice; C is the percentage of PAS-positive area of ​​renal tissue to the area of ​​glomerular tuft; D is the average glomerular basement membrane thickness of each group of mice under electron microscope; E is the measurement of basement membrane length and calculation of the number of foot processes per μm to evaluate the degree of foot process disappearance, n=6-10 in each group, and the inter-group comparison was performed using one-way ANOVA with Dunnett's multiple comparison test.

[0034] Fig. 20 The results showed that adenovirus-mediated overexpression of MYO1C could cause podocyte damage. A was the observation of MYO1C overexpression adenovirus transfection by cell immunofluorescence. B was the detection of MYO1C, NPHS2, WT-1, and Cleaved-capased-3 protein expressions in HPCs of each group by Western blot. NG: normal sugar culture group; NG+Ad-control: adenovirus empty load control group; NG+Ad-MYO1C: MYO1C adenovirus transfection group; bar=200μm.

[0035] Fig.21It showed that the p38 inhibitor SB 203580 could prevent podocyte injury caused by overexpression of MYO1C. A was detected by Western blot to detect the protein expression of MYO1C, SYNPO, p38, p-p38, p-CREB, and TNF-α in HPCs of each group, with GAPDH as the internal reference. B was the semi-quantitative analysis result of protein bands. The data were expressed as mean ± standard error. "*" indicated that compared with the NG group, "#" indicated that compared with the NG+Ad-control group. The Mann-Whitney U Test; ns, P ≥ 0.05, *P < 0.05, ** P< 0.01, #P<0.05, ## P< 0.01; n = 3 independent experiments.

[0036] Fig. 22 It showed that p38 agonist could partially prevent the protective effect of siRNA knockdown of MYO1C on podocyte injury. Western blot was used to detect the expression of MYO1C, SYNPO, p38, p-p38, p-CREB, and Cleaved-capased-3 proteins in HPCs of each group, with GAPDH as the internal reference. NG: normal sugar culture; HGHF: high sugar and high fat; siCtrl: siRNA empty control; siRNA: MYO1C-siRNA; p38 agonist: p38 agonist Dehydrocorydaline, n=3 independent experiments.

[0037] Fig.23 Adenoviral vector map. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0039] MYO1C is a member of the myosin superfamily and is involved in a series of cellular processes. However, whether it plays an important role in the pathogenesis and pathology of diabetic nephropathy, especially in the mechanism of podocyte injury, has not been reported. The present invention studies the relationship between MYO1C and diabetic nephropathy.

[0040] Example 1 The gene sequences used in the present invention are shown in Table 1: Table 1 Gene sequences 1. Verification of secretion of EVs and differential RNA expression in human podocytes under high glucose and high fat conditions The present invention discovered and verified uEVs-derived RNA with differential expression in DN patients, among which MYO1C mRNA Significantly differentially expressed in uEVs of T2DN patients.

[0041] (1) Establishment of a high-glucose and high-fat model of human podocytes cultured in vitro The present invention adds high-fat stimulation (palmitic acid, PA) to 30mM high glucose (HG) to construct DN in vitro and in vivo models. First, a human podocyte model was constructed by adding high-fat stimulation to the high-glucose model. Cell viability was tested at three stimulation time points: 12h, 24h, and 48h. The CCK-8 results were as follows: Figure 1 As shown, the cell activity of the HG+150μM PA, HG+300μM PA, and HG+600μM PA groups decreased significantly, with statistically significant differences compared with the control group ( P <0.01). Since the cell activity was too low at 300μM and 600μM concentrations, 30mM HG + 150μM stimulation for 48h was finally selected as the condition for subsequent experiments.

[0042] (2) Extraction and characterization of extracellular vesicles from human podocytes cultured in vitro TEM and NanoFCM analysis showed that the extracted podocyte-derived extracellular vesicles (HPC-driven EVs, hEVs) samples had cup-shaped vesicles with a characteristic distribution size of about 60-90 nm ( Figure 2 A, B). Western blot confirmed that the enriched hEVs samples expressed known EVs protein markers CD9, Alixs, and TSG101, while the negative control endoplasmic reticulum protein calnexin was expressed at a low level ( Figure 2 C).

[0043] (3) Differential RNA expression in extracellular vesicles secreted by podocytes and in podocytes themselves validates HPC-EV NG group: normal glucose culture group containing 5 mM glucose.

[0044] HM group: hyperosmotic control group containing 28.8 mM mannitol (Highmannitol, HM).

[0045] HGHF group: High glucose and high fat (HGHF) group containing 30 mM glucose and 150 μM palmitic acid.

[0046] Taqman probe RT-qPCR was used to verify the expression of candidate RNA in hEVs. Figure 3 The results showed that among the five candidate RNAs, MYO1C The expression trend of mRNA in hEVs was consistent with that in uEVs, that is, compared with the control group, the expression of mRNA in hEVs of the HGHF 48h group was significantly higher than that of the control group. MYO1C mRNA Significantly increased ( Figure 3 D, P= 0.0014)

[0047] Furthermore, the Taqman probe method RT-qPCR was used to verify the expression of the candidate RNA in HPC cells (Human podocyte cells). Figure 4 The results showed that compared with the control group, the HPCs in the HGHF 24h and HGHF 48h groups MYO1C mRNA Both significantly increased ( P= 0.0068; P= 0.0001, Figure 4 D).

[0048] 2. MYO1C is involved in podocyte injury caused by high sugar and high fat High glucose and high fat stimulation induces podocyte morphological changes, decreased expression of podocyte marker proteins, and overexpression of MYO1C like Figure 5 A, Ordinary light microscopy showed that HGHF stimulation induced shrinkage of HPC cells, with obvious damage. Western blot results showed that MYO1C was highly expressed in the HGHF group, HGHF induced downregulation of the expression of HPC podocyte marker proteins NPHS2, SYNPO, and WT-1, and upregulation of the expression of inflammatory factor TNF-α and apoptosis-related protein Cleave-caspased3 ( Figure 5 B). RT-qPCR further confirmed that the HGHF group MYO1C mRNA The expression of SYNPO and NPHS2 was increased (P<0.001), accompanied by decreased expression of SYNPO and NPHS2 and increased expression of TNF-α ( Figure 5 C). Cell immunofluorescence was consistent with the above results. There was no difference in the expression of SYNPO and MYO1C between HM and NG groups. Compared with NG, the expression of SYNPO in HGHF was significantly decreased, and the expression of MYO1C was significantly increased. Figure 6 shown.

[0049] 3. High-fat diet db / db Podocyte injury and MYO1C overexpression in diabetic nephropathy mice (1) High-fat feeding at different ages db / db General condition of mice The 5-week-old SPF male db / db Mice (n=24) and db / m Mice (n=10) were purchased from Jiangsu Jicui Pharmaceutical Technology Co., Ltd. db / db Mice were fed a high-fat diet (HFD) containing 60 kcal% fat. db / db +HFD; db / m Mice were fed a normal diet.

[0050] To further detect the expression of MYO1C in diabetic nephropathy in vivo, high-fat fed rats were used to db / db ( db / db +HFD) mice to establish a T2DN mouse model of podocyte injury. Figure 7 As shown, db / m Compared with the control group, 10-18 weeks old mice db / db +HFD group mice had a significant increase in body weight ( P< 0.01), blood glucose and UACR increased ( P< 0.05). Compared with the control group, the 18-week-old mice db / db +HFD group mice had a decreased kidney weight to body weight ratio ( P< 0.05), serum biochemistry results showed that serum BUN was elevated ( P< 0.05), Scr increased ( P< 0.001) and serum TG and CHO increased significantly ( P< 0.001).

[0051] (2) High-fat diet db / db Mouse kidneys show typical pathological manifestations of diabetic nephropathy The results of PAS and Masson staining of kidney tissue sections show that db / db The mice in the +HFD group showed obvious glomerular hypertrophy, mesangial cell proliferation, increased mesangial matrix, accompanied by glomerular lobulation and mild sclerosis ( Figure 8 A, B); quantitative evaluation shows db / db The average glomerular tuft area of ​​+HFD mice was greater than db / m Control group ( Figure 8 D), the percentage of PAS-positive area to glomerular tuft area was also greater than db / m Control group ( Figure 8 E). Electron microscopy shows that with the increase of age, db / db The kidneys of mice in the +HFD group showed severe podocyte foot process fusion, decreased podocyte number, and obvious basement membrane thickening ( Figure 8 C); quantitative evaluation shows that 18w db / db The mean glomerular basement membrane thickness of the mice in the +HFD group was significantly higher than that in the db / m Control group ( P< 0.001, Figure 8 F), the average number of podocytes per micrometer was significantly less than db / m Control group ( P< 0.001, Figure 8 G). Fig. 9 The results of tissue homogenate ELISA showed that compared with the control group mice, db / db +HFD group mice renal tissue homogenate inflammatory factor MCP-1 ( P< 0.01)、TNF-α( P< 0.001) and IL-1β ( P < 0.05) was significantly increased. In summary, the results show that high-fat diet db / db The mouse kidneys showed typical pathological manifestations of diabetic nephropathy.

[0052] (3) High-fat diet db / db MYO1C is overexpressed in the kidneys of diabetic nephropathy mice like Fig.10 The immunohistochemical staining experiments shown in A and B found that MYO1C was significantly expressed in the glomeruli of renal tissue, and was also expressed in the tubules and interstitium; db / m Compared with the control group mice, db / db The expression of MYO1C in the glomeruli and tubulointerstitium of mice in the +HFD group was increased (all P< 0.05). Western blot results also showed that db / m Compared with the control group mice, db / db The expression of MYO1C protein in the kidney tissue of mice in the +HFD group was significantly increased (all P< 0.001, Fig.10 C, D).

[0053] Further Western blot analysis revealed that ( Fig.11 A, B), compared with control db / m Compared with the mice in group db / db + MYO1C was significantly overexpressed in mice in the HFD group ( P< 0.01); In addition, the enrichment analysis of the present invention found that MYO1C may be involved in apoptosis-related pathways. The present invention also found that the expression of some inflammation-related factors in the tissue homogenate of the disease group mice was increased. Therefore, the present invention chose to further detect the protein expression of p38 and phosphorylated p-p38 in the p38MAPK pathway closely related to apoptosis and inflammation. The results showed that db / db In the +HFD group, p38 activity was enhanced, that is, the expression of phosphorylated p-p38 increased, and the p-p38 / p38 value increased ( P<0.05), accompanied by increased expression of the injury marker myofibril protein Desmin ( P< 0.05), podocyte marker protein WT-1 ( P< 0.05) and NPHS2 ( P< 0.05) decreased; RT-qPCR transcription level detection obtained consistent results, db / db +HFD group mice kidney tissue Myo1c , Tnf-α Increased expression ( P< 0.001), Nphs2 Reduced expression ( P< 0.01, see Fig.11 C); Immunofluorescence also confirmed that MYO1C db / db Overexpression in glomeruli of HFD mice ( Fig.12 ).

[0054] 4. Expression of MYO1C in kidney samples of DN patients To further confirm the expression of MYO1C in DN, the present invention collected adjacent control renal tissues from 6 patients without diabetes who underwent total nephrectomy for renal parenchymal tumors, as well as renal tissues from 8 patients with T2DN who underwent renal puncture biopsy for immunohistochemistry and RT-qPCR detection of MYO1C expression, and immunofluorescence staining was used to observe the expression of MYO1C in DN kidneys. PAS and immunohistochemistry results showed that compared with the control group, T2DN patients had obvious glomerular hypertrophy, mesangial hyperplasia, and sclerosis. Under the electron microscope, podocyte foot process fusion and basement membrane thickening were observed, which were consistent with the pathological characteristics of DN; immunofluorescence staining and semi-quantitative detection results showed that MYO1C expression was increased in T2DN ( P =0.013, Fig.13 AB), and RT-qPCR confirmed MYO1C mRNA Increased expression in T2DN ( P =0.001, Fig.12 C).

[0055] Example 2 SB 203580 was used as p38 inhibitor, purchased from MedChemExpress (HY-10256), at a final concentration of 30 μM.

[0056] 1. Effect of MYO1C knockdown on podocyte injury (1) siRNA can knock down the expression of MYO1C in human podocytes cultured in vitro The siRNA is MYO1C-siRNA-1, MYO1C-siRNA-2 or MYO1C-siRNA-3, as follows: MYO1C-siRNA-1: the sense strand is 5'-CCCAUUAUGAGCCAGUGCUUU-3', recorded as SEQ ID NO.13, and the antisense strand is 5'-AAAGCACUGGCUCAUAAUGGG-3', recorded as SEQ ID NO.14; MYO1C-siRNA-2: the sense strand is 5'-GCAGAGGAUUGAUUACGCCAA-3', recorded as SEQ ID NO.15, and the antisense strand is 5'-UUGGCGUAAUCAAUCCUCUGC-3', recorded as SEQ ID NO.16; MYO1C-siRNA-3: the sense strand is 5'-UGUAGCUCAAAGAAUCCCAUU-3', recorded as SEQ ID NO.17, and the antisense strand is 5'-AAUGGGAUUCUUUGAGCUACA-3', recorded as SEQ ID NO.18; CY3 siRNA NC: the sense strand is 5'-UUCUCCGAACGUGUCACGU-3', recorded as SEQ ID NO.19, and the antisense strand is 5'-ACGUGACACGUUCGGAGAA-3', recorded as SEQ ID NO.20.

[0057] When using SEQ ID NO. 13 to SEQ ID NO. 20 in the present invention, base TT is connected at the 3' position.

[0058] The Ad adenovirus used to overexpress MYO1C in an in vitro human podocyte model was constructed and synthesized by Shanghai GeneCare Gene. Fig.23 .

[0059] The present invention confirms that the candidate gene MYO1C is expressed in in vitro cultured podocytes stimulated by high sugar and high fat and in high fat-fed rats. db / db Overexpression in mouse kidney tissue is accompanied by decreased expression of podocyte marker proteins, podocyte damage, increased apoptosis, and increased proinflammatory cytokines. In order to explore the pathogenic role of MYO1C in diabetic nephropathy, three MYO1C small interfering RNAs were first transfected in HPCs to try to knock down MYO1C and verify it. Fig.14 A shows: Compared with the transfection control siControl group, after adding MYO1C small interfering RNA siRNA1, siRNA2, and siRNA3, the three groups of cells MYO1C mRNA The levels were significantly decreased, and the difference was statistically significant compared with the control group ( P< 0.05), among which MYO1C siRNA-1 group MYO1C mRNA The most obvious expression level ( Fig.14Therefore, MYO1C siRNA-1 was selected to transfect HGHF-induced HPCs for subsequent experiments.

[0060] (2) Knockdown of MYO1C can restore the expression of podocyte marker proteins, reduce podocyte apoptosis, and maintain cytoskeletal integrity The cells were divided into 4 groups: a control group with siRNA added in a normal sugar culture environment (NG+siControl group), a group with siRNA-MYO1C knockdown added in a normal sugar culture environment (NG+siRNA group), a control group with siRNA added in a HGHF culture environment (HGHF+siControl group), and a group with siRNA-MYO1C knockdown added in a HGHF culture environment (HGHF+siRNA group).

[0061] Using siRNA to knock down MYO1C, the Western blot results show that ( Fig.15 AB), compared with the NG+siControl group, the expression of MYO1C in the NG+siRNA group was significantly decreased ( P< 0.05), but did not affect podocyte marker proteins SYNPO, NPHS2 and WT-1 (all P >0.01); the expression of MYO1C in the HGHF+siControl group was increased, and the expression of podocyte marker proteins SYNPO, NPHS2 and WT-1 was decreased (all P< 0.05), accompanied by increased expression of inflammatory factor TNF-α and apoptosis-related protein Cleaved-caspased3 (both P< 0.05); similarly, transfection MYO1C After siRNA, the expression of MYO1C in the HGHF+siRNA group was significantly decreased (all P<0.05). P< 0.05), compared with the HGHF+siControl group, the expressions of podocyte marker proteins SYNPO, NPHS2, and WT-1 were increased (all P< 0.05), the expression of inflammatory factor TNF-α and apoptosis-related protein Cleaved-caspased3 was decreased (both P< 0.05), confirmed knockdown MYO1C It can protect podocytes from damage caused by high sugar and high fat stimulation. At the same time, cell immunofluorescence results can also be observed ( Fig.15 C) Compared with the NG+siRNA group and the HGHF+siControl group, transfection of siRNA reduced the expression of MYO1C in the NG+siRNA group and the HGHF+siRNA group. Using phalloidin to label filamentous actin (F-actin), knockdown MYO1CIt did not affect the integrity of the podocyte cytoskeleton, while the integrity of the podocyte cytoskeleton in the HGHF+siRNA group was higher than that in the HGHF+siControl group.

[0062] 2. AAV podocyte-specific knockdown of MYO1C alleviates renal injury in diabetic nephropathy mice (1) AAV-Myo1c can reduce proteinuria and improve renal function in DN mice The above in vitro cell results confirmed that MYO1C knockdown could alleviate HGHF-induced podocyte injury. To further explore the role of MYO1C in kidney and podocyte injury in DN mice, the present invention conducted a high-fat-fed DN mouse model. db / db Mice were injected with a single tail vein injection of serum 2 / 9 type MYO1C knockdown AAV (AAV- Myo1c ) or virus empty vector control (AAV-Ctrl), and 8 weeks after intervention was used as the observation endpoint. Fig.16 A, After intervention, the mice were weighed and their blood glucose and UACR levels were tested every two weeks. At 8 weeks, the mice in each group were killed and kidney tissue samples were collected. Fig.16 As shown in B and C, after the intervention, db / db group, AAV-Ctrl group and AAV- Myo1c There was no statistically significant difference in body weight and blood glucose levels between the groups at each observation time point ( P> 0.05). db / db Compared with the AAV-Ctrl group, the AAV- Myo1c The UACR of mice in the group was significantly reduced at 6 weeks after intervention, and the reduction was more obvious at 8 weeks ( Fig.16 D), the differences were statistically significant ( P< 0.05). db / db Compared with the AAV- Myo1c There was no difference in the ratio of kidney weight to body weight between the groups ( Fig.16 E), while BUN levels decreased significantly ( P =0.018, Fig.16 F), Scr level decreased significantly ( P =0.001, Fig.16 G). These results suggest that podocyte-specific knockdown of MYO1C expression can alleviate the effects of high-fat feeding independently of blood glucose levels and body weight. db / db Proteinuria and renal impairment in mice.

[0063] (2) Knockdown Myo1c Can reduce renal inflammation and pathological damage in diabetic nephropathy mice Immunohistochemistry and fluorescence staining showed that AAV- Myo1cThe expression of MYO1C in the 44 groups was significantly reduced. Fig.17 A, B), and db / db Compared with the control group, there was no significant difference in the expression of MYO1C in the AAV-Ctrl group, while Myo1c The expression of MYO1C in the glomeruli of mice in the group was significantly reduced, and the difference was statistically significant ( P< 0.001); Immunofluorescence results ( Fig.17 A) It can also be seen in AAV- Myo1c In the glomerular glomerulus of the mice, the expression of MYO1C (green) was reduced. db / db The expression of MYO1C in the renal tissue of mice was further verified by Western blot analysis. Fig.17 As shown in the results of C and D, AAV- Myo1c The expression level of MYO1C protein in the glomeruli of mice in group A was significantly lower than that in group B. db / db group and AAV-Ctrl group ( P< 0.05). In summary, podocyte specificity Myo1c -AAV for high-fat feeding db / db Knockdown of MYO1C in mouse kidney podocytes is effective.

[0064] From the results of immunoblotting experiments, we can further see that ( Fig.17 C, D), AAV-Ctrl group and db / db There was no significant difference in the expression of each protein detected compared with the db / db Compared with the AAV-Ctrl group, the AAV- Myo1c The p-p38 / p38 ratio of mice in the P< 0.01), indicating that p38 activity was reduced and the expression of downstream activated p-CREB was reduced ( P< 0.05), accompanied by an increase in the podocyte marker protein NPHS2 and a decrease in the expression of the apoptosis-related protein Cleaved-caspased-3 ( P< 0.05).

[0065] In addition, the results of tissue homogenate ELISA were Fig.18 As shown, db / db Compared with the mice in the AAV-Ctrl group, there were no significant differences in the levels of inflammatory factors MCP-1, TNF-α, IL-1β, IL-6, and IL-18 in the renal tissue homogenate of the mice in the AAV-Ctrl group (all P<0.05). P> 0.1); while AAV- Myo1c The inflammatory factor MCP-1 ( P=0.025), TNF-α ( P =0.004), and IL-6 ( P =0.026) was significantly reduced. Furthermore, the PAS results of kidney tissue sections showed that db / db The mice in the AAV-Ctrl and AAV-Ctrl groups showed obvious glomerular hypertrophy, mesangial cell proliferation, and mesangial matrix expansion, accompanied by glomerular lobulation and mild sclerosis ( Fig.19 A), while AAV- Myo1c The glomerular injury of the mice in the AAV- Myo1c The average glomerular tuft area of ​​mice in the group was less than db / db group and AAV-Ctrl group ( Fig.19 B), the percentage of PAS-positive area to glomerular tuft area was also less than db / db group and AAV-Ctrl group ( Fig.19 C). It can be seen under electron microscope. db / db The kidneys of mice in the AAV-Ctrl and AAV-Ctrl groups showed severe podocyte foot process fusion, decreased podocyte number, and obvious basement membrane thickening ( Fig.19 A); quantitative evaluation showed that AAV- Myo1c The average glomerular basement membrane thickness of mice in group db / db group and AAV-Ctrl group control ( Fig.19 D ，P< 0.001), the average number of podocytes per micrometer was significantly higher db / db group and AAV-Ctrl group control ( Fig.19 E ，P< 0.001). In summary, the above results show that podocyte-specific AAV knockdown Myo1c Can reduce high-fat diet db / db Renal inflammation, DN pathology, and podocyte injury in mice.

[0066] 3. MYO1C-p38 protein interaction prediction The AlphaFold prediction model of the full-length three-dimensional protein structure of MYO1C was obtained using UniProt and used as the receptor protein structure file. In addition, the experimentally confirmed ChainA structure of p38 was selected from the database as the ligand protein structure file. Protein-protein docking studies were performed using the Hdock tool. The results showed that the docking score of the best binding model, model_1, was -299.79 and the confidence score was 0.9524. The binding process of the two proteins was displayed in the form of Surface.

[0067] To further study the binding interface of the protein-protein complex, the results were imported into the LigPlot tool to generate a residue-residue 2D interaction map showing the entire interface. There are 7 pairs of hydrophobic interactions, 18 pairs of hydrogen bonds, and 4 pairs of salt bridges between the two proteins. There are possible hydrophobic interactions between the 633 and 644 sites of the receptor MYO1C protein and the known phosphorylation site 182 of the ligand p38 protein. At the same time, there is the possibility of hydrogen bonding between the 636 residue of the MYO1C protein and the known phosphorylation site 180 of the ligand p38 protein. In addition, using the in vitro podocyte model stimulated by high sugar and high fat and the immunoprecipitation technique, the expression of p38 protein can be detected after immunoprecipitating MYO1C with a specific antibody, further confirming the possible binding between the two.

[0068] 4. MYO1C overexpression may cause podocyte damage through p38 / p-CREB (1) Overexpression of MYO1C in HPCs using adenovirus transfection can cause podocyte damage After transient transfection of HPC with adenovirus, fluorescence microscopy revealed that the appropriate transfection efficiency was achieved at the selected titer ( Fig. 20 A). Western blot results show ( Fig. 20 B): Compared with the NG group, the expression of MYO1C and podocyte marker proteins in the NG+AD-control empty vector group was not affected; while the expression of MYO1C in the NG+Ad-MYO1C virus group was significantly increased, accompanied by a significant decrease in the expression of slit diaphragm-related protein NPHS2 and podocyte marker protein WT-1, and an upregulated protein expression level of Cleavedcaspase-3.

[0069] (2) Administration of p38 inhibitors can alleviate the damage of MYO1C overexpression to normal podocytes The cells were divided into 4 groups: normal sugar environment culture control group (NG group), Ad empty control group (NG+Ad-Control group), Ad-MYO1C overexpression group (NG+Ad-MYO1C group) and Ad-MYO1C overexpression plus SB203580 stimulation group (NG+Ad-MYO1C+SB203580 group).

[0070] In order to explore whether the p38 MAPK pathway plays a role in the relationship between MYO1C and podocyte injury, this part of the study used the p38 inhibitor SB 203580 for intervention. SB 203580 is an inhibitor of p38MAPK, which can inhibit the autophosphorylation activity of p38 and the phosphorylation activity of substrates, but does not inhibit the phosphorylation of p38 by upstream kinases. Western blot results showed ( Fig.21): Compared with the NG group, the expression of p38, p-p38 and downstream transcription factor protein p-CREB in the NG+Ad-control empty vector group was not affected; while the expression of p-p38 in the NG+Ad-MYO1C virus group was significantly upregulated ( P< 0.05), accompanied by a significant increase in the expression of p-CREB ( P< 0.05) and TNF-α expression was increased ( P< 0.05). Compared with the NG+Ad-MYO1C virus group, after the addition of the p38 inhibitor SB 203580, the expression of p-p38 / p38 in the NG+Ad-MYO1C+SB 203580 group was reduced, and the expression of downstream transcription factor proteins p-CREB and TNF-α was reduced ( P< 0.05), but did not affect the expression of MYO1C. The above results indicate that the podocyte injury caused by overexpression of MYO1C may be partly mediated by p38 / p-CREB.

[0071] 5. p38 agonist can prevent the alleviating effect of MYO1C knockdown on the damage of podocytes induced by high glucose and high fat The cells were divided into 6 groups: a control group with siRNA added in a normal sugar culture environment (NG+siCtrl group), a group with siRNA-MYO1C knockdown added in a normal sugar culture environment (NG+siRNA group), a group with siRNA-MYO1C and Dehdrocorydaline stimulation added in a normal sugar culture environment (NG+siRNA+p38 agonist group), a control group with siRNA added in a HGHF culture environment (HGHF+siCtrl group), a group with siRNA-MYO1C knockdown added in a HGHF culture environment (HGHF+siRNA group), and a group with siRNA-MYO1C and Dehdrocorydaline stimulation added in a HGHF culture environment (HGHF+siRNA+p38agonist group).

[0072] Previous experiments have confirmed that transfection of MYO1C-siRNA can reduce MYO1C overexpression caused by high sugar and high fat, and alleviate podocyte injury; to further understand whether the p38-related pathway plays a role in this process, this part of the study added the p38 agonist Dehydrocorydaline on the basis of transfection of siRNA. Western blot results showed ( Fig. 22): Compared with the NG+siControl group, the MYO1C expression in the NG+siRNA group and the NG+siRNA+p38 agonist group was significantly decreased; compared with the NG+siRNA group, after administration of p38 agonist, the p-p38 phosphorylated protein in the NG+siRNA+p38 agonist group increased, and the expression of the downstream transcription factor active p-CREB and the apoptosis-related protein Cleaved-caspased3 increased; on the other hand, under the stimulation of high glucose and high fat, the MYO1C expression in the HGHF+siControl group was upregulated, and the p-p38 phosphorylated protein was also significantly increased, accompanied by the increase of the downstream transcription factor p-CREB activity and the increase of the expression of Cleaved-caspased3. Compared with the HGHF+siControl group, the MYO1C expression in the HGHF+siRNA knockdown group was significantly reduced, accompanied by the alleviation of podocyte injury; after intervention with p38 agonist, compared with the HGHF+siRNA knockdown group, the MYO1C expression in the HGHF+siRNA+p38 agonist intervention group did not change significantly, but the expression of phosphorylated protein p-p38 increased, the expression of downstream transcription factors p-CREB and Cleaved-caspased3 increased, and the expression of podocyte marker SYNPO was downregulated. That is, intervention with p38 agonist prevented the protective effect of siRNA knockdown of MYO1C on podocyte injury caused by high glucose and high fat to a certain extent.

[0073] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0074] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of reagents for detecting MYO1C expression in the preparation of diabetic nephropathy detection products.

2. The use according to claim 1, characterized in that: Reagents for detecting the expression level of MYO1C include reagents for detecting the expression level of MYO1C in a sample using sequencing technology, probe hybridization technology, gene chip technology or fluorescent quantitative PCR technology.

3. The use according to claim 2, characterized in that: The reagent for detecting the expression level of MYO1C is the primer sequence shown in SEQ ID NO.1-2.

4. A diagnostic kit, characterized in that: The diagnostic kit comprises the reagent described in claim 3.

5. Use of an agent for inhibiting MYO1C expression in the preparation of a drug for treating diabetic nephropathy, characterized in that: The MYO1C is the MYO1C described in claim 1.

6. The use according to claim 5, characterized in that: The reagent for inhibiting MYO1C expression includes MYO1C-siRNA-1, MYO1C-siRNA-2 or MYO1C-siRNA-3; The sense strand of MYO1C-siRNA-1 is 5′-CCCAUUAUGAGCCAGUGCUUU-3′, and the antisense strand is 5′-AAAGCACUGGCUCAUAAUGGG-3′; The sense strand of MYO1C-siRNA-2 is 5′-GCAGAGGAUUGAUUACGCCAA-3′, and the antisense strand is 5′-UUGGCGUAAUCAAUCCUCUGC-3′; The sense strand of MYO1C-siRNA-3 is 5′-UGUAGCUCAAAGAAUCCCAUU-3′, and the antisense strand is 5′-AAUGGGAUUCUUUGAGCUACA-3′.

7. The use according to claim 6, characterized in that: The agent for inhibiting MYO1C expression also includes an adenovirus vector.

8. The use according to claim 5, characterized in that: The agent for inhibiting MYO1C expression is a p38 inhibitor.

9. The use according to claim 8, characterized in that: The p38 inhibitor is SB 203580.

10. The use according to claim 5, characterized in that: The reagent for inhibiting MYO1C expression is a serum 2 / 9 type adeno-associated virus with a specific NPHS2 promoter.

Images