Acute kidney injury biomarkers and their applications

Abstract

The present invention discloses an acute kidney injury biomarker and its application. The present invention first discovered that miR-323a-3p was differentially expressed in AKI model cells and normal HK-2 cells, specifically, miR-323a-3p was significantly highly expressed in AKI model cells; this indicates that miR-323a-3p can be used as a biomarker for AKI, for assisting the diagnosis or prognosis prediction of AKI, and provides a new approach for the diagnosis and prognosis prediction of AKI; at the same time, miR-323a-3p inhibitors can reduce the reactive oxygen content in cells by reducing the expression level of miR-323a-3p in AKI model cells, increase the mitochondrial membrane potential in cells, and then inhibit cell apoptosis and increase cell viability; this indicates that miR-323a-3p can be used as a biological target for the treatment of AKI, and provides new ideas and approaches for the treatment of AKI and mitochondrial damage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an acute kidney injury biomarker and application thereof. Background Art

[0002] Acute kidney injury (AKI) is considered a common clinical syndrome characterized by abnormalities in renal function and structure. AKI affects 30-60% of critically ill patients and has high morbidity and mortality, posing a huge clinical challenge worldwide.

[0003] More and more studies have shown that the burden of AKI has exceeded the acute phase, including recurrent AKI, increased risk of cardiovascular complications, and the development of chronic kidney disease. Mitochondria are organelles with many important functions and are an excellent source of energy for renal tubular cells. The reabsorption and secretion functions of the renal tubules rely on mitochondrial oxidative phosphorylation to provide energy. Studies have shown that mitochondrial damage is key to the progression of AKI. Therefore, improving mitochondrial function may be an effective intervention target for the treatment of AKI.

[0004] MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs that play a regulatory role in a variety of diseases. Recent studies have reported that miRNAs play an important regulatory role in the onset and development of AKI and mitochondrial damage, indicating that miRNAs can not only serve as a new way to explore the pathogenesis of AKI, but also may become a new target for the diagnosis, treatment and prognosis of AKI.

[0005] For example, the Chinese invention patent application with publication number CN112789356A discloses an acute kidney injury-specific biomarker, which is a MicroRNAs with low expression in patients with acute kidney injury: miRNA-5100; the technology also found that using a functional activity enhancer of miRNA-5100 to increase the expression of miRNA-5100 in the body can achieve the purpose of treating acute kidney injury.

[0006] MiR-323a-3p is also a type of MicroRNAs. Existing studies have found that MiR-323a-3p plays an important role in many types of diseases such as depression, osteosarcoma, pulmonary fibrosis, and breast cancer. However, there are currently no reports on the relationship between MiR-323a-3p and acute kidney injury. Summary of the invention

[0007] The purpose of the present invention is to provide an acute kidney injury biomarker and its application, which provides a new idea and new approach for the diagnosis and treatment of acute kidney injury.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0009] An acute kidney injury biomarker, the biomarker is miR-323a-3p.

[0010] The present invention discovered for the first time that miR-323a-3p is differentially expressed in acute kidney injury model cells and normal human renal cortical proximal tubule epithelial cells. Specifically, miR-323a-3p is significantly highly expressed in acute kidney injury model cells. This indicates that miR-323a-3p can be used as a biomarker for acute kidney injury to assist in the diagnosis or prognosis prediction of acute kidney injury, providing a new approach for the diagnosis and prognosis prediction of acute kidney injury.

[0011] Based on this, the present invention also provides the use of the above-mentioned acute kidney injury biomarker in the preparation of an acute kidney injury diagnostic reagent, and the use of the above-mentioned acute kidney injury biomarker in the preparation of an acute kidney injury prognosis prediction reagent.

[0012] Furthermore, the present invention also provides an acute kidney injury diagnostic reagent and an acute kidney injury prognosis prediction reagent, which can be a specific amplification primer of miR-323a-3p. As a specific embodiment, the present invention provides a group of specific amplification primers of miR-323a-3p, and the specific amplification primers include:

[0013] Upstream primer: 5′-TTACACGGTCGACCTCTGTC-3′;

[0014] Downstream primer: 5′-TGTCGTGGAGTCGGCAATTG-3′.

[0015] Furthermore, the present invention also provides an acute kidney injury diagnosis kit and an acute kidney injury prognosis prediction kit, wherein the kit contains the above-mentioned acute kidney injury diagnosis reagent or acute kidney injury prognosis prediction reagent.

[0016] At the same time, the present invention also provides the use of miR-323a-3p as a marker for human renal cortical proximal tubule epithelial cell damage.

[0017] Furthermore, the present invention also provides the use of a miR-323a-3p inhibitor in the preparation of a drug for treating acute kidney injury or a drug for treating human renal cortical proximal tubule epithelial cell injury;

[0018] And, the use of miR-323a-3p inhibitors as drugs for preventing and treating mitochondrial damage.

[0019] The present invention also found that the miR-323a-3p inhibitor can reduce the reactive oxygen content in the cells by reducing the expression level of miR-323a-3p in the acute kidney injury model cells, increase the mitochondrial membrane potential in the cells, and then inhibit cell apoptosis and increase cell viability; indicating that miR-323a-3p can be used as a biological target for the treatment of acute kidney injury, providing new ideas and approaches for the treatment of acute kidney injury and mitochondrial damage.

[0020] The miR-323a-3p inhibitor is a plasmid, virus, drug or other biological agent that can inhibit the expression of miR-323a-3p.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The present invention discovered for the first time that miR-323a-3p is differentially expressed in acute kidney injury model cells and normal human renal cortical proximal tubule epithelial cells. Specifically, miR-323a-3p is significantly highly expressed in acute kidney injury model cells. This indicates that miR-323a-3p can be used as a biomarker for acute kidney injury to assist in the diagnosis or prognosis prediction of acute kidney injury, providing a new approach for the diagnosis and prognosis prediction of acute kidney injury.

[0023] (2) The present invention also found that the miR-323a-3p inhibitor can reduce the reactive oxygen content in the cells by reducing the expression level of miR-323a-3p in the acute kidney injury model cells, increase the mitochondrial membrane potential in the cells, and then inhibit cell apoptosis and increase cell viability; indicating that miR-323a-3p can be used as a biological target for the treatment of acute kidney injury, providing new ideas and approaches for the treatment of acute kidney injury and mitochondrial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Comparison of the expression levels of miR-323a-3p in AKI model cells and normal HK-2 cells;

[0025] Among them, LPS refers to the AKI model cell group induced by lipopolysaccharide LPS, Control refers to the normal HK-2 cell group, and Relative expression refers to the relative expression level; the same below;

[0026] Figure 2 The effect of miR-323a-3p on cell viability in AKI model;

[0027] Among them, LPS+I represents the inhibition group transfected with MiR-323a-3p inhibitor plasmid, LPS+IC represents the inhibition reversal group transfected with inhibitor control plasmid; OD value represents OD value; the same below;

[0028] Figure 3 The effect of miR-323a-3p on cell apoptosis in AKI model (fluorescence microscopy observation results);

[0029] Figure 4 The effect of miR-323a-3p on cell apoptosis in AKI model (relative fluorescence intensity statistical results);

[0030] Among them, Relative fluorescence intensity (%) represents the relative fluorescence intensity (percentage) (with the AKI model cell group as a reference);

[0031] Figure 5 The effect of miR-323a-3p on intracellular reactive oxygen species in AKI model (fluorescence microscopy observation results);

[0032] Figure 6 The effect of miR-323a-3p on intracellular reactive oxygen species in AKI model (statistical results of relative levels of DCFH-DA dye);

[0033] Among them, Relative DCF fluorescence level (%) represents the relative level (percentage) of DCFH-DA dye (with the AKI model cell group as a reference);

[0034] Figure 7 The effect of miR-323a-3p on mitochondrial membrane potential of AKI model cells (observation results using laser scanning confocal microscopy);

[0035] Figure 8 The effect of miR-323a-3p on mitochondrial membrane potential of AKI model cells (statistical results of mitochondrial membrane potential);

[0036] Among them, Mitochondrial membrane potential represents mitochondrial membrane potential. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0038] Example 1 qRT-PCR experiment to detect the expression of miR-323a-3p in AKI model cells

[0039] Human renal cortical proximal tubule epithelial cells HK-2 were first cultured in a 6-well plate and treated with 50 μg / mL LPS for 24 hours to establish AKI model cells. The cells were then collected, and the cellular RNA was extracted using the TRIzol method, and then cDNA was synthesized using a reverse transcription kit. Finally, qRT-PCR experiments were performed using a qPCR kit and Real-time PCR System (U6 as an internal reference, nucleotide sequence number: NC_000015.10) to detect the expression of miR-323a-3p (nucleotide sequence number: NC_000014.9) in AKI model cells and normal HK-2 cells.

[0040] The primer information used for qRT-PCR is shown in Table 1.

[0041] Table 1

[0042]

[0043] The reaction system of qRT-PCR is shown in Table 2, and the reaction procedure is shown in Table 3.

[0044] Table 2 qRT-PCR reaction system

[0045]

[0046] Table 3 qRT-PCR reaction procedure

[0047] step temperature time Pre-denaturation 94℃ 30s transsexual 94℃ 5s Annealing / Extension 60℃ 30s Melting curve analysis 95℃ 15s 60℃ 10min 95℃ 15s 60℃ 15s

[0048] Note: Denaturation to annealing / extension phases were repeated for 40 cycles.

[0049] The experimental results of qRT-PCR are as follows Figure 1 shown.

[0050] Depend on Figure 1 It can be seen that miR-323a-3p is highly expressed in AKI model cells and lowly expressed in normal HK-2 cells, and there is a significant difference in the relative expression level of miR-323a-3p between AKI model cells and normal HK-2 cells (***P<0.001); this indicates that miR-323a-3p can be used as a biomarker for human renal cortical proximal tubule epithelial cell injury or acute kidney injury, and can be used to assist in the diagnosis or prognosis prediction of human renal cortical proximal tubule epithelial cell injury or acute kidney injury.

[0051] Example 2 CCK-8 assay to detect the effect of miR-323a-3p on cell viability in AKI model

[0052] The miR-323a-3p inhibitor plasmid and inhibitor control plasmid designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. were purchased, and the miR-323a-3p inhibitor plasmid and inhibitor control plasmid were transfected into HK-2 cells respectively. After 48 hours of transfection, the cells were cultured with LPS (50 μg / mL) for another 24 hours as the inhibition group and inhibition reversal group. At the same time, HK-2 cells transfected with empty plasmids were set as the AKI model cell group; then, CCK-8 reagent was added to the cells and incubated for 4 hours; finally, the optical density value at 450 nm was measured by a microplate reader to evaluate the viability of each group of cells; the test results are shown in Figure 2. Figure 2 shown.

[0053] Depend on Figure 2 It can be seen that the OD value of HK-2 cells in the AKI model cell group was lower after LPS induction, and the cell viability was lower; the OD value of the inhibition group HK-2 cells transfected with MiR-323a-3p inhibitor was significantly increased compared with the AKI model cell group after LPS induction (***P<0.001), and the OD value of the inhibition reversal group HK-2 cells transfected with inhibitor control was significantly decreased after LPS induction compared with the inhibition group, which was equivalent to the AKI model cell group; indicating that MiR-323a-3pin inhibitor can increase cell viability by inhibiting the expression of MiR-323a-3p in cells.

[0054] Example 3 TUNEL staining to detect the effect of miR-323a-3p on cell apoptosis in AKI model

[0055] MiR-323a-3p inhibitor plasmid and inhibitor control plasmid were constructed by the same method as in Example 2, and the MiR-323a-3p inhibitor plasmid and inhibitor control plasmid were transfected into HK-2 cells, respectively. After 48 hours, the cells were cultured with 50 μg / mL LPS for another 24 hours as the inhibition group and the inhibition reversal group. At the same time, HK-2 cells transfected with an empty plasmid were set as the AKI model cell group. Subsequently, the cells were fixed with paraformaldehyde fixative, permeabilized with immunostaining permeabilization solution, rinsed with phosphate buffer, and incubated with TUNEL staining solution. Finally, the apoptosis of cells in each group was observed under a fluorescence microscope. The results are shown in FIG. Figure 3 and Figure 4 shown.

[0056] like Figure 3 and Figure 4As shown in the figure, after LPS induction, the HK-2 cells in the AKI model cell group had a large number of apoptotic cells due to the high expression of MiR-323a-3p; while in the inhibition group cells transfected with MiR-323a-3p inhibitor plasmid, the expression of MiR-323a-3p was inhibited, and the number of apoptotic cells was significantly reduced compared with the AKI model cell group ( ++ P<0.01); in the inhibition reversal group cells transfected with inhibitor control plasmid, the function of MiR-323a-3p inhibitor was offset, resulting in the number of apoptotic cells being comparable to that of the AKI model cell group. The above experimental results indicate that miR-323a-3p inhibitor can inhibit apoptosis of AKI model cells.

[0057] Example 4 DCFH-DA staining to detect the effect of miR-323a-3p on intracellular reactive oxygen species in AKI model

[0058] MiR-323a-3p inhibitor plasmid and inhibitor control plasmid were constructed by the same method as in Example 2, and the MiR-323a-3p inhibitor plasmid and inhibitor control plasmid were transfected into HK-2 cells, respectively. After 48 hours, the cells were cultured with 50 μg / mL LPS for another 24 hours as the inhibition group and inhibition reversal group. At the same time, HK-2 cells transfected with an empty plasmid were set as the AKI model cell group. Subsequently, the cells were incubated with DCFH-DA dye for 30 minutes and washed with phosphate buffer. Finally, the changes in intracellular reactive oxygen species in each group were observed under a fluorescence microscope. The results are shown in FIG. Figure 5 and Figure 6 shown.

[0059] like Figure 5 and Figure 6 As shown, after LPS induction, the HK-2 cells in the AKI model cell group had a high level of intracellular reactive oxygen species due to the high expression of MiR-323a-3p; in the inhibition group cells transfected with the MiR-323a-3p inhibitor plasmid, the expression of MiR-323a-3p was inhibited, and the reactive oxygen species content was significantly reduced (++P<0.01); and in the inhibition reversal group cells transfected with the inhibitorcontrol plasmid, the function of the MiR-323a-3p inhibitor was offset, resulting in its reactive oxygen species content being comparable to that of the AKI model cell group. The above experimental results indicate that miR-323a-3p inhibitor can inhibit cell apoptosis by reducing the content of reactive oxygen species in AKI model cells.

[0060] Example 5 JC-1 staining to detect the effect of miR-323a-3p on mitochondrial membrane potential of AKI model cells

[0061] MiR-323a-3p inhibitor plasmid and inhibitor control plasmid were constructed by the same method as in Example 2, and the MiR-323a-3p inhibitor plasmid and inhibitor control plasmid were transfected into HK-2 cells respectively. After 48 hours, the cells were cultured with 50 μg / mL LPS for 24 hours as the inhibition group and inhibition reversal group. At the same time, HK-2 cells transfected with empty plasmid were set as the AKI model cell group; then, the cells were incubated with JC-1 dye for 20 minutes and washed with phosphate buffer. Finally, the changes in mitochondrial membrane potential of each group of cells were observed under a laser scanning confocal microscope; the results are shown in Figure 7 and Figure 8 .

[0062] like Figure 7 and Figure 8 As shown in the figure, after LPS induction, the mitochondrial membrane potential of HK-2 cells in the AKI model cell group was destroyed due to the high expression of MiR-323a-3p; while in the inhibition group cells transfected with MiR-323a-3p inhibitor plasmid, the mitochondrial membrane potential was significantly increased due to the inhibition of MiR-323a-3p expression ( + P<0.05); in the inhibition reversal group cells transfected with inhibitorcontrol plasmid, the function of MiR-323a-3p inhibitor was offset, resulting in the mitochondrial membrane potential in the cells being equivalent to that of the AKI model cell group. The above experimental results indicate that miR-323a-3p inhibitor can restore mitochondrial function by increasing mitochondrial membrane potential, thereby inhibiting cell apoptosis.

Claims

1. Application of reagents for detecting miR-323a-3p in the preparation of diagnostic kits for acute kidney injury.

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