Disease diagnosis and treatment with hsacircERI3 as target

Abstract

The invention belongs to the technical field of disease diagnosis and treatment, and particularly discloses disease diagnosis and treatment with hsacircERI3 as a target spot. The invention provides an application of a substance for detecting hsacircEIR3 in at least one of the following groups: preparing a product for diagnosing obstructive nephropathy; preparing a product for prognosis evaluation of obstructive nephropathy; a product for predicting the renal function recovery condition after obstruction of obstructive nephropathy is relieved is prepared; and preparing a product for evaluating the severity of obstructive nephropathy. It is found that the expression quantity of hsacircEIR3 in renal cortex tissue of an ON patient and the level of hsacircEIR3 in urine are obviously increased, and the level of hsacircEIR3 in urine is strongly related to the lateral residual renal function; the invention further finds that the overexpression of the hsacircEIR3 is helpful to slow down pathological changes of renal tubular epithelial cells and renal interstitial substances and inhibit renal fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of disease diagnosis and treatment, and in particular to disease diagnosis and treatment targeting hsa_circ_ERI3. Background Art

[0002] Obstructive nephropathy (ON) is often caused by upper urinary tract stone obstruction, and renal interstitial fibrosis is the late stage of chronic progression of ON, often followed by renal failure. The principle of treatment for ON is to make a clear diagnosis as early as possible and relieve the obstruction in time, but obstruction often has hidden symptoms and chronicity, with the characteristics of high incidence and low alertness. For patients diagnosed late, the judgment criteria for whether to remove the kidney or simply relieve the obstruction are still controversial. At present, there is no clear clinical guidance standard for this, and the recovery of renal function in each patient is also different, making it more difficult for doctors to make accurate judgments. Some patients still have irreversible renal damage after the obstruction is relieved. The reason is that the pathological process of chronic renal fibrosis has been initiated, but its regulatory mechanism has not been fully elucidated. The biomarkers currently used to diagnose and evaluate ON still have the disadvantages of low specificity and unstable test results, and drug treatment measures for ON also show limited therapeutic effects and side effects.

[0003] At present, some biomarkers have been used to evaluate renal injury caused by acute and chronic obstruction, including blood and urine neutrophil gelatinase-associated lipocalin (NGAL) levels, monocyte chemoattractant protein-1, kidney injury molecule-1, N-acetyl-bD-glucosaminidase, and cystatin C. Although they have certain sensitivity, their specificity is not high, and there is a lack of strong evidence to prove their value in predicting the recoverability of renal function. The most widely used serum creatinine level test cannot evaluate the situation of unilateral obstruction. Due to the lag and difficulty in judging the progression stage of ON, patients often suffer from renal atrophy and repeated urinary tract infections, mis-resection and missed renal resection, etc., which puts patients under great psychological, physiological and economic burdens. Therefore, it is very important to evaluate the degree of renal injury on the affected side, intervene early, and predict the recovery of renal function after the obstruction is relieved. Circular RNA (circRNA) is different from traditional linear RNA. CircRNA does not have a 5' end cap and a 3' end poly (A) tail. It is a non-coding RNA that forms a circular structure with a covalent bond. Studies have shown that circRNA has a closed circular structure and is widely present in various biological cells. It has the characteristics of high expression abundance, good conservation between species, and tissue and time-space specificity. Therefore, circRNA has broad prospects in the diagnosis of kidney disease and the development and application of its treatment methods.

[0004] Therefore, there is an urgent need to explore new highly sensitive and accurate diagnostic markers based on circRNA to assist in treatment decisions, and new therapeutic targets to intervene in the chronic progression of ON, so that doctors can more accurately and conveniently evaluate the relief of obstruction and the potential for renal function recovery in patients after surgery, and thus make a more accurate judgment on whether the surgery is successful. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides the use of a substance for detecting hsa_circ_EIR3.

[0006] The ERI1 Exoribonuclease Family Member 3 (ERI3) gene is expressed in the kidney and participates in a variety of metabolic processes. Its circular RNA: hsa_circ_EIR3 can regulate protein expression through a variety of pathways. The present invention found that the circular RNA (circbase ID: 0012138) hsa_circ_EIR3 of the ERI3 gene has good specificity and stability in the human kidney, and the expression of hsa_circ_EIR3 in the renal cortex tissue of ON patients is significantly increased compared with normal tissue; ON patients also have increased urinary hsa_circ_EIR3 levels during the course of the disease, and the hsa_circ_EIR3 level in urine and the residual renal function of the side show a significantly stronger correlation than the existing evaluation index NGAL level in urine and the residual renal function of the side; the ROC curve analysis results of the urinary hsa_circ_EIR3 level for the reproducibility judgment of renal function show that it can be used as a biomarker for judging the clinical outcome of ON renal fibrosis patients after surgery. The present invention further explored the role of hsa_circ_EIR3 in the regulation of ON renal fibrosis. The results unexpectedly found that overexpression of hsa_circ_EIR3 helps to slow down the pathological changes of renal tubular epithelial cells and renal interstitium, reduce ECM accumulation, and thus inhibit renal fibrosis, showing broad application prospects in the prevention and treatment of ON diseases.

[0007] The present invention also provides the use of hsa_circ_EIR3 or a substance that increases the abundance of hsa_circ_EIR3 in the preparation of a drug for preventing and / or treating obstructive nephropathy.

[0008] The invention also provides a pharmaceutical composition for treating obstructive nephropathy.

[0009] The invention also provides a diagnostic kit for obstructive nephropathy.

[0010] In a first aspect of the present invention, a substance for detecting hsa_circ_EIR3 is provided for use in at least one of (1) to (4);

[0011] (1) Preparation of products for the diagnosis of obstructive nephropathy;

[0012] (2) Prepare products for the prognosis assessment of obstructive nephropathy;

[0013] (3) Prepare a product for predicting the recovery of renal function after the obstruction is relieved in patients with obstructive nephropathy;

[0014] (4) Prepare a product for assessing the severity of obstructive nephropathy.

[0015] According to some embodiments of the present invention, the circbase ID of hsa_circ_EIR3 is hsa_circ_0012138.

[0016] According to a specific embodiment of the present invention, the present invention found that hsa_circ_EIR3 has good specificity and stability in human kidney, and has the potential to be used as a biomarker for detection; and found that the expression level of hsa_circ_EIR3 in the renal cortex tissue of ON patients was significantly increased compared with normal tissue, indicating that hsa_circ_EIR3 can be used as a marker for detecting ON; further experimental exploration found that ON patients will have an increase in the level of hsa_circ_EIR3 in urine during the course of the disease, and the level of hsa_circ_EIR3 in urine is significantly negatively correlated with the patient's lateral residual renal function, and the level of hsa_circ_EIR3 in urine and lateral residual renal function show a significantly stronger correlation than the existing evaluation index NGAL level in urine and lateral residual renal function; the present invention also studied the urine hs The ROC curve of a_circ_EIR3 level for reproducibility judgment of renal function showed that the judgment efficiency of hsa_circ_EIR3 was slightly higher than that of NGAL. Therefore, urine hsa_circ_EIR3 can be used as a biomarker to better judge the postoperative clinical outcome of patients with ON renal fibrosis, and has good application prospects. The present invention further explored the role of hsa_circ_EIR3 in the regulation of ON renal fibrosis. The results unexpectedly found that hsa_circ_EIR3 can be used as a protective factor to repair renal fibrosis, showing the effect of counteracting other factors that promote fibrosis. Its overexpression helps to slow down the pathological changes of renal tubular epithelial cells and renal interstitium, reduce ECM accumulation, and thus inhibit renal fibrosis. Therefore, regulating hsa_circ_EIR3 has broad application prospects in the prevention and treatment of ON diseases.

[0017] According to some embodiments of the present invention, the substance for detecting hsa_circ_EIR3 is a substance used in one or more detection techniques or methods selected from the following group: PCR, biochip method, Northern blotting method or RNA immunoprecipitation method.

[0018] According to some embodiments of the present invention, the substance for detecting hsa_circ_EIR3 is selected from at least one of: a PCR primer, a gene chip, an hsa_circ_EIR3-specific nucleic acid probe, or an hsa_circ_EIR3-specific protein.

[0019] According to some embodiments of the present invention, the PCR primer is an hsa_circ_EIR3-specific primer.

[0020] According to some embodiments of the present invention, the PCR primers are a primer pair consisting of DNA sequences shown as SEQ ID NO: 3 and SEQ ID NO: 4.

[0021] The second aspect of the present invention provides the use of hsa_circ_EIR3 or a substance that increases the abundance of hsa_circ_EIR3 in the preparation of a medicament for preventing and / or treating obstructive nephropathy.

[0022] The present invention found that the expression of hsa_circ_EIR3 in the kidneys of chronic ON patients at the tissue level was progressively upregulated as renal fibrosis worsened, and the expression level of hsa_circ_EIR3 at the cellular level increased with the increase in the time of TGF-β1 stimulation of HK2 cells, indicating that hsa_circ_EIR3 was significantly positively correlated with ON disease. However, the present invention also found that overexpression of hsa_circ_EIR3 by vector at the cellular level could reduce the collagen accumulation of cells, thereby alleviating cell fibrosis. Animal experiments also showed that overexpression of hsa_circ_EIR3 could alleviate tubular proliferation and widening of renal interstitium, reduce atrophy and death of renal tubular epithelial cells, thereby alleviating the pathological changes of renal tubular epithelial cells and renal interstitium caused by ureteral obstruction, and reduce the extracellular matrix (Extracellular matrix) at the renal tissue level. The research of the present invention shows that hsa_circ_EIR3 or substances that increase the level of hsa_circ_EIR3 have broad application prospects in the prevention and / or treatment of obstructive nephropathy and can be used in the preparation of drugs for the prevention and / or treatment of obstructive nephropathy.

[0023] According to some embodiments of the present invention, the substance that increases the abundance of hsa_circ_EIR3 includes at least one of a vector expressing hsa_circ_EIR3, an enzyme that promotes shearing or cyclization to form hsa_circ_EIR3, or an enzyme that inhibits the degradation of hsa_circ_EIR3.

[0024] In a third aspect of the present invention, a pharmaceutical composition for treating obstructive nephropathy is provided, wherein the active ingredient of the pharmaceutical composition includes at least one of hsa_circ_EIR3 or an agent that increases the expression level of hsa_circ_EIR3.

[0025] The present invention found that by increasing the level of hsa_circ_EIR3 in kidney cells or tissues, the collagen accumulation of cells can be reduced, thereby alleviating cell fibrosis, and can also alleviate tubular proliferation and widening of the renal interstitium, reduce tubular epithelial cell atrophy and death, thereby alleviating pathological changes in tubular epithelial cells and renal interstitium, and also reduce ECM accumulation in kidney tissue, thereby inhibiting renal fibrosis.

[0026] In a fourth aspect of the present invention, a diagnostic kit for obstructive nephropathy is provided, the kit comprising hsa_circ_EIR3-specific primers.

[0027] According to some embodiments of the present invention, the kit further comprises an RNA extraction reagent, an RNA reverse transcription reagent and a PCR amplification reagent.

[0028] According to some embodiments of the present invention, the hsa_circ_EIR3-specific primers are divergent primers.

[0029] According to some embodiments of the present invention, the hsa_circ_EIR3 specific primer may also be a conventional primer. After hsa_circ_EIR3 is first cut into linear RNA, hsa_circ_EIR3 can be detected using a conventional specific primer.

[0030] The present invention found that hsa_circ_EIR3 has good specificity and stability in human kidneys, and the results of RT-qPCR amplification using divergent primers designed for the hsa_circ_EIR3 cyclization site showed good specificity; in addition, urine detection using hsa_circ_EIR3 specific primers and RT-qPCR method showed that the urine hsa_circ_EIR3 level and the lateral residual renal function (R=0.756) showed a significantly stronger correlation than the existing evaluation index urine NGAL level and the lateral residual renal function (R=0.399), and the ROC curve analysis of the urine hsa_circ_EIR3 level for the reproducibility judgment of renal function showed that hsa_circ_EIR3 has a good judgment efficiency, and its judgment efficiency is slightly higher than NGAL.

[0031] According to some embodiments of the present invention, the hsa_circ_EIR3-specific primer is a primer pair consisting of a DNA sequence as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0032] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0034] Figure 1 This is a graph showing the expression specificity and stability of hsa_circ_EIR3 in kidney according to Example 1 of the present invention; wherein:

[0035] A: Melting curves of hsa_circ_EIR3 divergent primers and internal reference β-actin primers;

[0036] B: Relative expression of ERI3 linear RNA and circular RNA after R nuclease treatment;

[0037] C: Relative expression levels of ERI3 linear RNA and circular RNA at different time points under actinomycin D intervention.

[0038] Figure 2 This is a diagram showing the localization results of hsa_circ_EIR3 in human renal tubular epithelial cells according to Example 1 of the present invention.

[0039] Figure 3 This is a comparison chart of the correlation between the two biomarkers in Example 2 of the present invention and the residual renal function of the affected kidney.

[0040] Figure 4This is a comparison diagram of the ROC curves of the two biomarkers in Example 2 of the present invention.

[0041] Figure 5 This is a graph showing the expression and function study results of hsa_circ_EIR3 in chronic ON according to Example 3 of the present invention; wherein:

[0042] A: Statistical results of the relationship between hsa_circ_EIR3 and the severity of ON disease;

[0043] B: Statistical results of the relationship between hsa_circ_EIR3 and the time of TGF-β1 stimulation of HK2 cells;

[0044] C: The effect of hsa_circ_EIR3 on TGF-β1-induced HK2 cell fibrosis.

[0045] D: Graph showing the effect of hsa_circ_EIR3 on UUO model mice. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0047] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0048] The tissue biopsy specimens in the following examples of the present invention were ethically collected from male and female patients of different ages, and their informed consent was obtained. The study was approved by the Review Committee of the Second Xiangya Hospital of the People's Republic of China, followed the principles of the Declaration of Helsinki, and complied with all relevant ethical regulations for research involving human participants. In addition, the study complied with the Guidelines for the Review and Approval of Human Genetic Resources of the Ministry of Science and Technology.

[0049] The animals in the present invention were kept in the Specific Pathogen-Free Animal Facility of the Experimental Animal Center of Central South University (Changsha, China). All animals had free access to water and food, and were kept under a 12-h light-dark cycle at a constant temperature and humidity with good ventilation and cleanliness. All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Central South University, and all relevant ethical regulations for animal testing and research were followed.

[0050] Unless otherwise specified, in the result graphs of the embodiments of the present invention, * indicates a significance level of p<0.05, ** indicates a significance level of p<0.01, and *** indicates a significance level of p<0.001.

[0051] Unless otherwise specified, the immunology, biochemistry, chemistry, molecular biology, cell biology, genomics and recombinant DNA employed in the present invention are conventional skills in the art.

[0052] Example 1

[0053] This example studies the specificity and stability of hsa_circ_EIR3 expression in human kidney.

[0054] 1. Experimental methods:

[0055] 1) First, search for the CDS sequence of the β-actin gene from the NCBI official website, and obtain the full-length sequence and cyclization site of hsa_circ_EIR3 from the circBase database. Use the online primer design function of the official website of Shanghai Sangon Biotechnology Co., Ltd. in China to design β-actin and hsa_circ_EIR3 primers according to the SYBRGreen dye method fluorescence quantitative PCR primer design principle, all of which were synthesized by Sangon Biotechnology. β-actin was used as a calibration gene for the internal control of hsa_circ_EIR3. The primer sequences are shown in Table 1.

[0056] Table 1 RT-qPCR primers

[0057]

[0058] (1) RNA extraction:

[0059] Total RNA was extracted according to the instructions of TrizolTM Reagent Kit (Invitrogen, Carlsbad, CA, USA). All experimental tools used for total RNA extraction were disposable sterile enzyme-free consumables or were treated with RNase+180°C dry baking.

[0060] (2) Identification of total RNA:

[0061] The RNA concentration and purity were detected by UV spectrophotometer and diluted with enzyme-free water to control the RNA concentration to 600-900 μM.

[0062] (3) Reverse transcription to obtain cDNA:

[0063] The RNA obtained above was used to remove genomic DNA and reverse transcribe using the Evo M-MLV reverse transcription kit from AG to obtain a cDNA template for RT-qPCR.

[0064] (4) RT-qPCR:

[0065] AG's SYBR Green Pro Taq HS premixed qPCR kit and the cDNA template obtained above were used for RT-qPCR reaction. Each sample was tested three times, and the melting curve and amplification curve were drawn to compare the melting curve of hsa_circ_EIR3 with that of the internal reference.

[0066] 2) The total RNA of HK2 cells was treated with ribonuclease R, and the linear mRNA expression abundance of hsa_circ_EIR3 and its parent gene ERI3 was compared. Actinomycin D was used to intervene in HK2 cells at different times, and the half-life of hsa_circ_EIR3 and linear ERI3 mRNA was detected by RT-qPCR at 0h, 4h, 8h, 12h, and 24h.

[0067] 3) RNA-FISH detection of tissue frozen sections:

[0068] Renal cortex tissue of hydronephrosis kidney and normal renal cortex tissue adjacent to renal cancer were obtained and hybridized with red fluorescent-labeled hsa_circ_EIR3 probe to observe its subcellular localization.

[0069] (1) Tissue probe hybridization:

[0070] ①Fixed tissue specimens: Wash the renal cortical tissue with PBS buffer and then fix it with 4% paraformaldehyde for about 1-2 hours.

[0071] ② Tissue dehydration: First, place the kidney specimen in a gradient concentration of sucrose solution for dehydration, first dehydrate at a concentration of 15% for 8 hours, and then dehydrate at a concentration of 30% for 12 hours.

[0072] ③ Frozen sections: Use a freezing microtome to make frozen sections and store them at -80℃ for later use.

[0073] ④ Fixation: Take out the frozen sections and place them at room temperature to dry, add an appropriate amount of 4% paraformaldehyde, wash the sections with PBS buffer after 8-10 minutes, continue shaking and washing for 3-5 minutes, and wash 2-3 times in total.

[0074] ⑤Digestion: Use a gene pen to circle the area to be hybridized, add an appropriate amount of proteinase K solution to the area to digest the tissue, and rinse the area to be hybridized 3 times with distilled water after 3 minutes, each time for 3-5 minutes.

[0075] ⑥ Pre-hybridization: Mix 99% volume of Pre-hybridization Buffer and 1% volume of Blocking Solution to prepare pre-hybridization solution, incubate in a 37℃ constant temperature water bath to 37℃, then add to the area to be hybridized, and continue to incubate at 37℃ for 1h. At this time, in a light-proof environment, take 2.5μL of hsa_circ_EIR3 probe storage solution and mix it with the hybridization solution to prepare a hybridization solution containing the probe. The appropriate amount of probe added needs to be explored through preliminary experiments.

[0076] ⑦ Hybridization: Remove the pre-hybridization solution completely, add 100 μL of hybridization solution to the area to be hybridized on the slice in a light-proof environment, and place it in a 37°C constant temperature water bath overnight.

[0077] ⑧Washing: Wash the sections three times with 2×SSC hybridization solution in a 37°C water bath in a dark environment, gently shaking for 5 minutes each time. Then wash the sections with 1×SSC hybridization solution in a 37°C water bath for 5 minutes. Finally, wash the sections with 0.5×SSC hybridization solution at room temperature for 5 minutes.

[0078] (2)DNA staining:

[0079] ① In a light-proof environment, add an appropriate amount of 1×DAPI staining solution to the slices, shake gently to cover the bottom of the dish, and stain at room temperature for 10 minutes.

[0080] ② In a light-proof environment, add appropriate amount of PBS buffer to the slices and wash them three times, shaking on a shaker for 5 minutes each time.

[0081] (3) Sealing:

[0082] Cover with a coverslip in a dark environment and perform fluorescence detection using a laser confocal microscope as soon as possible or store at 4°C for a short period of time before detection.

[0083] 2. Experimental results:

[0084] The results are as follows Figure 1 and Figure 2 shown.

[0085] in, Figure 1 Figure A shows the melting curves of the divergent primers for hsa_circ_EIR3 and the internal reference β-actin primer. The results show that the melting curve of hsa_circ_EIR3 is a single peak like the internal reference when the divergent primers designed for the hsa_circ_EIR3 cyclization site are used for RT-qPCR amplification, which reflects the specificity of the amplified product. Figure 1Figure B shows the relative expression of ERI3 linear RNA and circular RNA after ribonuclease R treatment. The results show that after the total RNA of HK2 cells was treated with R nuclease, the abundance of hsa_circ_EIR3 was significantly higher than that of ERI3 linear mRNA, indicating its circular existence; Figure 1 Figure C shows the relative expression of ERI3 linear RNA and circular RNA at different time points under the intervention of actinomycin D. The results show that when actinomycin D is used to intervene HK2 at different times, its half-life is found to exceed 24 hours.

[0086] The above experimental results and data indicate that hsa_circ_EIR3 has good specificity and stability in human kidneys and has the potential to be used as a biomarker for detection.

[0087] Figure 2 This is the localization map of hsa_circ_EIR3 in human renal tubular epithelial cells observed using a laser confocal microscope. It can be seen from the figure that hsa_circ_EIR3 is mainly distributed in the cytoplasm of human renal tubular epithelial cells, and the fluorescence intensity in the renal cortical tissue (Obstructive) of ON patients is significantly stronger than that in normal renal cortical tissue (Normal), indicating that the expression of hsa_circ_EIR3 in the kidneys of ON patients is significantly increased.

[0088] Example 2

[0089] This example studies the clinical correlation between hsa_circ_EIR3 in urine and ON renal fibrosis.

[0090] 1. The population included in the study of this embodiment is patients with upper urinary tract stones who visited the Department of Urology of the Second Xiangya Hospital of Central South University from June 1, 2022 to August 31, 2022, and clinical information was collected:

[0091] 1) 50 patients with different degrees of ON progression were prospectively enrolled:

[0092] ① Inclusion criteria: patients aged over 18 years, with symptoms of hydronephrosis including abdominal mass, abdominal distension or pain for more than 1 week, upper urinary tract stones combined with hydronephrosis found by CT or ultrasound examination, and who are scheduled for surgical treatment.

[0093] ②Exclusion criteria: patients with other renal diseases and whose initial treatment was nephrectomy.

[0094] ③Indicators collected: age, gender, course of disease, body mass index (BMI), bilateral / unilateral obstruction, whether there is urinary tract infection, lateral glomerular filtration rate (GFR), serum creatinine level, urine NGAL concentration, renal cortical thickness under CT, renal artery resistance index (RI), maximum anterior-posterior diameter of renal pelvis, urine pH on the affected side, surgical method, urine specimen. NGAL is a mature biomarker for renal injury and is used as a reference level for the prognostic value of urinary hsa_circ_EIR3 in this example.

[0095] ④ Clinical outcome: divided into successful renal preservation and renal preservation failure. The renal image was rechecked 3 months after surgery. If the GFR of each side increased by more than 5 mL / min compared with that before surgery, it was considered a successful renal preservation. Otherwise, it was considered a renal preservation failure. Patients who underwent secondary nephrectomy were directly judged as renal preservation failure.

[0096] 2) Ten patients with non-renal diseases were prospectively enrolled during the same period:

[0097] Urine and urine specimens were collected, and urine specimens were used solely to determine the normal level of hsa_circ_EIR3 and the normal concentration of NGAL.

[0098] The level of hsa_circ_EIR3 was determined by real-time quantitative polymerase chain reaction (RT-qPCR), and the concentration of NGAL was determined by latex-enhanced immunoturbidimetry (Nanjing Novozyme Biotechnology Co., Ltd., B1501C).

[0099] 2. Statistical analysis:

[0100] 1) The above clinical indicators were included in univariate and logistic regression analysis to analyze the influencing factors of reversibility of renal function and whether urinary hsa_circ_EIR3 concentration was an independent factor affecting the clinical outcome of ON.

[0101] 2) Compare the changes in urinary hsa_circ_EIR3 levels in patients without renal disease and patients with different degrees of chronic progression of ON, and explore the changing trend of urinary hsa_circ_EIR3 levels with the progression of chronic obstruction.

[0102] 3) Using the changes in urine NGAL concentration as a reference, the correlation between urine hsa_circ_EIR3 levels and residual renal function was detected. Finally, the receiver operating characteristic (ROC) curves of urine hsa_circ_EIR3 and NGAL were drawn, and the sensitivity and specificity of urine NGAL and urine hsa_circ_EIR3 for determining the clinical outcome of ON were calculated and compared.

[0103] 3. Results:

[0104] The results of the analysis of influencing factors related to the recoverability of renal function in patients are shown in Table 2. As shown in Table 2, the mean urine NGAL level of the 50 ON patients prospectively enrolled was higher than the normal value of 8.88 ng / mL, and the mean urine hsa_circ_EIR3 relative concentration was higher than the normal value (ΔCq was less than 3.90 of the normal value), indicating that ON patients will have increased urine NGAL and urine hsa_circ_EIR3 levels during the course of the disease. Among these 50 ON patients, 35 cases successfully saved the kidney during the review process, and 15 cases failed to save the kidney, with a success rate of 70%. Between the two groups of patients who succeeded and failed to save the kidney, old age, long course of disease, bilateral obstruction, combined urinary tract infection, low residual GFR, low renal cortical thickness, high RI index, high maximum anterior-posterior diameter of the renal pelvis, high ipsilateral urine pH, high urine NGAL level and high urine hsa_circ_EIR3 level were risk factors for failure of renal preservation surgery (P<0.05).

[0105] Table 2 Analysis results of factors affecting the renal function recoverability of patients

[0106]

[0107]

[0108] Notes to Table 1: a. Renal artery vascular resistance index, reflecting renal artery vascular resistance, used to evaluate renal perfusion; b. CT images measured in the electronic film reading system, reflecting the dilation of hydronephrosis; c. Urine drainage through the renal puncture channel during percutaneous nephroscopic surgery, and urine drainage through the ureteral catheter in the ureteroscope channel during ureteroscopic surgery; d. The difference in the number of cycles between the target RNA and the internal reference RNA in the PCR system, and the ΔCq value is inversely proportional to the target RNA concentration.

[0109] Factors with p<0.001 were included in the multivariate unconditional logistic analysis, and it was found that advanced age and high maximum anterior-posterior diameter of the renal pelvis were independent risk factors for renal preservation failure. Although urine NGAL level and urine hsa_circ_EIR3 level were not independent influencing factors for renal preservation failure in this statistical analysis, correlation analysis showed that (such as Figure 3As shown in the results, urinary NGAL levels and urinary hsa_circ_EIR3 levels were significantly negatively correlated with the residual renal function of each side (p<0.05), and the urinary hsa_circ_EIR3 levels showed a significantly stronger correlation with the residual renal function of each side (R=0.756) than with the urinary NGAL levels (R=0.399).

[0110] To further explore the potential value of urine hsa_circ_EIR3 as a predictor of postoperative clinical outcomes in patients with ON renal fibrosis, this example plotted the ROC curves of urine NGAL level and urine hsa_circ_EIR3 level for reversibility of renal function, and calculated the area under the curve (AUC) (Figure Figure 4 The results showed that both urine NGAL level and urine hsa_circ_EIR3 had good judgment efficacy, and the efficacy of hsa_circ_EIR3 was slightly higher than that of NGAL, although the difference between the two was not statistically significant in this sample (p=0.589). Combined with the fact that urine hsa_circ_EIR3 level had a significantly stronger correlation with the residual renal function of each side, it was shown that urine hsa_circ_EIR3 could be used as a biomarker to better judge the postoperative clinical outcome of patients with ON renal fibrosis.

[0111] Example 3

[0112] This example studies the potential value of hsa_circ_EIR3 in the regulation of ON renal fibrosis.

[0113] 1. Research methods used in this example:

[0114] 1) In this example, the expression of hsa_circ_EIR3 was detected in normal renal cortical tissues adjacent to the cancer resected from 4 renal cancer patients (healthy group) and renal cortical tissues of nephrectomy specimens from 8 ON patients (4 ON groups and 4 severe ON groups). The general characteristics of the patients are shown in Table 3:

[0115] Table 3 General characteristics of the healthy group, ON group and severe ON group

[0116]

[0117] Notes: *Symptoms include abdominal mass, bloating or pain; ON group: symptom duration <2 weeks, lateral glomerular filtration rate (GFR)>10mL / min, renal cortical thickness>3mm; severe ON group: symptom duration >2 weeks, lateral GFR<10mL / min, renal cortical thickness<3mm.

[0118] 2) In this example, the level of hsa circ EIR3 in HK2 cells after TGF-1 stimulation for increasing time was detected by RT-qPCR.

[0119] 3) Cell experiments:

[0120] The human renal proximal tubule (HK2) cells cultured in vitro were divided into 4 groups, namely, double negative control group, transforming growth factor β1 (TGF-β1) intervention group, hsa_circ_EIR3 overexpression plasmid transfection group, and TGF-β1+hsa_circ_EIR3 overexpression plasmid transfection double treatment group; the expression levels of collagen Ⅰ (Col Ⅰ), collagen Ⅲ (Col Ⅲ), and fibronectin (FN) in each group of cells were detected by Western Blot experiments.

[0121] The specific steps for each group processing are as follows:

[0122] (1) Cells were passaged one day before transfection, and transfection was performed the next day when the cells had grown to about 70%.

[0123] (2) 6 h before transfection, replace the serum-free medium to perform cell starvation treatment to make the cells homogenous.

[0124] (3) Solution A (2 mL per medium dish): Add 5 μL of the pre-packaged overexpression plasmid (add empty plasmid to the double negative control group and intervention group) to 120 μL of Opti-MEM medium, mix well, and place at room temperature for 10 min.

[0125] (4) Solution B (2 mL in a medium dish): Add 5 μL of Lip-2000 liposomes to 120 μL of Opti-MEM medium, mix well, and leave at room temperature for 5 min.

[0126] (5) Mix equal volumes of Solution A and Solution B, shake gently to mix well, and place at room temperature for 20 min.

[0127] (6) Use a pipette to disperse the AB mixture into the cells in the dish and shake it evenly in a "cross" shape on the table.

[0128] (7) The medium dish was placed in a cell culture incubator and cultured. After 6 hours, the medium was replaced with normal complete culture medium. After 24 hours, TGF-β1 saline solution with a final concentration of 5 ng / mL was added to the intervention group and the double-treatment group (the double negative control group and the overexpression plasmid transfection group were added with the same volume of saline).

[0129] Among them, the hsa_circ_EIR3 overexpression plasmid uses the pLenO-GTP blank vector and the insert fragment SEQ ID NO:7, and is constructed using the conventional overexpression plasmid construction method in the art; the sequence of the insert fragment SEQ ID NO:7 is:.

[0130] 4) Animal experiments:

[0131] 8-10-week-old C57BL / 6 male mice were divided into a control group, a unilateral ureteral obstruction model group (UUO group), an hsa_circ_EIR3 overexpression plasmid intervention group, and an UUO+hsa_circ_EIR3 overexpression plasmid intervention group, with 3 mice in each group.

[0132] The specific experimental methods are as follows:

[0133] The UUO group and the UUO+hsa_circ_EIR3 overexpression plasmid intervention group were treated with UUO, and the control group and the hsa_circ_EIR3 overexpression plasmid intervention group were treated with sham surgery (the sham surgery did not suture the ureter after separation but put it back in situ, and the other steps were consistent with the classic UUO steps). Then, the mice in the hsa_circ_EIR3 intervention group were injected with 25μg hsa_circ_EIR3 overexpression plasmid (the control group and UUO group were injected with the corresponding blank vector) through the tail vein, once a day for three consecutive days. After the model was constructed, the mice were killed and the kidneys were taken for paraffin sections for HE staining and Masson staining, and the renal tissue lesions were observed under a microscope.

[0134] 2. Experimental results:

[0135] The results are as follows Figure 5 shown.

[0136] in, Figure 5 Figure A shows that at the tissue level, the expression of hsa_circ_EIR3 in the kidneys of chronic ON patients is progressively upregulated as renal fibrosis worsens.

[0137] Figure 5 Figure B shows that the expression level of hsa_circ_EIR3 at the cellular level increases with the increase of TGF-β1 stimulation time in HK2 cells.

[0138] Figure 5 The results of Western Blot band analysis in Figure C showed that TGF-β1 caused collagen accumulation in HK2 cells, while overexpression of hsa_circ_EIR3 alleviated cell fibrosis, indicating that overexpression of hsa_circ_EIR3 can alleviate TGF-β1-induced HK2 cell fibrosis.

[0139] Depend on Figure 5The HE and Masson staining results of Figure D (scale bar in the figure: 50μm) show that: corresponding to the in vitro experiment, UUO caused tubular dilatation, renal cortical atrophy and extracellular matrix (ECM) accumulation, and the overexpression of hsa_circ_EIR3 weakened the pathological changes induced by UUO; the HE staining results showed that UUO caused tubular dilatation, widening of the renal interstitium, atrophy and death of tubular epithelial cells, and the pathological changes of tubular epithelial cells and renal interstitium were alleviated after the injection of hsa_circ_EIR3 plasmid; the Masson staining results of collagen showed that the overexpression of hsa_circ_EIR3 reduced ECM accumulation. It shows that the overexpression of hsa_circ_EIR3 by tail vein injection of hsa_circ_EIR3 overexpression plasmid in c57BL / 6 mice can inhibit renal fibrosis in UUO mice.

[0140] In summary, the present invention has found through experimental exploration that the circular RNA hsa_circ_EIR3 of the ERI3 gene has good specificity and stability in the human kidney, and has the potential to be used as a biomarker for detection; and it was found that the expression level of hsa_circ_EIR3 in the renal cortex tissue of ON patients was significantly increased compared with that in normal tissue, indicating that hsa_circ_EIR3 can be used as a marker for detecting ON; further experimental exploration found that the level of hsa_circ_EIR3 in urine of ON patients will increase during the course of the disease, and the level of hsa_circ_EIR3 in urine is significantly negatively correlated with the residual renal function of the patient, and the level of hsa_circ_EIR3 in urine and the residual renal function of the side show a significantly stronger correlation than the existing evaluation index NGAL level in urine and the residual renal function of the side; the present invention also studied the expression of hsa_circ_ The ROC curve of EIR3 level for judging the reversibility of renal function showed that the judgment efficiency of hsa_circ_EIR3 was slightly higher than that of NGAL. Therefore, urine hsa_circ_EIR3 can be used as a biomarker to better judge the postoperative clinical outcome of patients with ON renal fibrosis, and has good application prospects. The present invention further explored the role of hsa_circ_EIR3 in the regulation of ON renal fibrosis. It was unexpectedly found that hsa_circ_EIR3 can be used as a protective factor to repair renal fibrosis, showing the effect of counteracting other factors that promote fibrosis. Its overexpression helps to slow down the pathological changes of renal tubular epithelial cells and renal interstitium, reduce ECM accumulation, and thus inhibit renal fibrosis. Therefore, the present invention discovered the application prospect of regulating hsa_circ_EIR3 in the treatment of ON diseases.

[0141] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a substance for detecting hsa_circ_EIR3 in at least one of (1) to (4); (1) Preparation of products for the diagnosis of obstructive nephropathy; (2) Prepare products for the prognosis assessment of obstructive nephropathy; (3) Prepare products for predicting the recovery of renal function after the obstruction is relieved in patients with obstructive nephropathy; (4) Prepare a product for assessing the severity of obstructive nephropathy.

2. The use according to claim 1, characterized in that: The circbase ID of the hsa_circ_EIR3 is hsa_circ_0012138.

3. The use according to claim 2, characterized in that: The substance for detecting hsa_circ_EIR3 is a substance used in one or more detection techniques or methods selected from the following group: PCR, biochip method, Northern blotting method or RNA immunoprecipitation method.

4. The use according to claim 2, characterized in that: The substance for detecting hsa_circ_EIR3 is selected from: at least one of: PCR primers, gene chips, hsa_circ_EIR3-specific nucleic acid probes or hsa_circ_EIR3-specific proteins.

5. The use according to claim 4, characterized in that: The PCR primers are hsa_circ_EIR3 specific primers; Preferably, the PCR primers are a primer pair consisting of the DNA sequences shown in SEQ ID NO: 3 and SEQ ID NO:

4.

6. Use of hsa_circ_EIR3 or substances that increase the abundance of hsa_circ_EIR3 in the preparation of drugs for preventing and / or treating obstructive nephropathy.

7. The use according to claim 6, characterized in that: The substance that increases the abundance of hsa_circ_EIR3 includes at least one of a vector expressing hsa_circ_EIR3, an enzyme that promotes shearing or cyclization to form hsa_circ_EIR3, or an enzyme that inhibits the degradation of hsa_circ_EIR3.

8. A pharmaceutical composition for treating obstructive nephropathy, characterized in that: The active ingredient of the pharmaceutical composition includes at least one of hsa_circ_EIR3 or an agent that increases the expression level of hsa_circ_EIR3.

9. A diagnostic kit for obstructive nephropathy, characterized in that: The kit includes hsa_circ_EIR3-specific primers.

10. The kit according to claim 9, characterized in that The hsa_circ_EIR3-specific primers are divergent primers.

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