Application of mitochondrial double-stranded RNA as lupus nephritis serum biomarker

Abstract

The invention discloses application of mitochondrial double-stranded RNA (Ribonucleic Acid) as a lupus nephritis serum biomarker. The invention discloses application of a reagent for detecting mitochondrial double-stranded RNA in preparation of a lupus nephritis serum auxiliary diagnosis reagent and / or a prognosis auxiliary diagnosis reagent. The mitochondrial double-stranded RNA is selected from any one or more of ND1, ND4, ND4L, COX2, ATP6 and ATP8. The Mt-dsRNA combination provided by the invention can evaluate the kidney activity state or prognosis of an LN patient, and can improve the sensitivity and specificity of a single index when being combined with biochemical indexes for use. Therefore, by combining the mt-dsRNA with the existing biochemical indexes, the occurrence and development process of the LN can be evaluated more accurately and efficiently, and then personalized treatment schemes suitable for different LN patients are formulated according to the occurrence and development process of the LN.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis, and relates to the application of preparing a kit for evaluating the activity and prognosis of lupus nephritis by measuring mitochondrial double stranded RNA (mt-dsRNA) in serum. Background Art

[0002] Lupus nephritis (LN) is a renal injury caused by the autoimmune disease systemic lupus erythematosus (SLE) and is the most common and important visceral complication of SLE. [1] LN is the most common secondary glomerular disease in my country and the main cause of death in patients. [2] Despite overall improvements in the care of LN patients and improvements in 5- and 10-year renal survival, the prognosis remains unsatisfactory. [3] The assessment and continuous monitoring of renal activity status in LN are of great significance to the prognosis of LN patients.

[0003] Although renal biopsy remains the gold standard for LN diagnosis, assessment of renal activity and prognosis, its invasiveness and potential complications make it unsuitable for continuous monitoring. [4] However, existing conventional laboratory markers have some problems in evaluating the renal activity status and prognosis of LN. [5] Studies have shown that 24-hour proteinuria and estimated glomerular filtration rate (eGFR) are the main biomarkers for evaluating LN patients and are significantly correlated with the renal prognosis of LN patients. [3] However, when it comes to the assessment of renal activity in LN, the correlation between the two is not significant, so they cannot be considered the most reliable marker of renal activity in LN. [6] In addition, an increase in serum anti-double-stranded DNA antibody levels often heralds the onset of LN. [7] When antibody levels are significantly elevated, implementing preventive treatment measures for patients may help reduce the incidence of LN. However, there is no significant correlation between serum anti-dsDNA antibody levels and renal activity status. [8]. Therefore, it is very important to find a biomarker that can help determine the renal activity status and prognosis of LN patients. The ideal LN biomarker should have the following characteristics: (1) It can evaluate the renal activity status; (2) It can define the recurrence and remission of LN to monitor the clinical effect of LN intervention; (3) The test specimens are easy to obtain, non-invasive, easy to perform at the bedside or clinical standard laboratory, and it is fast, convenient, and low-cost. Currently, no single biomarker meets these standards, and multiple biomarkers need to be used in combination. [8] With the development of functional genomics and proteomics, researchers have screened out some new biomarkers of LN with promising clinical applications from serum samples, such as serum VCAM-1. [9] , serum ICAM-1 [9] , serum MCP-1

[10] . Although the expression levels of these markers are increased in the serum of LN patients compared with healthy controls, the expression levels of these markers are not sufficiently correlated with LN renal activity or renal pathology. In addition, these biomarkers need to be further evaluated and verified in practice in terms of their sensitivity and specificity, the time limit of their appearance after renal damage, and their effects on long-term and short-term prognosis. Conducting research on new molecular markers is of great significance for monitoring the efficacy and evaluating the prognosis of LN. At the same time, it also provides new clues for the study of pathogenesis and the exploration of therapeutic targets, and is more conducive to the realization of targeted and individualized precision medicine.

[0004] Mitochondria are the product of endosymbiotic bacteria and are an organelle that retains basic prokaryotic characteristics.

[11] , is an important place for the production of reactive oxygen species (Mitochondrial reactive oxygen species, mtROS)

[12] , oxidative stress induced by reactive oxygen species can lead to lipid peroxidation (LPO) in renal tissue, ultimately leading to ferroptosis of renal cells. In addition, given the microbial origin of mitochondria, this organelle has long been an attractive source of LN autoantigens.

[13] Anti-mitochondrial antibodies (AMA) have been described in LN. The antigens they target include mitochondrial outer membrane (MOM), mitochondrial DNA (mtDNA), mitochondrial RNA (mtRNA), and heat shock protein 60 (Hsp60). These anti-mitochondrial antibodies can bind to the corresponding antigens to form immune complexes, which are deposited in the glomerular mesangium or in the subendothelial and subepithelial spaces, leading to the appearance of crescents mediated by immune complexes in the glomeruli.

[13] In addition, these immune complexes also lead to the chemotaxis and polarization of macrophages, which in turn lead to the infiltration of renal interstitial inflammatory cells.

[14] Moreover, these immune complexes can activate complement through a series of cascade reactions through the classical pathway, induce the production of membrane attack complexes, and ultimately lead to kidney tissue damage.

[15] These studies suggest that targeting mitochondria is a potential intervention to protect mitochondrial structure and function and improve kidney injury.

[0005] In mammals, mitochondrial DNA is bidirectionally transcribed, producing overlapping transcripts to form mitochondrial double stranded RNA (mt-dsRNA).

[16] As part of the innate immune response, mt-dsRNA is the most important source of endogenous dsRNA

[17] Three types of dsRNA pattern recognition receptors have been reported, including retinoic acid inducible gene-I (RIG-1), melanoma differentiation related gene 5 (MDA5),

[18] and Toll-like receptor 3 (TLR3)

[19] These mt-dsRNAs are degraded by related degradation complexes, such as the complex formed by polyribonucleotide phosphorylase (Polyribonucleotide nucleotidyltransferase 1, PNPT1) and mitochondrial RNA helicase (SUPV3L1, SUV3), to avoid the activation of immune response.

[20] PNPT1 and SUV3 form an RNA degradation complex in mitochondria and colocalize with dsRNA and dsDNA

[21] PNPT1 and SUV3 are essential for the degradation of mt-dsRNA. The loss of PNPT1 and SUV3 will lead to an increase in the half-life of mt-dsRNA and the accumulation of mt-dsRNA degradation intermediates. Silencing the expression of SUV3 in cells will increase the stability of mature mRNA and cause the accumulation of RNA that is difficult to observe under normal conditions due to low abundance or rapid removal, including mt-dsRNA decay intermediates, abnormally formed RNA, RNA processing byproducts, and a large number of non-coding antisense RNAs.

[22] .

[0006] Therefore, it is not difficult to speculate that once mt-dsRNAs leak out of mitochondria, they have strong immunogenicity and promote the occurrence and development of diseases. In addition, different mt-dsRNAs have different functions, for example: COX3 deficiency exists in mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome and recurrent myoglobinuria; COX2 is constitutively expressed in some tissues under physiological conditions, such as endothelium, kidney and brain, and is upregulated under pathological conditions, such as in cancer and inflammation. Its upregulation is associated with increased cell adhesion, phenotypic changes, anti-apoptosis and tumor angiogenesis. ND4L is associated with Leber hereditary optic neuropathy and diabetes;. CYTB is involved in electron transport-coupled proton transport and response to glucagon, and is associated with ovarian and bladder cancers; ATP6 is essential for the acidification of various cellular components, including endosomes, lysosomes, clathrin-coated vesicles, secretory vesicles, and chromaffin granules; ATP8 may be involved in the pathogenesis of multiple sclerosis; ND1 may be involved in the electron transfer of ubiquinone, and its mutations may be associated with a variety of diseases, including Leber hereditary optic neuropathy, Alzheimer's disease, and Parkinson's disease.

[0007] In summary, as one of the pathogenic molecules, mt-dsRNA has significant application potential in the fields of disease activity status and prognosis assessment. Summary of the invention

[0008] The object of the present invention is to provide serum mt-dsRNA biomarkers related to the renal activity status and prognosis evaluation of LN.

[0009] Another object of the present invention is to provide applications of the marker.

[0010] The technical solution of the present invention is obtained by the following method:

[0011] 1) Use eukaryotic transcriptome resequencing technology and independent sample RT-qPCR technology to analyze the expression level of mt-dsRNA in serum samples of healthy controls and LN patients, and explore the potential of mt-dsRNA as a serum biomarker for LN.

[0012] 2) Using RT-qPCR technology, the serum mt-dsRNA expression levels of healthy controls (HC), inactive LN patients (iLN) and active LN patients (aLN) were detected to screen out mt-dsRNA with great potential in distinguishing iLN from aLN.

[0013] 3) Using RT-qPCR technology, the serum mt-dsRNA of iLN patients and aLN patients was detected in a larger LN sample set to further screen out mt-dsRNA with great potential in distinguishing iLN from aLN.

[0014] 4) The Mann-Whitney u test and LASSO regression were used to perform differential expression analysis on the mt-dsRNA expression levels between patients with low pathological activity and patients with high pathological activity, and to screen out mt-dsRNA that performed well in distinguishing LN patients with different degrees of pathological activity.

[0015] 5) The differential expression analysis of serum mt-dsRNA expression levels in LN patients with different prognoses was performed by Mann-Whitney u test and LASSO regression, and the mt-dsRNA with excellent performance in distinguishing LN patients with different prognoses was screened out.

[0016] The purpose of the present invention can be achieved through the following technical solutions:

[0017] The invention relates to a reagent for detecting mitochondrial double-stranded RNA in the preparation of a serum auxiliary diagnostic reagent for lupus nephritis and / or a prognosis auxiliary diagnostic reagent, wherein the mitochondrial double-stranded RNA is selected from any one or more of ND1, ND4, ND4L, COX2, ATP6 and ATP8.

[0018] As a preferred embodiment of the present invention, the reagent for detecting mitochondrial double-stranded RNA is a specific primer for PCR amplification of mitochondrial double-stranded RNA; the specific primer sequences for PCR amplification of ND1 are shown in SEQ ID NO.1 and SEQ ID NO.2, the specific primer sequences for PCR amplification of ND4 are shown in SEQ ID NO.3 and SEQ ID NO.4, the specific primer sequences for PCR amplification of ND4L are shown in SEQ ID NO.5 and SEQ ID NO.6, the specific primer sequences for PCR amplification of COX2 are shown in SEQ ID NO.7 and SEQ ID NO.8, the specific primer sequences for PCR amplification of ATP6 are shown in SEQ ID NO.9 and SEQ ID NO.10, and the specific primer sequences for PCR amplification of ATP8 are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0019] As a further preferred embodiment of the present invention, the reagent for detecting mitochondrial double-stranded RNA ND4L and COX2 is used in the preparation of a serum-assisted diagnostic reagent having any of the following functions:

[0020] 1) Differentiate between healthy people and patients with inactive LN;

[0021] 2) Differentiate between healthy people and patients with active LN;

[0022] 3) Differentiate between patients with inactive LN and those with active LN.

[0023] As a further preference of the present invention, reagents for detecting mitochondrial double-stranded RNA ND1, ND4L and ATP8 are used in the preparation of serum-assisted diagnostic reagents for distinguishing patients with low pathological activity LN from patients with high pathological activity LN; preferably, reagents for detecting mitochondrial double-stranded RNA ND1, ND4L and ATP8 are combined with reagents for detecting blood creatinine, serum albumin and 24h urine protein in the preparation of serum-assisted diagnostic reagents for distinguishing patients with low pathological activity LN from patients with high pathological activity LN.

[0024] As a further preference of the present invention, a reagent for detecting at least three mitochondrial double-stranded RNAs among mitochondrial double-stranded RNAs ND1, ND4, ND4L, COX2, ATP6 and ATP8 is used in combination with or without a reagent for detecting at least two indicators among serum creatinine, serum albumin and 24-hour urine protein in the preparation of a serum-assisted diagnostic reagent for distinguishing the prognosis of lupus nephritis.

[0025] As a further preferred embodiment of the present invention, the reagent for detecting mitochondrial double-stranded RNA ND1, ND4, ND4L, COX2, ATP6 and ATP8 is used in the preparation of serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis with any of the following functions:

[0026] 1) Differentiate between LN patients with complete remission and LN patients with partial remission;

[0027] 2) distinguish LN patients with complete remission from those with relapsed LN;

[0028] 3) Differentiate between LN patients with partial remission and LN patients with relapse.

[0029] In a specific embodiment of the present invention, a reagent for detecting mitochondrial double-stranded RNA ND1, ND4 and COX2 is combined with a reagent for detecting blood creatinine and 24-hour urine protein in the preparation of a serum-assisted diagnostic reagent for distinguishing the prognosis of lupus nephritis. The serum-assisted diagnostic reagent for distinguishing the prognosis of lupus nephritis is used to distinguish between LN patients with complete remission and LN patients with partial remission.

[0030] In a specific embodiment of the present invention, a reagent for detecting mitochondrial double-stranded RNA ND1, ND4 and ND4L is combined with a reagent for detecting blood creatinine and 24-hour urine protein to prepare a serum-assisted diagnostic reagent for distinguishing the prognosis of lupus nephritis. The serum-assisted diagnostic reagent for distinguishing the prognosis of lupus nephritis is used to distinguish between patients with complete remission of LN and patients with recurrent LN.

[0031] In a specific embodiment of the present invention, a reagent for detecting mitochondrial double-stranded RNA ATP6, ATP8 and ND4 is combined with a reagent for detecting blood creatinine, serum albumin and 24-hour urine protein in the preparation of a serum-assisted diagnostic reagent for distinguishing the prognosis of lupus nephritis. The serum-assisted diagnostic reagent for distinguishing the prognosis of lupus nephritis is used to distinguish between patients with partial remission of LN and patients with recurrent LN.

[0032] A lupus nephritis serum auxiliary diagnostic reagent and / or prognosis auxiliary diagnostic reagent, comprising a reagent for detecting the mitochondrial double-stranded RNA; preferably comprising PCR specific primers for detecting any one or more of ND1, ND4, ND4L, COX2, ATP6 and ATP8; and further preferably comprising the primers.

[0033] Beneficial effects:

[0034] This patent selects LN patients with different activities and prognoses, provides the application of mt-dsRNA in detecting and diagnosing the activity and prognosis of LN patients, and obtains mt-dsRNA as a LN serum biomarker. The mt-dsRNA provided by the present invention as a LN serum biomarker has the following functions:

[0035] 1) Differentiate between iLN and aLN

[0036] The results of human serum eukaryotic transcriptome resequencing and independent sample RT-qPCR showed that the relative expression levels of most mt-dsRNAs encoding functions in the serum of LN patients were significantly increased compared with healthy controls, including COX2, ND4, COX3, ND4L, ATP8, COX1, ATP6, ND2, ND3 and ND6. The training set consisting of a small batch of HC and LN samples showed that 6 mt-dsRNAs (ND1, ND4 , ND4L, COX2, ATP6 and ATP8) performed well in distinguishing HC, iLN and aLN. The validation set consisting of a large number of LN samples showed that two mt-dsRNAs (COX2 and ND4) performed well in distinguishing iLN from aLN. When mt-dsRNAs were combined to distinguish iLN from aLN, the area under the curve was 0.905.

[0037] Differentiate between low and high pathological activity LN

[0038] We found that ND1, ND4L, and ATP8 performed well in distinguishing patients with low pathological activity from those with high pathological activity. The combined use of biochemical information and mt-dsRNA was even more effective, with an area under the curve of 0.95 when used together to distinguish patients with low pathological activity from those with high pathological activity.

[0039] Differentiate between LN with different prognoses

[0040] The results of the prognostic study showed that six genes (ND1, ND4, ND4L, COX2, ATP6 and ATP8) performed well in distinguishing lupus patients with different prognoses. When biochemical information was combined with mt-dsRNA to distinguish LN patients with complete remission from those with partial remission, the area under the curve was 0.938; when distinguishing LN patients with complete remission from those with relapse, the area under the curve was 0.988; when distinguishing LN patients with partial remission from those with relapse, the area under the curve was 0.979.

[0041] Summarize:

[0042] The Mt-dsRNA combination of the present invention can evaluate the renal activity state or prognosis of LN patients, and when used in combination with biochemical indicators, the sensitivity and specificity of a single indicator can be improved. In addition, Mt-dsRNA can define the recurrence and remission of LN, and when used in combination with biochemical indicators, the sensitivity and specificity of a single indicator can be improved. Therefore, by combining mt-dsRNA with existing biochemical indicators, we can more accurately and efficiently evaluate the occurrence and development of LN, and then formulate personalized treatment plans suitable for different LN patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 . Analysis of differential expression of serum mt-dsRNA between healthy controls and LN patients

[0044] Analysis of differential expression of serum mt-dsRNA between healthy controls and LN patients.

[0045] A: Eukaryotic transcriptome resequencing was performed on two groups of serum samples, and the relative quantitative method was used for analysis. The samples were from mixed samples of 10 healthy controls and 10 LN patients, and the reference genes were the average values ​​of RNR1, RNR2, GAPDH, and β-Actin. B: qPCR detection was performed on two groups of serum samples, and the relative quantitative method was used for analysis. The samples were from independent samples of 10 healthy controls and 10 LN patients, and the reference gene was RNR1.

[0046] Figure 2 .Analysis of differential expression of serum mt-dsRNA in healthy controls, inactive LN patients and active LN patients in the training set A: qPCR detection was performed on the three groups of training set samples, and the relative quantitative method was used for analysis. The samples came from 50 healthy controls, 12 inactive LN patients and 39 active LN patients, and the internal reference gene was RNR1. Inactive LN patients were defined as those who met the following criteria: baseline serum creatinine <1.24mg / dl, 24h urine protein <0.4g / 24h, serum creatinine <1.24mg / dl, 24h urine protein <0.4g / 24h, eGFR>90ml / min / 1.73m2, and antinuclear antibodies <1:100 when the samples were collected. The baseline value was the test result 180 days before the sample was collected. Active LN patients were defined as those who did not meet the above criteria.

[0047] BD: Analysis of the diagnostic value of mt-dsRNA expression in distinguishing healthy controls, inactive LN patients from active LN patients. The mt-dsRNAs included in the analysis included those with significantly increased expression levels in the serum of active LN patients compared with inactive LN patients in the training set, including ATP8, ATP6, COX2, ND6, COX3, ND1, ND4, and ND4L. B: ROC curves for distinguishing healthy controls from inactive LN patients were drawn by mt-dsRNA. The genes with excellent performance were COX2 and ND4. C: ROC curves for distinguishing healthy controls from active LN patients were drawn by mt-dsRNA. The genes with excellent performance were ATP8, ATP6, COX2, ND1, ND4, and ND4L. D: ROC curves for distinguishing inactive LN patients from active LN patients were drawn by mt-dsRNA. The genes with excellent performance were ATP8, ATP6, ND1, and ND4.

[0048] Figure 3 . Analysis of mt-dsRNA differential expression between inactive LN patients and active LN patients in the test set

[0049] A: Two sets of training set samples were tested by qPCR and analyzed by relative quantitative method. The samples came from 39 inactive LN patients and 132 active LN patients, and the internal reference gene was RNR1.

[0050] BC: Analysis of the diagnostic value of mt-dsRNA expression in distinguishing inactive LN patients from active LN patients. The mt-dsRNAs included in the analysis include COX2, ATP8, ND4, and ND4L. Among them, B: The above four mt-dsRNAs are used to draw ROC curves for distinguishing inactive from active LN patients. C: The above four mt-dsRNAs are used to draw ROC curves for distinguishing inactive from active LN patients. The genes with excellent performance are COX2 and ND4.

[0051] Figure 4 . Analysis of differential expression of mt-dsRNA between patients with low pathological activity and patients with high pathological activity

[0052] A: Analysis of mt-dsRNA expression in LN patients with different pathological activities. Samples were obtained from 42 patients with low pathological activity and 25 patients with high pathological activity. The degree of pathological activity was defined by the NIH-AI score. Patients with low pathological activity were defined as those with an NIH-AI score ≤ 7. Patients with high pathological activity were defined as those with an NIH-AI score > 7. BC: Analysis of the diagnostic value of mt-dsRNA expression in distinguishing patients with low pathological activity from those with high pathological activity. The mt-dsRNAs included in the analysis included ATP8, ATP6, COX2, ND1, ND4, and ND4L. Among them, B: ROC curves for distinguishing patients with low pathological activity from those with high pathological activity were drawn for ND1 alone. C: ROC curves for distinguishing patients with low pathological activity from those with high pathological activity were drawn for the combination of biochemical indicators, mt-dsRNA, and biochemical indicators and mt-dsRNA. The genes with excellent performance were ND1, ND4L, and ATP8.

[0053] Figure 5 Analysis of differential expression of serum mt-dsRNA in LN patients with different prognoses

[0054] A: The expression of mt-dsRNA in LN patients with different prognoses was analyzed. The samples were from 55 LN patients with complete remission, 16 LN patients with partial remission, and 29 LN patients with relapse. The prognosis was divided into three groups: complete remission, partial remission, and relapse. LN patients with complete remission were defined as those who met the following criteria: urine protein quantification <0.5g / 24h, serum albumin ≥35g / L, and blood creatinine <1.24mg / dl. LN patients with partial remission were defined as those who met the following criteria: urine protein decreased by more than 50% compared with the baseline value and urine protein quantification <3.0g / 24h, serum albumin >30g / L, and blood creatinine increased by no more than 25% of the baseline value. Recurrent LN patients were defined as those who met any of the following criteria: Urine protein: if the baseline value was <0.5g / 24h, it increased to ≥1g / 24h; if the baseline value was 0.5-1.0g / 24h, it increased to ≥2g / 24h; or if the baseline value was >1g / 24h, it increased ≥2 times; Serum creatinine: if the baseline value was <2mg / dl, it increased by more than 0.2mg / dl; if the baseline value was ≥2mg / dl, it increased by more than 0.4mg / dl. The baseline value was the test result 360 days before the sample was collected. Biochemical indicators included: 24h urine protein, serum creatinine, and serum albumin. BG: The diagnostic value of analyzing the expression of mt-dsRNA in distinguishing different prognostic situations of LN patients. The mt-dsRNAs included in the analysis included ATP8, ATP6, COX2, ND1, ND4, and ND4L. Among them, B: The above 6 mt-dsRNAs were separately drawn to distinguish the ROC curves of LN patients with complete remission and partial remission. C: The ROC curves for distinguishing LN patients with complete remission and partial remission were drawn by combining biochemical indicators, the above 6 mt-dsRNAs, and the above 6 mt-dsRNAs. The genes with excellent performance are COX2, ND4, ND4L, and ND1. D: The above 6 mt-dsRNAs were used to draw ROC curves for distinguishing LN patients with complete remission and relapse. E: The ROC curves for distinguishing LN patients with complete remission and relapse were drawn by combining biochemical indicators, the above 6 mt-dsRNAs, and the above 6 mt-dsRNAs. The genes with excellent performance are ND1, ND4L, and ND4. F: The ROC curves for distinguishing LN patients with partial remission and relapse were drawn by combining the above 6 mt-dsRNAs. G: The ROC curves for distinguishing LN patients with partial remission and relapse were drawn by combining biochemical indicators, the above 6 mt-dsRNAs, and the above 6 mt-dsRNAs. The genes with excellent performance are ATP8, ATP6, and ND4. DETAILED DESCRIPTION

[0055] Example 1 Elevated serum mt-dsRNA expression in LN patients

[0056] 1) In order to explore the potential of mt-dsRNA as a serum biomarker for LN, we mixed the sera of 10 LN patients and 10 sex- and age-matched healthy controls, extracted total RNA for sequencing, and performed relative quantitative analysis using the mean read counts of RNR1, RNR2, GAPDH, and β-Actin as internal references. The inclusion criteria for LN patients were: a) aged 12-70 years; b) LN patients confirmed by conventional light microscopy, immunofluorescence, and electron microscopy. The results showed that compared with the sera of healthy controls, the relative expression levels of most mt-dsRNAs in the sera of LN patients were significantly increased, including ATP6, COX1, COX2, CYTB, ND3, ND4, ND5, ND6, TA, TD, TE, TF, TG, TH, TI, TM, TN, TP, TQ, TS1, TS2, TT, TV, TW, and TY( Figure 1 A).

[0057] 2) To verify the sequencing results, we then performed independent RT-qPCR tests on the serum samples of the above 10 LN patients and 10 healthy controls, using RNR1 as an internal reference for relative quantitative analysis. Compared with healthy controls, the relative expression levels of most mt-dsRNAs with mRNA functions in LN patients were significantly increased, including COX2, ND4, COX3, ND4L, ATP8, COX1, ATP6, ND2, ND3, and ND6 ( Figure 1 B). This suggests the potential of serum mt-dsRNA as a biomarker for LN.

[0058] Example 2 Large sample validation found that COX2 and ND4 combined analysis performed well in distinguishing active and inactive LN patients

[0059] 1) Serum samples of 50 healthy controls, 12 inactive LN patients (iLN) and 39 active LN patients (aLN) were collected as training sets. iLN patients were defined as those who met the following criteria: baseline serum creatinine <1.24 mg / dl, 24h urine protein <0.4 g / 24h, serum creatinine <1.24 mg / dl, 24h urine protein <0.4 g / 24h, eGFR>90 ml / min / 1.73 m 2 , antinuclear antibody <1:100. The baseline value is the biochemical information 180 days before the sample was collected. aLN is defined as LN patients who do not meet one or more of the above criteria.

[0060] 2) The three groups of samples in the training set were tested by RT-qPCR and analyzed by relative quantitative method. Compared with the serum of healthy controls, the expression level of mt-dsRNA in the serum of LN patients was significantly increased. In the serum of LN patients, compared with iLN, the expression level of some mt-dsRNA in aLN was significantly increased, including ATP8, ATP6, COX2, ND6, COX3, ND1, ND4 and ND4L ( Figure 2 A). The results of the training set suggest that the above eight genes have great potential in distinguishing iLN from aLN compared with other mt-dsRNAs.

[0061] 3) Next, in order to analyze the performance of mt-dsRNA expression levels in distinguishing HC, iLN and aLN, we combined the above 8 genes and drew ROC curves for distinguishing HC from iLN, HC from aLN, and iLN from aLN ( Figure 2 BD). Through the combined analysis of the above 8 mt-dsRNAs, the combined formula for distinguishing HC from iLN was: Y = -1.762 + 0.836 * COX2 + 0.819 * ND4, and the area under the curve was 0.979; the combined formula for distinguishing HC from aLN was Y = 1.004 + 4.016 * ATP8 + 4.143 * ATP6 + 0.837 * COX2 + 0.745 * ND1 + 3.434 * ND4 + 1.408 * ND4L, and the area under the curve was 0.984; the combined formula for distinguishing iLN from aLN was Y = 4.308 + 3.749 * ATP8 + 1.944 * ATP6 + 1.403 * ND1 + 1.55 * ND4, and the area under the curve was 0.952. This suggests that in the training set, ND1, ND4, ATP6 and ATP8 performed well in distinguishing iLN from aLN, and ND4L and COX2 also performed well in distinguishing HC from iLN, and HC from aLN. This suggests that ND1, ND4, ND4L, COX2, ATP6 and ATP8 have the potential to become LN serum biomarkers, and the above six genes were included in subsequent experiments.

[0062] 4) In order to explore the potential of the above six mt-dsRNAs that performed well in distinguishing HC, iLN and aLN as LN serum biomarkers, we performed RT-qPCR on two groups of samples in the test set and analyzed them by relative quantification. The samples came from 40 iLN patients and 130 aLN patients, and the internal reference gene was RNR1. Compared with iLN patients, the expression levels of some mt-dsRNAs in aLN patients were significantly increased, including ATP6, COX2, ND1 and ND4 ( Figure 3A). This indicates that in the test set, the above four genes have great potential in distinguishing iLN from aLN. Next, in order to analyze the performance of mt-dsRNA expression levels in distinguishing iLN from aLN, we plotted the ROC curves of the above four genes for distinguishing iLN from aLN individually and jointly ( Figure 3 BC). The results showed that when COX2, ATP8, ND4 and ND4L were used to distinguish iLN from aLN, the areas under the curve were 0.667, 0.635, 0.613 and 0.588 respectively. When the above four genes were used for joint differentiation, COX2 and ND4 performed best, with an area under the curve of 0.905, and the joint formula was Y = 1.281 + 0.005 * ND4 + 0.402 * COX2. This shows that after verification of the training set and the test set, the joint analysis of COX2 and ND4 performed well in distinguishing iLN from aLN, and is expected to become a new biomarker to assist in the judgment of LN activity.

[0063] Example 3 Large sample validation found that ND1, ND4L and ATP8 performed well in distinguishing LN patients with different degrees of pathological activity

[0064] To verify the potential of mt-dsRNA as a serum biomarker for LN, we further performed differential expression analysis on the expression levels of mt-dsRNA in patients with low pathological activity and those with high pathological activity. First, we scored the pathological activity of LN patients using the National Institutes of Health's Lupus Nephritis Activity Index (NIH-AI) scoring system. Patients with low pathological activity LN were defined as patients with an NIH-AI score of ≤7, with a total of 42 patients. Patients with high pathological activity LN were defined as patients with an NIH-AI score of >7, with a total of 25 patients.

[0065] The results showed that compared with patients with low pathological activity LN, the expression level of ND1 in patients with high pathological activity LN was significantly increased ( Figure 4 A). Next, to analyze the performance of mt-dsRNA expression levels in distinguishing patients with low pathological activity from patients with high pathological activity, we plotted ROC curves for ND1 alone, biochemical information combined, mt-dsRNA combined, and biochemical information and mt-dsRNA combined to distinguish patients with low pathological activity from patients with high pathological activity ( Figure 4BC). The biochemical information included in the analysis included hemoglobin, serum creatinine (Scr), serum albumin, eGFR, 24h urinary protein (represented by proteinuria in the following formula), anti-C1q antibody, complement C3, complement C4, CD3 lymphocytes and CD4 lymphocytes. The mt-dsRNA included in the analysis included ND1, ND4, ND4L, COX2, ATP6 and ATP8 screened from the training set.

[0066] The results showed that when distinguishing patients with low pathological activity and high pathological activity of LN, the combined differentiation of biochemical information and mt-dsRNA performed best, with the combined formula of Y = -0.3030-0.31*proteinuria-0.036*Scr+0.004serum albumin+0.062*ND1+0.252*ND4L-0.728*ATP8, and the area under the curve was 0.95. ND1 alone distinguished, and the area under the curve was 0.705. The area under the curve of biochemical information combined differentiation was 0.8, and the area under the curve of ND1, ND4L and ATP8 three mt-dsRNA combined differentiation was 0.94. This suggests that the combined use of mt-dsRNA and biochemical information can enhance the ability to use serum samples to judge the pathological activity of LN. By integrating the expression levels of mt-dsRNA and changes in biochemical markers, we can more accurately evaluate the pathological activity status of LN patients and provide more accurate and personalized guidance for clinical treatment, which is of great significance for improving the treatment effect and quality of life of LN patients.

[0067] Example 4 Large sample validation found that ND1, ND4L, COX2, ND4, ATP6 and ATP8 performed well in distinguishing LN patients with different prognoses

[0068] To verify the potential of mt-dsRNA as a serum biomarker for LN prognosis, we performed differential expression analysis on the serum mt-dsRNA expression levels of LN patients with different prognoses. The samples were from 55 LN patients with complete remission, 16 LN patients with partial remission, and 29 LN patients with relapse.

[0069] Complete remission of LN patients was defined as those who met the following criteria: urine protein quantitative <0.5g / 24h, serum albumin ≥35g / L, and serum creatinine <1.24mg / dl. Partial remission of LN patients was defined as those who met the following criteria: urine protein decreased by more than 50% compared with the baseline value and urine protein quantitative <3.0g / 24h, serum albumin >30g / L, and serum creatinine increased by no more than 25% of the baseline value. Relapse of LN patients was defined as those who met any of the following criteria: urine protein: if the baseline value is <0.5g / 24h, it increases to ≥1g / 24h; if the baseline value is 0.5-1.0g / 24h, it increases to ≥2g / 24h; or if the baseline value is >1g / 24h, it increases by ≥2 times; serum creatinine: if the baseline value is <2mg / dl, it increases by more than 0.2mg / dl; if the baseline value is ≥2mg / dl, it increases by more than 0.4mg / dl. The baseline value is the test result 360 days before the sample is collected.

[0070] The results showed that compared with LN patients with complete remission, the expression levels of ATP8, ATP6, COX2, ND4, ND4L and ND1 were significantly increased in LN patients with partial remission and relapse. Compared with LN patients with partial remission, the expression levels of ATP8, ATP6, COX2, ND4 and ND4L in LN patients with relapse were significantly increased ( Figure 5 A). Next, in order to analyze the performance of mt-dsRNA expression levels in distinguishing LN patients with different prognoses, we plotted the ROC curves of mt-dsRNA alone, biochemical information combined with mt-dsRNA, and biochemical information and mt-dsRNA combined with different prognostic conditions ( Figure 5 BG). The biochemical information included in the analysis included hemoglobin, serum creatinine, serum albumin, eGFR, 24h urine protein, anti-C1q antibody, complement C3, complement C4, CD3 lymphocytes and CD4 lymphocytes. The mt-dsRNA included in the analysis included ND 1, ND4, ND4L, COX2, ATP6 and ATP8 screened from the training set.

[0071] The results showed that the combined differentiation of biochemical information and mt-dsRNA performed best in distinguishing LN patients with complete remission and partial remission. The combined formula was Y = -2.145 + 2.102 * proteinuria + 1.411 * Scr + 0.385 * ND4 + 0.193 * COX2 + 0.066 * ND4L + 0.277 * ND1, and the area under the curve was 0.938. When ND1, ND4, ND4L, COX2, ATP6 and ATP8 were used for differentiation separately, the areas under the curve were 0.716, 0.636, 0.639, 0.712, 0.729 and 0.721, respectively. When biochemical information was used for joint differentiation, the area under the curve was 0.869. When ND1, ND4, ND4L, COX2, ATP6 and ATP8 were used for joint differentiation, the area under the curve was 0.902.

[0072] When distinguishing LN patients with complete remission and relapse, the combined distinction of biochemical information and mt-dsRNA performed best, the combined formula was Y = -1.927 + 2.339 * proteinuria + 0.932 * Scr + 0.202 * ND4 + 0.311 * ND4L + 0.092 * ND1, and the area under the curve was 0.988. When ND1, ND4, ND4L, COX2, ATP6 and ATP8 were used for differentiation separately, the areas under the curve were 0.723, 0.694, 0.875, 0.868, 0.856, and 0.810, respectively. When biochemical information was used for joint differentiation, the area under the curve was 0.788. When ND1, ND4, ND4L, COX2, ATP6 and ATP8 were used for joint differentiation, the area under the curve was 0.944.

[0073] When distinguishing patients with partial remission and relapse of LN, the combined distinction of biochemical information and mt-dsRNA performed best, with the combined formula of Y=0.245+0.296*proteinuria+0.042*Scr-0.184*Serum albumin+1.32*ATP8+0.336*ATP6+0.206*ND4, and the area under the curve was 0.979. When ND1, ND4, ND4L, COX2, ATP6 and ATP8 were distinguished separately, the areas under the curve were 0.504, 0.583, 0.78, 0.804, 0.761, and 0.701, respectively. When biochemical information was used for joint distinction, the area under the curve was 0.802. When ND1, ND4, ND4L, COX2, ATP6 and ATP8 were used for joint distinction, the area under the curve was 0.894.

[0074] The above results show that the combined use of mt-dsRNA and biochemical information can enhance the ability to use serum samples to judge the prognosis of LN. This combined detection method provides doctors with more comprehensive and accurate information. By accurately judging the prognosis of LN, doctors can take intervention measures earlier, thereby delaying the progression of LN disease, reducing the incidence of complications, and improving the quality of life and survival rate of patients. Therefore, the combined use of mt-dsRNA and biochemical information has broad application prospects and important clinical value in the diagnosis and treatment of LN.

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Claims

1. Use of a reagent for detecting mitochondrial double-stranded RNA in the preparation of a serum-assisted diagnostic reagent for lupus nephritis and / or a prognosis-assisted diagnostic reagent, characterized in that: The mitochondrial double-stranded RNA is selected from any one or more of ND1, ND4, ND4L, COX2, ATP6 and ATP8.

2. The use according to claim 1, characterized in that The reagent for detecting mitochondrial double-stranded RNA is a specific primer for PCR amplification of mitochondrial double-stranded RNA; The specific primer sequences for PCR amplification of ND1 are shown in SEQ ID NO.1 and SEQ ID NO.2, the specific primer sequences for PCR amplification of ND4 are shown in SEQ ID NO.3 and SEQ ID NO.4, the specific primer sequences for PCR amplification of ND4L are shown in SEQ ID NO.5 and SEQ ID NO.6, the specific primer sequences for PCR amplification of COX2 are shown in SEQ ID NO.7 and SEQ ID NO.8, the specific primer sequences for PCR amplification of ATP6 are shown in SEQ ID NO.9 and SEQ ID NO.10, and the specific primer sequences for PCR amplification of ATP8 are shown in SEQ ID NO.11 and SEQ ID NO.

12.

3. The use according to any one of claims 1-2, characterized in that Use of a reagent for detecting mitochondrial double-stranded RNA ND4L and COX2 in the preparation of a serum auxiliary diagnostic reagent having any of the following functions: 1) Differentiate between healthy people and patients with inactive LN; 2) Differentiate between healthy people and patients with active LN; 3) Differentiate between patients with inactive LN and those with active LN.

4. The use according to any one of claims 1-2, characterized in that The use of reagents for detecting mitochondrial double-stranded RNA ND1, ND4L and ATP8 in the preparation of serum-assisted diagnostic reagents for distinguishing patients with low pathological activity LN from patients with high pathological activity LN; preferably the use of reagents for detecting mitochondrial double-stranded RNA ND1, ND4L and ATP8 in combination with reagents for detecting blood creatinine, serum albumin and 24-hour urine protein in the preparation of serum-assisted diagnostic reagents for distinguishing patients with low pathological activity LN from patients with high pathological activity LN.

5. The use according to any one of claims 1-2, characterized in that The use of a reagent for detecting at least three mitochondrial double-stranded RNAs among mitochondrial double-stranded RNA ND1, ND4, ND4L, COX2, ATP6 and ATP8, combined with or not combined with a reagent for detecting at least two indicators among serum creatinine, serum albumin and 24-hour urine protein in the preparation of a serum auxiliary diagnostic reagent for distinguishing the prognosis of lupus nephritis.

6. The use according to claim 5, characterized in that Use of reagents for detecting mitochondrial double-stranded RNA ND1, ND4, ND4L, COX2, ATP6 and ATP8 in the preparation of serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis with any of the following functions: 1) Distinguish LN patients with complete remission from those with partial remission; 2) distinguish LN patients with complete remission from those with relapsed LN; 3) Differentiate between LN patients with partial remission and LN patients with relapse.

7. The use according to claim 5, characterized in that The use of reagents for detecting mitochondrial double-stranded RNA ND1, ND4 and COX2 combined with reagents for detecting blood creatinine and 24-hour urine protein in the preparation of serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis. The serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis are used to distinguish between completely remitted LN patients and partially remitted LN patients.

8. The use according to claim 5, characterized in that The use of reagents for detecting mitochondrial double-stranded RNA ND1, ND4 and ND4L combined with reagents for detecting blood creatinine and 24-hour urine protein in the preparation of serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis. The serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis are used to distinguish between completely remitted LN patients and recurrent LN patients.

9. The use according to claim 5, characterized in that The use of reagents for detecting mitochondrial double-stranded RNA ATP6, ATP8 and ND4 combined with reagents for detecting blood creatinine, serum albumin and 24-hour urine protein in the preparation of serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis. The serum auxiliary diagnostic reagents for distinguishing the prognosis of lupus nephritis are used to distinguish between patients with partial remission of LN and patients with recurrent LN.

10. A serum-assisted diagnostic reagent and / or prognosis-assisted diagnostic reagent for lupus nephritis, characterized in that A reagent for detecting the mitochondrial double-stranded RNA as described in claim 1; preferably containing PCR-specific primers for detecting any one or more of ND1, ND4, ND4L, COX2, ATP6 and ATP8; and further preferably containing the primers as described in claim 2.

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