Application of LncRNA KIFAP3-5:1 in the preparation of drugs for preventing and treating diabetic nephropathy

Abstract

The present invention relates to the application of LncRNA KIFAP3-5:1 in drugs for the prevention and treatment of diabetic nephropathy. Experimental studies have demonstrated that, compared with the healthy control group, the expression of LncRNA KIFAP3-5:1 in the plasma of patients with diabetic nephropathy is significantly down-regulated. In vitro cell experiments have shown that up-regulating LncRNA KIFAP3-5:1 can significantly inhibit the EMT process of renal tubular epithelial cells. Therefore, LncRNA KIFAP3-5:1 can be used as a molecular target for the preparation of drugs for the application in the prevention and treatment of drugs, providing a new idea for the research and development of drugs for the prevention and treatment of diabetic nephropathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of LncRNA KIFAP3-5:1 in the preparation of drugs for preventing and treating diabetic nephropathy. Background Art

[0002] Diabetic nephropathy is proteinuria and progressive decrease in glomerular filtration rate (GFR) caused by long-term diabetes. Diabetic nephropathy is one of the most important complications of diabetic patients. The incidence rate in China is also on the rise, and it has become the second cause of end-stage renal disease, second only to various glomerulonephritis. Due to its complex metabolic disorders, once it develops into end-stage renal disease, it is often more difficult to treat than other kidney diseases. Therefore, timely prevention and treatment are of great significance for delaying diabetic nephropathy.

[0003] LncRNA is a non-coding RNA with a length greater than 200 nucleotides. Among the RNAs contained in cells, the proportion of lncRNA far exceeds that of well-known mRNA, miRNA, etc. The proportion of lncRNA is as high as more than 90%, while mRNA only accounts for about 2%. In recent years, studies have shown that lncRNA plays an important role in many physiological and pathological processes such as cell proliferation and differentiation, apoptosis and inflammation, and has become a research hotspot. More and more studies have shown that lncRNA is closely related to kidney diseases, including AKI and chronic kidney diseases. Therefore, lncRNA related to renal injury in diabetic nephropathy is of great significance for the preparation of related prevention and treatment drugs. It has far-reaching significance for the early diagnosis of DN, the research and development of molecular targeted drugs, the improvement of treatment methods, and the improvement of patient survival rate. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of LncRNA KIFAP3-5:1 in the preparation of drugs for preventing and treating diabetic nephropathy on the basis of the existing technology.

[0005] The second purpose of the present invention is to provide the application of lentivirus overexpressing LncRNA KIFAP3-5:1 in the preparation of drugs for preventing and treating diabetic nephropathy.

[0006] The third purpose of the present invention is to provide a pharmaceutical composition, which uses the above lentivirus overexpressing LncRNA KIFAP3-5:1 as an active ingredient or the main active ingredient, supplemented with a pharmaceutically acceptable carrier.

[0007] The technical solution of the present invention is as follows:

[0008] Application of LncRNA KIFAP3-5:1 in the preparation of drugs for preventing and treating diabetic nephropathy. The sequence of LncRNA KIFAP3-5:1 is SEQ ID NO.1, which is specifically as follows: CAGGC CCATT TTGCC ATACA TCTTT GAATA CAAATTCCTC ATTCC TATAT CCCTG CCTTC ACCCC TTCTTCTCCA TGCCC TCACT TGAAA AGCCA TCACCTGTTC TTCAC CTGTCCGGAT CCTTG CTGAT AAAAT TCAAG TCCCA GTTCC TCTGT AAAACTGGTCTCCCA CTTCA ACTAA CAGCA TGTTC TCTCT CTTCC TCTTCTGAAC TCCTT TTATC TGTTC TGTTTGATAC TTATT AATAT ATTATCTTGA ACTGT TGCTT.

[0009] When LncRNA KIFAP3-5:1 is used in the preparation of drugs for preventing and treating diabetic nephropathy, the drugs can be made into solid preparations or liquid preparations. Further, based on the technical solution provided by the present invention, the drugs can be made into injections, oral liquids, granules, powders, tablets or capsules.

[0010] In a preferred embodiment, LncRNA KIFAP3-5:1 uses lentivirus (LV) as a vector to prepare LncRNA KIFAP3-5:1 overexpressing lentivirus.

[0011] The present invention also provides the application of LncRNA KIFAP3-5:1 overexpressing lentivirus in the preparation of drugs for preventing and treating diabetic nephropathy.

[0012] In a preferred embodiment, the LncRNA KIFAP3-5:1 overexpressing lentivirus (rLV-EF1a-lncRNA(LOC102552829-201)-bGH polyA-CMV-mCherry-hGH polyA, abbreviated as LV-LncRNA KIFAP3-5:1) mentioned in the present invention, the preparation method comprises the following steps:

[0013] 1) Plasmid construction: ① Vector linearization: Using GV492 as the backbone (pCMV-Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin), digest it with BamHI and AgeI to obtain the linearized vector fragment. ② Obtaining the target fragment: Digest the chemically synthesized plasmid containing the target gene with BamHI / AgeI to obtain the target gene LncRNA KIFAP3-5:1 sequence, and PCR amplify the target gene fragment. ③ Ligation of the backbone and the target gene: Mix the linearized vector with the fragment, and under the action of T4 ligase, achieve the ligation of the fragment and the vector. ④ Transform Escherichia coli Trans1-T1, pick monoclonal colonies, and identify positive clones by PCR detection, enzyme digestion and sequencing.

[0014] 2) Co-transfect the constructed viral vector, viral packaging helper plasmid (Helper 1.0) and viral packaging helper plasmid (Helper2.0) into 293T packaging cells to package lentivirus. Amplify and collect the virus, and use ultracentrifugation for concentration and purification to obtain a high-concentration lentivirus stock solution, and measure the virus titer; Co-transfect 293T cells with the empty vector and the lentiviral vector according to the above steps to package the negative control lentivirus.

[0015] This invention was screened by gene chip and verified by qRT-PCR. The results showed that: The expression of LncRNA KIFAP3-5:1 in the plasma of diabetic nephropathy patients was significantly decreased, indicating that the down-regulation of LncRNA KIFAP3-5:1 was related to the formation of DN. Further, in vitro experiments, transfect the LncRNA KIFAP3-5:1 knockdown virus into human and murine renal tubular epithelial cells, and the results showed that: Down-regulating LncRNA KIFAP3-5:1 induced the EMT process in renal tubules. After overexpressing LncRNA KIFAP3-5:1 in renal tubular epithelial cells, the results showed that: Overexpressing LncRNA KIFAP3-5:1 could improve the EMT process. Thus, the role of LncRNA KIFAP3-5:1 in the EMT process of DN renal tubular cells was clarified from both positive and negative aspects. Next, constructing a diabetic nephropathy mouse model further confirmed that LncRNA KIFAP3-5:1 could improve renal interstitial fibrosis and renal tubular EMT, providing a new idea for the research and development of drugs for the prevention and treatment of DN.

[0016] The present invention also provides a pharmaceutical composition, which uses the above-mentioned LncRNA KIFAP3-5:1 overexpressing lentivirus as the active ingredient or the main active ingredient, supplemented with a pharmaceutically acceptable carrier. The pharmaceutical composition can be a solid preparation or a liquid preparation, especially an injection, oral liquid, granule, powder, tablet or capsule. Pharmaceutically acceptable excipients play a key role in the entire development process of formulation technology. In many formulations, excipients account for the majority, so to a large extent, the properties of excipients determine the properties of the formulation. Excellent excipients can enhance the stability of the main drug and extend the validity period of the medicament; they can regulate the release rate of the main drug in vivo and in vitro; they can change the absorption of the drug in the body and increase the bioavailability. The specific type of excipient is not limited.

[0017] Adopting the technical solution of the present invention, the advantages are as follows:

[0018] The present invention has confirmed the important role of LncRNA KIFAP3-5:1 in EMT of DN renal tubular cells, and upregulating LncRNA KIFAP3-5:1 can significantly improve renal tubular EMT. Therefore, LncRNA KIFAP3-5:1 can be used as a molecular target for preparing drugs for preventing and treating DN, providing a new idea for the research and development of drugs for preventing and treating DN. Description of the Drawings

[0019] Figure 1 shows the expression of LncRNA KIFAP3-5:1 in plasma samples of patients in the N group, T2DM group and DN group;

[0020] Figure 2 shows the expression change of LncRNA KIFAP3-5:1 in renal tubular epithelial cells induced by high glucose; among them, Figure 2 in (A) is the expression of LncRNA KIFAP3-5:1 in hRPTECs cells; Figure 2 in (B) is the expression of LncRNA KIFAP3-5:1 in mRTEC cells;

[0021] Figure 3 shows the verification of stable overexpression and knockdown of LncRNA KIFAP3-5:1; among them, Figure 3 in (A) and (B) are the expression conditions of LncRNA KIFAP3-5:1 in hRPTECs cells; Figure 3 in (C) and (D) are the expression conditions of LncRNA KIFAP3-5:1 in mRTEC cells;

[0022] Figure 4 shows the effect of knocking down KIFAP3-5:1 on EMT of hRPTECs cells and mRTEC cells under normal glucose culture; among them, Figure 4In (A) is the expression of EMT-related proteins in hRPTECs cells; Figure 4 In (B) is the statistical result of EMT-related proteins; Figure 4 In (C) is the expression of EMT-related proteins in mRTCE cells; Figure 4 In (D) is the statistical result of EMT-related proteins;

[0023] Figure 5 is the effect of overexpressing KIFAP3-5:1 on EMT of hRPTECs cells and mRTEC cells under high glucose culture; among them, Figure 5 In (A) is the expression of EMT-related proteins in hRPTECs cells; Figure 5 In (B) is the statistical result of EMT-related proteins; Figure 5 In (C) is the expression of EMT-related proteins in mRTCE cells; Figure 5 In (D) is the statistical result of EMT-related proteins;

[0024] Figure 6 is the effect of overexpressing KIFAP3-5:1 on renal function indexes of diabetic mice; among them, Figure 6 In (A) is the statistical result of BUN levels of mice in each group; Figure 6 In (B) is the statistical result of UP levels of mice in each group; Figure 6 In (C) is the statistical result of mAlb levels of mice in each group; Figure 6 In (D) is the statistical result of β2-MG levels of mice in each group; Figure 6 In (E) is the statistical result of NAG levels of mice in each group; data are expressed as Mean±SEM, n = 6; * P<0.05, ** P<0.01, compared with the db / m group; # P<0.05, ## P<0.01, compared with the db / db+LV-Control group;

[0025] Figure 7 is the effect of overexpressing KIFAP3-5:1 on renal interstitial fibrosis and renal EMT of diabetic mice; Figure 7 In (A) is the expression of EMT-related proteins in mice in each group; Figure 7 In (B) is the statistical result of EMT-related proteins in mice in each group; Figure 7 In (C) is HE staining, PAS staining, Masson staining, Sirius red staining in the renal cortex of mice in each group; Figure 7 In (D) is the positive area of Masson staining in the renal cortex of mice in each group; Figure 7 In (E) is the statistical result of the positive area of Sirius red staining in the renal cortex of mice in each group; Figure 7Statistical results of the positive areas of PAS staining in the renal cortex of mice in each group (F); data are expressed as Mean±SEM, n = 6; **P<0.01, compared with the db / m group; ##P<0.01, compared with the db / db+LV-Control group. Detailed implementation mode

[0026] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0027] Example 1 Differential expression of LncRNA KIFAP3-5:1 in the plasma of DN patients

[0028] 1. Sample source

[0029] According to the criteria of the Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes, 30 patients diagnosed with type 2 diabetes (T2DM) admitted to the hospital were collected, including 15 T2DM patients (normal albuminuria, urinary albumin creatinine ratio (UACR) < 30 mg / g) and 15 DN patients. At the same time, 15 non-diabetic healthy volunteers (N) were included as the control group. The clinical pathological data of the patients were recorded in detail. We obtained ethylenediaminetetraacetic acid (EDTA) whole blood coagulation from all patients and healthy controls. All blood samples were centrifuged at 4000 rpm for 10 min and then at 12000 rpm for 10 min in sequence. The free plasma in the supernatant was stored at -80 °C for further analysis. The collection of specimens and experimental operations complied with the operating procedures and ethical norms of clinical experiments.

[0030] 2. Extraction of total plasma RNA

[0031] (1) Thaw 200 μl of plasma samples;

[0032] (2) Add 1 ml of QIAzol Lysis reagent and pipette up and down to mix;

[0033] (3) Incubate at room temperature for 5 min;

[0034] (4) Add chloroform with the same volume as the plasma, tighten the ep tube, and shake vigorously for 15 s;

[0035] (5) Incubate at room temperature for 5 min; centrifuge at 4 °C, 12000 g, for 15 min;

[0036] (6) Transfer the upper aqueous phase to a new collection tube and add anhydrous ethanol with 1.5 times the volume of the aqueous phase;

[0037] (7) Take 700 μl of the sample and add it to the RNeasy MinElute spin column. Close the lid of the EP tube and centrifuge at 8500 g for 15 s at room temperature; Discard the liquid that passed through the spin column and repeat step (7) using the remaining sample;

[0038] (8) Add 700 μl of RWT buffer, 500 μl of RPE buffer, and 500 μl of 80% ethanol to the RNeasy MinElute spin column in sequence. Close the lid of the EP tube and centrifuge at 8500 g for 15 s at room temperature respectively. Discard the liquid that passed through the spin column;

[0039] (9) Place the RNeasy MinElute spin column into a new 2 ml collection tube; Open the lid of the spin column and centrifuge at full speed for 5 min to dry the membrane. Discard the liquid that passed through the spin column and the collection tube;

[0040] (10) Place the RNeasy MinElute spin column into a new 1.5 ml collection tube; Add 14 μl of nuclease-free water to the middle of the membrane in the spin column. Gently close the lid and centrifuge at 13000 r for 1 min to elute the RNA;

[0041] (11) Detect the RNA concentration and OD260 / 280 using NanoDrop.

[0042] 3. Plasma RNA reverse transcription reaction

[0043] (1) For genomic gDNA removal reaction, prepare the reaction system according to Table 1 below:

[0044] Table 1 gDNA removal reaction system

[0045]

[0046] (2) Incubate at 45 °C for 2 min, then immediately take it out and place it on ice.

[0047] (3) Prepare the reverse transcription mixture on ice according to Table 2 below. After mixing, place it on ice.

[0048] Table 2 Reverse transcription reaction system

[0049]

[0050] (4) Incubate at 25 °C for 3 min; Incubate at 45 °C for 10 min; Incubate at 85 °C for 5 min to inactivate the reverse transcriptase.

[0051] 4. The primer sequences used in the plasma qRT-PCR part are as follows:

[0052] KIFAP3-5:1 (human source)

[0053] Upstream: 5’-CAGGCCCATTTTGCCATACATCTT-3’

[0054] Downstream: 5’-CAGCAAGGATCCGGACAGGTGA-3’

[0055] β-actin (human origin)

[0056] Upstream: 5’-GCAAAGACCTGTACGCCAAC-3’

[0057] Downstream: 5’-AGTACTTGCGCTCAGGAGGA-3’

[0058] 5. Real-time fluorescence quantitative PCR of plasma samples

[0059] Add each reagent according to the reaction system in Table 3 below, mix well and centrifuge, then add to the PCR-96 well plate, seal the film and centrifuge again.

[0060] Table 3 qRT-PCR reaction system

[0061]

[0062] After adding the samples, place the PCR-96 well plate in the real-time fluorescence quantitative PCR instrument. The PCR program is as follows:

[0063] Pre-denaturation: 95°C, 2 min;

[0064] PCR reaction: 95°C, 15 s; 60°C, 30 s, 50 cycles;

[0065] Melting curve analysis: 95°C, 15 s; 60°C, 60 s; 95°C, 15 s;

[0066] Cooling: 40°C, 30 s.

[0067] After the reaction is completed, use Roche LightCycle 480 software to calculate the Ct value of the target gene in the sample, export the data, and calculate the relative level of KIFAP3-5:1 by the ΔΔCt method.

[0068] 6. Statistical analysis

[0069] Use SPSS 21.0 software to perform statistical analysis on the experimental data. Measurement data are expressed as mean ± standard deviation (mean ± SD). Paired T-test is used for comparison between two groups, one-way ANOVA is used for comparison of three groups and above, and LSD-t test is used for multiple comparisons. All experiments are repeated three times, and P < 0.05 is considered statistically significant.

[0070] 7. Experimental results

[0071] The results of QPCR are shown in Figure 1 , and the results showed that the expression level of LncRNA KIFAP3-5:1 in the plasma of DN patients was significantly lower than that of the control group (6.968±0.559 vs 1.037±0.202), and the difference was statistically significant (P<0.05).

[0072] Example 2 Expression changes of LncRNA KIFAP3-5:1 in high glucose-induced renal tubular epithelial cells.

[0073] 1. Cell culture

[0074] Human proximal tubular epithelial cells (hRPTECs; human sv40-transfected human proximal tubular epithelial cell line HK-2) were purchased from the American Type Culture Collection (Manassas, VA, USA), and mouse renal tubular epithelial cells (mRTECs) were purchased from Shanghai Lianmai Biotechnology Co., Ltd. All cells were cultured in a humid environment with 5% carbon dioxide at 37°C. The cells were cultured in DMEM medium containing 5.56 mmol / L d-glucose (normal glucose, NG), supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum (Grand Island, NY). These cells were passaged at 80%-90% confluence. To induce the DN model, we treated the cells with high glucose (HG) medium containing 30 mmol / L D-glucose for 48 h.

[0075] 2. Detection of the expression of LncRNA KIFAP3-5:1 in cells by fluorescence quantitative PCR

[0076] To detect the effect of high glucose on the expression of LncRNA KIFAP3-5:1 in renal tubular epithelial cells, the total RNA of each group of cells was extracted by the Trizol method, and fluorescence quantitative PCR was performed on the cells after 48 h of high glucose culture.

[0077] 3. Statistical analysis

[0078] SPSS 21.0 software was used to perform statistical analysis on the experimental data, and the measurement data were expressed as mean±standard deviation (mean±SD). The paired T-test was used for comparison between two groups, the one-way ANOVA was used for comparison of three groups and above, and the LSD-t test was used for multiple comparisons. All experiments were repeated three times, and P<0.05 was considered statistically significant.

[0079] 4. Experimental results

[0080] As Figure 2As shown, compared with the NG group, the expression level of LncRNA KIFAP3-5:1 in hRPTECs of the HG group was significantly decreased (P<0.05), suggesting that the downregulation of LncRNA KIFAP3-5:1 may be involved in the formation of DN.

[0081] Example 3 Effect of LncRNA KIFAP3-5:1 on EMT of Renal Tubular Epithelial Cells

[0082] 1. Cell culture

[0083] hRPTECs and mRTECs cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% P / S in an incubator at 37°C, 5% CO 2 , with a relative humidity of 90%. The medium was changed once every 2-3 days, and routine digestion and passage were carried out using 0.25% trypsin containing EDTA.

[0084] 2. Construction of LV-KIFAP3-5:1 and sh-KIFAP3-5:1

[0085] Lentiviruses for overexpressing and knocking down the expression of lncRNA KIFAP3-5:1 were purchased from Shanghai GeneChem Co., Ltd.

[0086] Specific process:

[0087] Plasmid construction: ① Vector linearization: For overexpression, using GV492 as the backbone (pCMV-Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin), digestion was carried out with BamHI and AgeI to obtain a linearized vector fragment; for knockdown, using GV248 as the backbone (pCMV-hU6-MCS-Ubiquitin-EGFP-IRES-puromycin), digestion was carried out with AgeI and EcoRI to obtain a linearized vector fragment; ② Obtaining of target fragments: Digest the chemically synthesized plasmid containing the target gene with BamHI / AgeI or AgeI / EcoRI to obtain the LncRNA KIFAP3-5:1 sequence of the target gene, and PCR amplify the target gene fragment. ③ Ligation of the backbone and the target gene: Mix the linearized vector and the fragment, and under the action of T4 ligase, achieve the ligation of the fragment and the vector. ④ Transform Escherichia coli Trans1-T1, pick monoclonal colonies, and identify positive clones by PCR detection, enzyme digestion and sequencing.

[0088] The constructed viral vector, viral packaging helper plasmid (Helper 1.0), and viral packaging helper plasmid (Helper 2.0) were co-transfected into 293T packaging cells to package lentivirus. The virus was amplified and collected, and then concentrated and purified by ultracentrifugation to obtain a high-concentration lentivirus preservation solution. The virus titer was measured. The empty vector and the lentiviral vector were co-transfected into 293T cells according to the above steps to package negative control lentivirus.

[0089] 3. Cell Transfection

[0090] To confirm the role of KIFAP3:5-1 in renal tubular lesions, we conducted in vitro experiments using hRPTECs and mRTECs cells simultaneously. In the KIFAP3:5-1 overexpression experiment, the two types of renal tubular epithelial cells cultured in HG were divided into two groups: (1) HG + vector group, in which cells were transfected with the control lentiviral vector; (2) HG + KIFAP3:5-1 group, in which cells were transfected with the KIFAP3:5-1 overexpression lentiviral vector. More specifically, hRPTECs were cultured to 40-50% confluence and then infected with the plasmid carrying the KIFAP3:5-1 gene in a humid atmosphere of 5% carbon dioxide for 48 h. Plasmid transfection was carried out using Lipofectamine 3000 (ThermoFisher, USA), and the vector was used as a control.

[0091] We constructed a KIFAP3-5:1 gene knockout cell model using KIFAP3-5:1 shRNA lentivirus. Specifically, hRPTECs and mRTECs cultured in NG were also divided into two groups: (1) NG + vector group, in which cells were transfected with the control lentiviral vector; (2) NG + KIFAP3:5-1 shRNA group, in which cells were transfected with the KIFAP3:5-1 shRNA lentiviral vector. Forty-eight hours after cell transfection, the subsequent experiments of this example of the present invention were carried out.

[0092] 4. Fluorescent Quantitative PCR Detection of the Expression of LncRNA KIFAP3-5:1 in Cells

[0093] To detect whether the expression of LncRNA KIFAP3-5:1 was successfully interfered, the present invention used the Trizol method to extract the total RNA of each group of cells, and fluorescent quantitative PCR detection was performed on the cells 48 h after transfection.

[0094] 5. Western Blot Analysis

[0095] Total proteins in renal cortex and cells were collected in lysis buffer and measured by bicinchoninic acid protein assay (BCA Protein Assay Kit, Pierce Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Equal amounts of proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane saturated with 1% bovine serum albumin at 37 °C for 1 h. The primary antibodies used were as follows: β-actin (USA, 1:10000), ZO-1 (USA, 1:1000), E-cadherin (BioWorld, China, 1:400), vimentin (Affinity, China, 1:1000), α-SMA (Affinity, China, 1:1000), PRRX-1 (USA, 1:5000). The gray scale of the bands was quantified by Odyssey instrument (Gene Company, USA) and normalized to the β-actin internal control.

[0096] 6. Statistical analysis

[0097] Data were expressed as Mean±SEM. The t-test was used for comparison between two groups, one-way analysis of variance (ANOVA) was used for comparison among multiple groups, and the LSD test or Dunnett's T3 test was used for pairwise comparison. The test level α = 0.05, and P < 0.05 was considered statistically significant.

[0098] 7. Experimental results

[0099] (1) Construction of lncRNA KIFAP3-5:1 overexpression and knockdown stable expression strains

[0100] The results of fluorescence quantitative PCR showed that in the two kinds of cells, compared with the normal group (NG), the expression of KIFAP3-5:1 in the empty vector knockdown group (NG / sh-Control) and the empty vector overexpression group (NG / LV-Control) did not change significantly, indicating that the lentiviral vector had no effect on the expression of KIFAP3-5:1. As Figure 3 shown, compared with the empty vector group (NG / sh-Control), the expression of KIFAP3-5:1 in the knockdown group (NG / sh-KIFAP3-5:1) decreased significantly. Compared with the empty vector group (NG / LV-Control), the expression of KIFAP3-5:1 in the overexpression group (NG / LV-KIFAP3-5:1) increased significantly. The above results showed that the construction of KIFAP3-5:1 knockdown and overexpression stable expression strains was successful.

[0101] (2) Effect of lncRNA KIFAP3-5:1 on EMT of hRPTECs cells

[0102] To observe the effect of KIFAP3-5:1 on EMT in hRPTECs and mRTECs cells, under NG (5.56 mmol / L) culture conditions, the expression of KIFAP3-5:1 was knocked down in hRPTECs and mRTECs cells, and we detected EMT-related proteins. The Western Blot results of the two types of cells are shown in Figure 4 As shown, compared with the empty vector group (NG / sh-Control), the expressions of vimentin and α-smooth muscle actin (α-SMA) in the knockdown group (NG / sh-KIFAP3-5:1) were significantly increased, while the expressions of tight junction protein ZO-1 and E-cadherin were significantly decreased. The above results indicate that knocking down KIFAP3-5:1 under NG culture conditions can increase the expression of mesenchymal marker proteins, reduce the expression level of epithelial marker proteins, and promote EMT in renal tubular epithelial cells.

[0103] To study the effect of overexpressing KIFAP3-5:1 on EMT in renal tubular epithelial cells induced by high glucose, after obtaining the stable overexpression strain of KIFAP3-5:1 and the empty vector, they were treated with HG (30 mmol / L) for 48 h, and then the expression levels of EMT-related proteins were detected. The Western blot results of the two types of cells are shown in Figure 5 As shown, compared with the empty vector group (HG / LV-Control), the expressions of Vimentin and α-SMA proteins in the overexpression group (HG / LV-KIFAP3-5:1) were significantly decreased, while the expressions of ZO-1 and E-Cadherin proteins were significantly increased. The above results indicate that overexpressing KIFAP3-5:1 can reduce the expression of mesenchymal marker proteins in renal tubular epithelial cells induced by high glucose, promote the expression of epithelial marker proteins, and improve EMT in renal tubular epithelial cells induced by high glucose.

[0104] Example 4 Effect of Overexpressing KIFAP3-5:1 on Renal Function and Renal Interstitial Fibrosis in db / db Mice

[0105] 1. Experimental animals:

[0106] (1) Healthy male SPF-grade db / m and db / db mice were provided by the Experimental Animal Center of Xuzhou Medical University. All animal experiment operations complied with the regulations of the Animal Experiment Ethics Committee.

[0107] (2) Randomly select at least 6 db / m mice as the normal control group (db / m group), and randomly divide the db / db mice into a model group (db / db group), a blank vector group (db / db + LV-Control group), and an overexpression group (db / db + LV-KIFAP3-5:1 group), with at least 6 mice in each group. Regularly raise them until they are 12 weeks old. Inject normal saline into the tail veins of the mice in the db / m group and db / db group, and inject lentiviral blank vector and KIFAP3-5:1 overexpression lentivirus into the tail veins of the mice in the blank vector group and overexpression group respectively. The virus injection dose is 6×107 TU per mouse, and regularly raise them until they are 16 weeks old.

[0108] 2. Collection of mouse blood and urine and detection of biochemical indicators

[0109] Use a capillary to collect 0.3 mL of blood from the orbital venous plexus of the mouse, let it stand for 2 hours, centrifuge at 2500 rpm for 15 minutes, take the serum and store it at 4°C for subsequent detection of indicators such as serum urea nitrogen. Use a mouse metabolic cage to collect the 24-hour urine of the mouse, centrifuge at 2500 rpm for 10 minutes, take the supernatant and store it at 4°C for subsequent detection of indicators such as β2-microglobulin (β2-MG), urinary protein concentration (UP), microalbuminuria (mAlb), N-acetyl-β-D-glucosaminidase (NAG), etc.

[0110] 3. Renal tissue pathological detection

[0111] The 4-μm sections of paraffin-embedded renal tissue are stained with HE, Sirius Red, Masson, and periodic acid-Schiff (PAS). After drying at 65°C for 30 minutes, the sections are dewaxed twice in xylene, 10 minutes each time, and rehydrated successively with 100% ethanol (I), 100% ethanol (II), 95% ethanol, 90% ethanol, 80% ethanol, and deionized water, 10 minutes each time. Subsequently, the sections are stained with HE, Masson, and PAS solutions. Randomly collect 20 pictures, and perform quantification by two blinded researchers to calculate the percentage of mesangial matrix occupying each glomerulus.

[0112] 4. Western blot:

[0113] Western blot is used to detect the protein expression levels of ZO-1, E-Cadherin, Vimentin, and α-SMA in renal tissue.

[0114] 5. Statistical analysis:

[0115] Data are expressed as Mean±SEM. The t-test was used for comparison between two groups, one-way analysis of variance (ANOVA) was used for comparison among multiple groups, and the LSD test or Dunnett’s T3 test was used for pairwise comparison. The significance level was set at α = 0.05, and P<0.05 was considered statistically significant.

[0116] 6. Experimental results:

[0117] The kit was used to detect blood urea nitrogen (BUN), urinary protein (UP), urinary microalbumin (mAlb), urinary β2-microglobulin (β2-MG) and urinary N-acetyl-β-D-glucosaminidase (NAG) in mice. The results showed ( Figure 6 ), compared with the db / m group, the levels of BUN, UP, mAlb, β2-MG and NAG in the db / db group were significantly increased, suggesting that the renal function of diabetic mice was significantly impaired. Compared with the db / db+LV-Control group, the above indexes in the db / db+LV-KIFAP3-5:1 group were significantly decreased. The above results indicate that overexpression of KIFAP3-5:1 has a certain improvement effect on renal function injury in diabetic mice.

[0118] To further observe the effect of overexpressing KIFAP3-5:1 on renal fibrosis and EMT in diabetic mice, Western Blot was used to detect EMT-related proteins. The results showed ( Figure 7 A, B), compared with db / db mice, the expressions of ZO-1 and E-Cadherin in the renal cortex of db / db mice treated with overexpression of KIFAP3-5:1 were significantly increased, while the expressions of Vimentin and α-SMA were significantly decreased. The above results indicate that overexpression of KIFAP3-5:1 can improve renal EMT in diabetic mice.

[0119] The pathological changes of mouse kidneys were evaluated by H&E, Masson, PAS and Sirius red staining ( Figure 7 C~F). The results showed that in the db / db mice, more tubular epithelial cell edema and degeneration were visible at the corticomedullary junction, the cells were swollen, and glycogen and collagen deposition were obvious. After overexpression of KIFAP3-5:1, the morphological structure of the kidney was improved, and the accumulation of collagen and glycogen was significantly reduced. The above results indicate that overexpression of KIFAP3-5:1 has a certain inhibitory effect on renal fibrosis in diabetic mice.

[0120] In summary, the present invention identified the differentially expressed LncRNA KIFAP3-5:1 in the plasma of DN patients and confirmed its involvement in the process of EMT in renal tubular cells from both positive and negative aspects. Therefore, LncRNA KIFAP3-5:1 can be used as a molecular target for the preparation of drugs for the prevention and treatment of diabetic nephropathy.

[0121] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of LncRNA KIFAP3-5:1 in the preparation of drugs for preventing and treating diabetic nephropathy, characterized in that, the sequence of the LncRNA KIFAP3-5:1 is as shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that, the LncRNA KIFAP3-5:1 uses lentivirus as a vector.

3. The use according to claim 1, characterized in that, the drug is made into a liquid preparation or a solid preparation.

4. The use according to claim 3, characterized in that, the drug is made into an injection, oral liquid, granule, powder, tablet or capsule.

5. Use of lentivirus overexpressing LncRNA KIFAP3-5:1 in the preparation of drugs for preventing and treating diabetic nephropathy, the sequence of the LncRNA KIFAP3-5:1 is as shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that, the drug inhibits the process of renal tubular EMT.

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