Use of shRNA specifically inhibiting expression of lncAI662270 in preparation of drugs for treating renal fibrosis

Abstract

The application discloses application of shRNA for specifically inhibiting expression of long-chain non-coding RNA AI662270 in preparation of a medicine for treating renal fibrosis or chronic kidney disease. Overexpression of non-coding RNA AI662270 in a mouse kidney can promote occurrence of renal fibrosis of the mouse. Inhibition of expression of lncRNA AI662270 in a ligated mouse kidney can relieve occurrence of renal fibrosis of the mouse, and provides a new target for treatment of renal fibrosis.

Description

Technical Field

[0001] This invention relates to the use of shRNA that specifically inhibits the expression of lncRNA AI662270 in the preparation of drugs for treating renal fibrosis. Background Technology

[0002] More than 10% of the world's adult population suffers from chronic kidney disease. Although chronic kidney disease progresses slowly, it becomes a serious health hazard and a drain on social medical resources once it progresses to end-stage renal disease. This also indicates that current treatments have limited effectiveness and can only slow disease progression. Renal fibrosis is a common pathological feature of various chronic kidney diseases, mainly manifested as glomerular sclerosis, tubular atrophy, tubulointerstitial fibrosis, inflammatory cell infiltration, and tubular microvascular loss. Fibrosis leads to excessive deposition of extracellular matrix and increased expression of fibronectin in the glomerular mesangium, tubulointerstitium, and Bowman's capsule [1,2]. The area of ​​fibrotic interstitial lesions is closely related to renal function and long-term prognosis. In renal tissue, fibrosis can occur in the glomeruli, known as glomerular sclerosis, or in the tubules, known as tubulointerstitial fibrosis [3,4]. Currently, there is a lack of effective treatments for renal fibrosis in clinical practice; therefore, there is an urgent need to develop new treatment methods to reverse disease progression. Summary of the Invention

[0003] The purpose of this invention is to provide the application of shRNA that specifically inhibits the expression of lncRNA AI662270 (long non-coding RNA AI662270) in the preparation of drugs for treating renal fibrosis, wherein the positive strand sequence of lncRNA AI662270 is shown in SEQ ID No. 1. The sequence of shRNA that specifically inhibits the expression of lncRNA AI662270 is shown in SEQ ID No. 2.

[0004] Another object of the present invention is to provide the use of shRNA that specifically inhibits the expression of lncRNA AI662270 in the preparation of drugs for treating chronic kidney disease. The sequence of the shRNA that specifically inhibits the expression of lncRNA AI662270 is shown in SEQ ID No. 2.

[0005] Beneficial effects: Overexpression of lncRNA AI662270 in mouse kidneys promotes renal fibrosis. Inhibition of lncRNA AI662270 expression in ligated mouse kidneys alleviates renal fibrosis, providing a novel target for the treatment of renal fibrosis. Attached Figure Description

[0006] Figure 1 qRT-PCR was used to verify the expression of lncRNA AI662270 in a mouse fibrosis model.

[0007] Figure 2 lncRNAAI662270 was overexpressed in mouse kidney tissue.

[0008] Figure 3 Knockdown of lncRNA AI662270 expression in a mouse model of renal fibrosis.

[0009] The symbols in the diagram are explained as follows:

[0010] UUO (Unilateral Uretera Obstruction) model refers to a model of unilateral ureteral obstruction.

[0011] IRI (Ischemia-reperfusion Injury) model refers to the ischemia-reperfusion model.

[0012] DN (Diabetic Nephropathy) model, diabetic nephropathy model

[0013] Sham refers to the sham surgery group.

[0014] The letters 'd' and 'day' indicate the number of days in the date.

[0015] 12W and 14W refer to 12 weeks and 14 weeks, respectively.

[0016] HE (hematoxylin-eosin staining) refers to the hematoxylin-eosin staining method.

[0017] Masson staining, also known as Masson staining.

[0018] pCI refers to empty carrier plasmid.

[0019] pCI-AI662270 refers to overexpression of the AI662270 plasmid.

[0020] Acta2 refers to an actin protein with several aliases, including α-actin, α-actin-2, aortic smooth muscle, or α-smooth muscle actin (α-SMA).

[0021] ColⅠ refers to type I collagen

[0022] GAPDH refers to internal reference.

[0023] UUO+shNC refers to the delivery of NC plasmids into the kidney tissue of mice with renal fibrosis.

[0024] UUO+AI662270shRNA refers to the delivery of the AI662270 knockdown plasmid into the kidney tissue of mice with renal fibrosis. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] Primer list

[0027]

[0028]

[0029] Antibody list

[0030]

[0031] Materials Methods

[0032] 1. Ultrasound-mediated plasmid delivery to the kidney

[0033] To construct the lncAI662270 overexpression plasmid, total RNA was first extracted from mouse tissues and reverse transcribed into cDNA for later use. Primers were designed based on the lncAI662270 cDNA sequence from NCBI to amplify lncAI662270. The size of the target fragment was verified by agarose gel electrophoresis and the fragment was recovered. The target fragment was ligated into the eukaryotic expression vector pCI containing the CMV promoter using homologous recombination. Colony PCR was performed on the obtained colony plates for verification. After successful verification, the plasmid was extracted by shaking and sent to Sangon Biotech Co., Ltd. for sequencing, finally yielding the pCI-AI662270 plasmid. The lncAI662270 shRNA / shNC plasmid vector was pGPU6 / GFP / Neo, and the target sequence was 5'-gctgtagattgctggtaatgt-3' (SEQ ID No. 9), synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0034] The sequence of lncAI662270 is shown in SEQ ID No. 1, and the sequence of shDNA constructed into the lncAI662270shRNA plasmid is shown in SEQ ID No. 2. The plasmids were directly delivered to the kidneys via ultrasound-mediated delivery to achieve overexpression and knockdown of the target gene in the kidneys. pCI, pCI-AI662270, lncAI662270shRNA, and lncAI662270shNC plasmids were prepared at concentrations of 2 μg / μL. The plasmids and injection-grade sulfur hexafluoride microbubbles were mixed at a volume ratio of 1:1. 200 μL of the mixture was injected via the tail vein. Simultaneously, the left kidney area of ​​the mice was shaved clean to expose the surface skin. After applying coupling agent, ultrasound was continuously administered for 5 minutes at an output power of 1W. The pCI and pCI-AI662270 plasmids were delivered to the left kidneys of wild-type mice weekly for two months. In the experiment of knocking down lncAI662270 in a mouse renal fibrosis model induced by UUO, lncAI662270 shNC and lncAI662270 shRNA plasmids were delivered to the kidneys immediately after surgery, and samples were collected 7 days later.

[0035] 2. Western blot assay for proteins

[0036] (1) Extraction of tissue protein (2) Preparation of SDS-polyacrylamide gel (3) Blocking and antibody incubation.

[0037] 3. Real-time quantitative PCR experiment

[0038] (1) RNA extraction (2) Real-time qPCR experiment: Take 1 μg of RNA and use Novizan III. The 1st Strand cDNA Synthesis Kit (+gDNA wiper) reverse transcription system was used to reverse transcribe RNA into cDNA. Novizan was used for qPCR experiments. The qPCR Master Mix (High ROX Premixed) dye-based quantitative PCR detection kit is used to detect changes in gene expression levels.

[0039] 4. Pathological analysis

[0040] First, mouse kidney tissue was collected and preserved by paraffin embedding. (1) HE staining: The tissue samples were first dewaxed and hydrated. The hydrated sections were placed in hematoxylin staining solution for 3 min, then washed twice with ddH2O, and then filtered through a gradient of ethanol and alcohol-soluble eosin for 1 min. The stained sections were dehydrated, mounted, and dried before images were captured using a microscope. (2) Masson staining: After dewaxing, the tissue samples were stained using the Simbrosen Masson staining kit. The stained sections were dehydrated and mounted.

[0041] 5. Plasmid extraction

[0042] All plasmids used in the experiment were extracted according to the kit instructions. The kits used were the Plasmid Mini-Prep Kit (Kit Catalog No.: DP103) and the Endotoxin-Free Plasmid Large-Scale Prep Kit (Kit Catalog No.: DP117).

[0043] Example 1: Expression of lncRNA AI662270 in normal and fibrotic kidney tissues

[0044] Materials: Healthy male ICR (20±2g) wild-type mice, SPF grade. Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. TRIZOL purchased from Thermo Fisher Scientific. ChamQ Universal SYBRqPCR Master Mix real-time quantitative PCR reagent purchased from Novizumi Biotechnology Co., Ltd. Sodium pentobarbital purchased from Nanjing Shengxing Biotechnology Co., Ltd. Routine chemical reagents used in the experiment were purchased from Nanjing Wanqing Chemical Glassware Instrument Co., Ltd. Anti-fluorescence quenching mounting solution purchased from Beyotime Biotechnology Co., Ltd.

[0045] Two methods

[0046] (1) Establishment of an animal model of renal interstitial fibrosis (UUO)

[0047] Healthy male ICR (20±2g) wild-type mice, SPF grade. This experiment was divided into a model group and a sham-operated group, with 6 mice randomly assigned to each group. All mice were intraperitoneally injected with sodium pentobarbital (50mg / kg). After anesthesia and stable respiration, the operating table was disinfected with 75% alcohol, and the mice were fixed in place. The abdomen was shaved, and then disinfected with 75% alcohol. The outer layer of skin was incised along the midline of the abdomen, and the linea alba was carefully cut open. Two 4-0 silk sutures were tied approximately 15mm below the renal pelvis, and the wound was sutured using a layer-by-layer suture method. In the sham-operated group, only the abdominal cavity was opened, but the ureter was not ligated; the wound was sutured layer by layer.

[0048] (2) Establishment of an animal model of diabetic nephropathy (DN)

[0049] Healthy male C57BL / 6J (20±2g) wild-type mice, SPF grade. The specific method for constructing a streptozotocin (STZ)-induced type 1 diabetic nephropathy model is as follows: First, prepare a sodium citrate buffer solution with a pH of 4.5. STZ was administered intraperitoneally at a dose of 50 mg / kg for 5 consecutive days. The experimental control group received an equal volume of sodium citrate buffer solution. After 10 days, tail clipping was performed, and fasting blood glucose levels were measured. Mice with blood glucose levels ≥11.1 mmol / L were selected for diabetic nephropathy.

[0050] (3) Establishment of animal models of ischemia-reperfusion (IRI)

[0051] Healthy male C57BL / 6J (20±2g) wild-type mice, SPF grade. This experiment was divided into a model group and a sham-operated group, with at least 6 mice randomly assigned to each group. All mice were intraperitoneally injected with sodium pentobarbital (50mg / kg). After the anesthesia stabilized, the operating table was disinfected with 75% alcohol, and the mice were fixed in place. Abdominal hair was shaved, and the area was disinfected with 75% alcohol. The outer layer of abdominal skin was cut, and the suture linea alba was further cut. The renal arteries of both kidneys were quickly located using sterile cotton swabs and clamped with sterile arterial clamps. The abdomen was covered with sterile gauze and suspended in a 37.3℃ water bath. After 30 minutes of ischemia, the arterial clamps were removed to restore blood flow, and the color change of the kidneys was observed. After repositioning, 300μL of physiological saline was injected, and the wound was sutured layer by layer. The mice were returned to clean cages after regaining consciousness. In the sham-operated group, only the abdominal cavity was opened; the renal arteries were not clamped, and the wound was sutured layer by layer.

[0052] (4) Real-time quantitative PCR was used to verify the expression changes of lncRNA AI662270.

[0053] After RNA extraction using the Trizol method, 1 μg of RNA was used with Novizan. III. The 1st Strand cDNA Synthesis Kit (+gDNA wiper) reverse transcription system was used to reverse transcribe RNA into cDNA. Novizan was used for qPCR experiments. The qPCR Master Mix (High ROX Premixed) dye-based quantitative PCR detection kit was used to detect changes in the expression level of lncRNA AI662270.

[0054] Genomic DNA removal:

[0055] RNase-free ddH2O to 16μl4×gDNA wiper Mix 4μl

[0056] Template RNA 1μg

[0057] Mix thoroughly by pipetting, 42℃ / 2min

[0058] Reverse transcription reaction: 5×HiScriptⅢqRT SuperMix 4μl

[0059] 16 μl of reaction solution from step 1

[0060] Reverse transcription conditions: 37℃ for 15 min, 85℃ for 5 s

[0061] The cDNA obtained from reverse transcription is used in the next step of the reaction; qPCR system and reaction conditions.

[0062]

[0063] qPCR reaction conditions: 95℃ for 5 min, 95℃ for 15 s, 60℃ for 30 s, 72℃ for 45 s, 40 cycles.

[0064] Three results

[0065] lncRNA AI662270 was significantly upregulated in the UUO model starting from day 3. Figure 1 A). Starting from day 3 of the IRI model, the expression level of lncAI662270 also showed a significant upward trend. Figure 1 B). In the DN model, the expression level of lncAI662270 was upregulated after weeks 12 and 14 of streptozotocin injection. Figure 1 C). Therefore, from the perspective of lncRNA, we continue to study the function and molecular mechanism of lncAI662270 in the process of renal fibrosis.

[0066] Figure 1 The results shown in Figures A and C indicate that lncRNA AI662270 was significantly upregulated in a mouse model of renal fibrosis. (A) Real-time quantitative PCR was used to detect changes in the expression of lncRNA AI662270 in kidney tissue of a UUO model, with the sham group serving as a control. (B) Real-time quantitative PCR was used to detect changes in the expression of lncRNA AI662270 in kidney tissue of an IRI model, with the sham group serving as a control. (C) Real-time quantitative PCR was used to detect changes in the expression of lncRNA AI662270 in kidney tissue of a DN model, with the sham group serving as a control.

[0067] Example 2: Expression of lncAI662270 in mouse kidney tissue induces renal fibrosis.

[0068] Total RNA was extracted from mouse kidney tissue, and the cDNA sequence of lncAI662270 was amplified by RT-PCR. This cDNA sequence was cloned into the eukaryotic expression vector pCI (pCI plasmid promoter is CMV) to obtain the pCI-AI662270 recombinant plasmid. The plasmid was encapsulated in sulfur hexafluoride microbubbles (ultrasound contrast agent) and delivered to the left kidney of mice via ultrasound-mediated delivery. The experiment was conducted for two months, with plasmid delivery once a week to maintain lncAI662270 expression. The left kidney of mice was harvested eight weeks after plasmid delivery for real-time quantitative PCR detection. Figure 2 B). Mice were sacrificed two months after plasmid delivery. HE and Masson staining showed that after overexpression of lncAI662270, extracellular collagen deposition, tubular atrophy, and renal interstitial fibrosis occurred in mouse kidney tissue. Figure 2 A). Western blot results showed that Collagen I expression was increased in kidney tissue after overexpression of lncAI662270. Figure 2 D). Activation of myofibroblasts is a key factor leading to renal interstitial fibrosis. qRT-PCR results showed a significant upregulation of α-SMA expression. Figure 2 C). The above results indicate that the pCI-AI662270 plasmid, after being delivered to mouse kidney tissue, can promote the activation of fibroblasts, thereby inducing renal interstitial fibrosis.

[0069] Figure 2 The results shown in Figures A and D indicate that lncRNA AI662270 expression induces fibrosis in mouse kidney tissue. (A) HE and Masson assays detect the pathological state and collagen deposition in kidney tissue. Scale bar: 100 μm. (B) Real-time quantitative PCR detects the expression changes of lncRNA AI662270 after delivery of the overexpression plasmid pCI-AI662270 to kidney tissue. (C) Real-time quantitative PCR detects the expression changes of α-SMA in kidney tissue after overexpression of lncRNA AI662270. (D) Western blotting detects changes in ColⅠ protein expression levels. pCl is the empty vector plasmid in the figure.

[0070] Example 3: Knockdown of lncAI662270 in the UUO model inhibits the occurrence of renal fibrosis.

[0071] The above results indicate that lncAI662270 is closely related to the occurrence of renal fibrosis. To further explore the relationship between the two, a plasmid capable of expressing lncAI662270 shRNA in mammals was constructed. Immediately after UUO surgery, the lncAI662270 shRNA plasmid was delivered to the ligated left kidney via ultrasound. Seven days later, the mice were sacrificed, and left kidney tissue was collected. qRT-PCR showed a decrease in the expression level of lncRNAAI662270. Figure 3 C). After paraffin embedding, HE and Masson staining were performed. The staining results showed that the UUO model group and the plasmid control group had severe renal fibrosis, while the degree of renal fibrosis in mice with knockdown of lncAI662270 was somewhat alleviated and the collagen deposition area was reduced. Figure 3 A). Western blot results also showed that knocking down lncAI662270 reduced the degree of fibrosis, and Collagen I expression was subsequently downregulated. Figure 3 B). Quantitative analysis of immunofluorescence staining area showed that knocking down lncAI662270 significantly reduced the fluorescent staining area of ​​α-SMA protein. Figure 3 D). This shows that the expression changes of lncAI662270 during UUO-induced renal fibrosis in mice are closely related to the proliferation and activation of interstitial fibroblasts and macrophage infiltration.

[0072] In a UUO-induced renal fibrosis model, knocking down lncAI662270 expression reduced fibroblast activation and decreased extracellular matrix collagen secretion, thus alleviating renal fibrosis. These results suggest that lncAI662270 may regulate the development and progression of renal interstitial fibrosis by promoting fibroblast activation and proliferation.

[0073] Figure 3 The results shown in AD indicate that knockdown of lncRNA AI662270 inhibits fibrosis in the UUO model. (A) HE and Masson assays were used to detect the pathological state and collagen deposition in renal tissue. Scale bar: 100 μm. (B) Western blotting was used to detect changes in ColI expression in mice after delivery of lncAI662270 shRNA plasmid to the UUO model. (C) Real-time quantitative PCR was used to analyze changes in lncAI662270 expression in mice after delivery of lncAI662270 shRNA plasmid to the UUO model. (D) Image-Pro-Plus was used to quantitatively analyze the α-SMA fluorescence staining area.

[0074] References

[0075] [1]AJDixon,J.Burns,MSDunnill,JOMcGee,Distribution of fibronectin in normal and diseased human kidneys,J Clin Pathol 33(11)(1980)1021-8.

[0076] [2] A. Van Vliet, HJ Baelde, LJ Vleming, E. de Heer, JA Bruijn, Distribution of fibronectin isoforms in human renal disease, J Pathol 193(2)(2001)256-62.

[0077] [3] K. Reidy, HM Kang, T. Hostetter, K. Susztak, Molecular mechanisms of diabetic kidney disease, J Clin Invest 124(6)(2014)2333-40.

[0078] [4] JSDuffield, Cellular and molecular mechanisms in kidney fibrosis, J Clin Invest 124(6)(2014)2299-306.

[0079] The above-mentioned experimental examples are merely preferred embodiments of the present invention and do not limit the present invention in any way. It should be noted that for those skilled in the art, any technical solutions obtained by equivalent substitution or equivalent transformation, or any improvements and modifications made without departing from the principle of the present invention, are all within the protection scope of the present invention.

Claims

1. The application of shRNA that specifically inhibits the expression of lncRNA AI662270 in the preparation of drugs for treating renal fibrosis, wherein the sequence of the shRNA that specifically inhibits the expression of lncRNA AI662270 is shown in SEQ ID No. 2.

Images