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Application of circular RNAhsa_circ_0007015 in preparation of medicine for preventing and treating chronic kidney disease

A chronic kidney disease, circular technology, applied in the direction of single-stranded DNA virus, DNA / RNA fragments, gene therapy, etc., to achieve the effect of protecting damage, preventing and treating renal fibrosis, and strong specificity

Active Publication Date: 2021-12-03
THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

完整序列为:5'-CTATCTTCCAGAGGGCCACACTGGGCATGGACACCCTTTTCCCTGCCTGGAGGAGCACAGGTGATAGTGTAATTTTCCAGTCACGAAACTGCTAAGGCCATCTCAGGGGCGTGTGCGCCAGGATAGGCGGGCGGCGTCCGAGGACCACATAGCCATGCCTTTTGGTCTGAAGCTCCGCCGGACACGGCGCTACAACGTCCTGAGCAAGAACTGCTTTGTCACACGGATTCGCCTGCTGGACAGCAATGTTATCGAGTGCACGCTGTCGGTGGAAAGCACAGGGCAAGAATGCCTGGAGGCTGTGGCCCAGAGGCTGGAGCTGCGAGAGACGCACTACTTTGGCCTTTGGTTTCTCAGCAAGAGCCAGCAAGCACGATGGGTGGAGCTGGAGAAACCTCTGAAGAAACATCTGGACAAATTCGCTAATGAGCCTTTGCTTTTCTTTGGAGTCATGTTCTATGTGCCAAATGTGTCATGGCTTCAGCAAGAGGCCACAAG-3'目前hsa_circ_0007015基因在肾脏纤维化发生发展中的作用级相关机制还未见报道,在肾脏纤维化基因治疗中的应用还欠明确,因此,研究一种hsa_circ_0007015 Gene-related drugs are necessary and unique for the treatment of renal fibrosis

Method used

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  • Application of circular RNAhsa_circ_0007015 in preparation of medicine for preventing and treating chronic kidney disease
  • Application of circular RNAhsa_circ_0007015 in preparation of medicine for preventing and treating chronic kidney disease
  • Application of circular RNAhsa_circ_0007015 in preparation of medicine for preventing and treating chronic kidney disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Circular RNA sequencing identification of hsa_circ_0007015 gene

[0033] By designing PCR primers on both sides of the hsa_circ_0007015 reverse junction site, the sequences on both sides of the hsa_circ_0007015 circularization site were amplified, and the accurate circularization site of hsa_circ_0007015 was obtained by sanger DNA sequencing. The PCR primer sequences are as follows:

[0034] hsa_circ_0007015_F:5’-TCGGAGTCATGTTCTATGTGC-3’

[0035] hsa_circ_0007015_R:5’-CCTGAGATGGCCTTAGTAGTTTT-3’

[0036] PCR uses mouse kidney cDNA as a template, amplification system and conditions are: 2×PCR MIX (Vazyme Company) 20ul, upstream and downstream primers (10mM) 1ul each, cDNA template 2ul, sterilized water to make up 40ul system; 94°C for 2min Pre-denaturation, within 35 cycles: denaturation at 98°C for 30s, annealing at 58°C for 20s, extension at 68°C for 30s, extension at 68°C for 5min after the cycle, and storage at 4°C. Purify the PCR product, perform sanger...

Embodiment 2

[0038] Example 2: Overexpression of the hsa_circ_0007015 gene in renal fibrosis

[0039] qPCR method was used to detect hsa_circ_0007015 gene in normal mouse kidney, two kinds of fibrosis model mouse kidney (mouse ischemia-reperfusion and unilateral ureteral obstruction model), human renal tubular epithelial cell line HK2, HK2 stimulated by TGFβ expression in . Use the RNA extraction kit (Yishan Company) to extract total RNA from tissues or cell lines, take 1ug of RNA and reverse-transcribe it into cDNA, and then use the cDNA as a template for SYBR fluorescent dye qPCR detection, and use the above PCR primer sequence to detect the number of amplification cycles. Using GAPDH as an internal reference gene for standardization and comparison, the results are shown in figure 2 . The results showed that the expression of hsa_circ_0007015 gene was increased in the kidneys of two fibrosis model mice, suggesting the correlation between hsa_circ_0007015 gene and renal fibrosis. At t...

Embodiment 3

[0040] Example 3: Preparation of targeted inhibition AAV-9 targeting hsa_circ_0007015 gene

[0041] 1. Preparation of AAV9-hsa_circ_0007015-shRNA

[0042] AAV9-hsa_circ_0007015-shRNA vector was purchased from Hanbio Technology (Shanghai) Co., Ltd. The virus type used for adeno-associated virus packaging is AAV9, and the shRNA sequence of hsa_circ_0007015 (sense strand:

[0043] 5'-AATTCGCAGGAGGCCACAAGCTGTCTTCAAGAGAGACAGCTTGTGGCCTCCTGTTTTTTG-3'; Antisense strand: 5'-GATCCAAAAAACAGGAGGCCACAAGCTGTCTCTCTTGAAGACAGCTTGTGGCCTCCTGCG-3') to obtain the target vector plasmid, and then co-transfected with pAAV-RC vector plasmid and pHelper vector plasmid into 293T cells, and the virus was collected and purified to obtain Adeno-associated virus AAV9-hsa_circ_0007015-shRNA.

[0044] 2. In vivo injection of AAV9-hsa_circ_0007015-shRNA

[0045] SPF grade 8-10 week old C57BL / 6J male mice were selected for virus injection. The mice were anesthetized by intraperitoneal injection of 1% pentoba...

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Abstract

The invention discloses an application of circular RNAhsa_circ_0007015 in preparation of a medicine for preventing and treating a chronic kidney disease. The target inhibitor of the hsa_circ_0007015 gene can be used for inducing the silencing of the hsa_circ_0007015 gene through RNA (Ribonucleic Acid) interference, so that the occurrence of renal fibrosis is reduced. According to the gene therapy method, expression of fibrosis-related proteins such as [alpha]-SMA, Collagen I and FN in renal tubular epithelial cells can be remarkably reduced, and meanwhile, the renal fibrosis degree after ischemia-reperfusion injury and unilateral ureteral obstruction of adult mice is remarkably improved. Therefore, a targeted inhibitor of the hsa_circ_0007015 gene can play a role in protecting the kidney, and the hsa_circ_0007015 gene serving as a novel gene medicine can be applied to the aspects of treating chronic kidney diseases, relieving the renal fibrosis degree, improving the renal function and the like.

Description

(1) Technical field [0001] The present invention relates to the application of circular RNA hsa_circ_0007015 in the preparation of medicines for preventing and treating chronic kidney disease. (2) Background technology [0002] Chronic kidney disease (chronic kidney disease, CKD) is defined as chronic renal structural or functional damage caused by various reasons, which progresses slowly over a period of months to years, eventually leading to chronic renal failure or even death. The incidence of CKD is increasing year by year, and it is a major public health problem faced by all countries. CKD can be secondary to hypertension, diabetes, obesity, and kidney damage from aging. In China, the prevalence of CKD has reached 10.8%. Patients with CKD stage 4 or above need to receive renal replacement therapy, including peritoneal dialysis, hemodialysis, and kidney transplantation, which brings enormous pressure to patients and society. Therefore, effective prevention and treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N7/01C12Q1/6883G01N33/58G01N33/68C12Q1/6841A61K48/00A61P13/12
CPCC12N15/113C12N7/00C12Q1/6883G01N33/582G01N33/68C12Q1/6841A61K48/005A61K48/0008A61P13/12C12N2310/14C12N2310/532C12N2750/14121Y02A50/30
Inventor 韩飞聂琬云林伟强王毓茜张颖
Owner THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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