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Application of PYCR1 gene as target in preparation of esophageal cancer product and related product

A technology of esophageal cancer and genes, applied in the field of biomedical research, can solve the problem of unknown role of PYCR1, and achieve the effect of promoting apoptosis, inhibiting proliferation, and inhibiting invasion and metastasis of esophageal cancer cells

Pending Publication Date: 2022-03-08
LANZHOU UNIV SECOND HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the role of PYCR1 in esophageal cancer is unknown

Method used

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  • Application of PYCR1 gene as target in preparation of esophageal cancer product and related product
  • Application of PYCR1 gene as target in preparation of esophageal cancer product and related product
  • Application of PYCR1 gene as target in preparation of esophageal cancer product and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1 Preparation of RNAi lentivirus against human PYCR1 gene

[0113] 1. Screening for effective siRNA targets against the human PYCR1 gene

[0114] Retrieve PYCR1 (NM_001282281) gene information from Genbank; design effective siRNA targets for PYCR1 gene. According to the principle of RNA interference sequence design, multiple 19-21nt RNA interference target sequences were designed using the CCT7 gene as a template. After evaluation and determination by design software, the following sequences were selected as interference targets (Table 1-1).

[0115] Table 1-1 is targeted at the siRNA target sequence of human PYCR1 gene

[0116]

[0117] 2. Preparation of lentiviral vector

[0118] Synthesize double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I restriction endonucleases at both ends of the siRNA target site; act on the GV493-gcGFP vector (Shanghai (provided by Jikai Gene Chemical Technology Co., Ltd.) to make it linearized, and agar...

Embodiment 2

[0138] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0139] Human esophageal cancer KYSE-150 cells and human esophageal cancer TE-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection value (MOI, A549:10), an appropriate amount of PYCR1-siRNA lentivirus prepared in Example 1 was added to the experimental group, and the control lentivirus was added to the control group, and the culture medium was replaced after 24 hours of cultivation, until the infection time reached 5 Days later, cells were collected. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 2-1 for the reverse...

Embodiment 3

[0148] Example 3 Western Blotting method to detect gene silencing efficiency

[0149] 1. Extraction of total cell protein

[0150] (1) Preparation of virus-infected cells: prepare human esophageal cancer target cells KYSE-150 and TE-1 in the logarithmic growth phase and trypsinize them respectively to make cell suspensions (the number of cells is about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, A549:10), an appropriate amount of the RNAi virus (PYCR1-siRNA lentivirus) prepared in Example 1 against the PYCR1 interference target was added to the experimental group, and the control lentivirus was added to the control group, and replaced after 24 hours of cultivation medium, infected for 5 days.

[0151] Then, the cell samples were received and washed twice with PBS. Take an appropriate amount of RIPA lysate, add PMSF within a few minutes before use, so that the final concen...

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Abstract

The invention belongs to the field of biological medicines, and relates to application of a PYCR1 gene as a target in preparation of a product for treating and / or diagnosing esophageal cancer. After expression of the PYCR1 gene is down-regulated by adopting an RNAi method, proliferation, growth, migration and infiltration of esophageal cancer cells can be remarkably inhibited, apoptosis of cancer cells is promoted, and the growth process of esophageal cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of esophageal cancer cells, promote apoptosis of the esophageal cancer cells, inhibit cloning of the esophageal cancer cells, inhibit invasion of the esophageal cancer cells, inhibit metastasis of the esophageal cancer cells and inhibit growth of the esophageal cancer, so that the siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus can be used for treating and detecting the esophageal cancer; wide application prospects are realized.

Description

technical field [0001] The invention belongs to the field of biomedical research, and relates to the use of human PYCR1 gene as a target in preparing esophageal cancer products and related products. Background technique [0002] Esophageal cancer is the seventh most common type of cancer in the world and the sixth highest cancer-related death rate. Esophageal squamous cell carcinoma (ESCC) is the main histopathological subtype of esophageal cancer, with a high incidence in my country 2 . Over the past 20 years, clinical diagnosis and treatment strategies have improved, but the overall 5-year survival rate of ESCC patients is still unsatisfactory due to recurrence, extensive invasion, and metastasis. Furthermore, the comprehensive molecular mechanisms underlying ESCC recurrence and metastasis remain unclear. [0003] Proline is one of the most abundant amino acids in the cellular microenvironment, and its metabolism and synthesis are necessary for tumor cells. Pyrroline-5...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K31/713A61P35/00A61P35/04C12N15/113C12N15/867C12N7/01C12Q1/6886
CPCA61K45/00A61K31/713A61P35/00A61P35/04C12N15/1137C12N15/86C12N7/00C12Q1/6886C12N2310/14C12N2320/30C12N2740/15021C12N2740/15043C12Q2600/136C12Q2600/158
Inventor 李斌孟于琪
Owner LANZHOU UNIV SECOND HOSPITAL
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