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Application of human PSMD7 gene and related product

A gene and application technology, applied in the application of human PSMD7 gene and related products, can solve the problems of PSMD7 gene and other problems

Inactive Publication Date: 2020-10-23
AFFILIATED HOSPITAL OF JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there is no report about the use of PSMD7 gene in the treatment of pancreatic cancer

Method used

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  • Application of human PSMD7 gene and related product
  • Application of human PSMD7 gene and related product
  • Application of human PSMD7 gene and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1 Preparation of RNAi lentivirus against human PSMD7 gene

[0120] 1. Screening for effective siRNA targets against the human PSMD7 gene

[0121] Retrieve PSMD7 (NM_002811) gene information from Genbank; design effective siRNA targets for PSMD7 gene. Table 1-1 lists the screened effective siRNA target sequences against the PSMD7 gene.

[0122] Table 1-1 is targeted at the siRNA target sequence of human PSMD7 gene

[0123] SEQ ID NO TargetSeq(5'-3') 1 TGAGGAAGTTGGAGTTGAA

[0124] 2. Preparation of lentiviral vector

[0125] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Medical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragments.

[0126] Table 1-2 Double-stranded DNA Oligo with sticky ends ...

Embodiment 2

[0145] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0146] Human pancreatic cancer ASPC-1 cells and PANC-1 cells in the logarithmic growth phase were digested with trypsin to make a 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, ASPC-1:20, PANC-1:20), add an appropriate amount of the lentivirus prepared in Example 1, replace the medium after 24 hours of cultivation, and collect the cells after the infection time reaches 5 days . 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 transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 minutes to inactivate ...

Embodiment 3

[0153] Example 3 Western Blot detection target reduces PSMD7 gene protein level expression

[0154] 1. Extraction of total cell protein

[0155] (1) Infect the target cells (human pancreatic cancer ASPC-1 cells, pancreatic cancer PANC-1 cells, MOI, ASPC-1:20, PANC-1) with the control virus and the RNAi virus targeting PSMD7 interference target respectively :20). 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 concentration of PMSF is 1mM. (Use RIPA lysate, link to instructions: http: / / www.beyotime.com / ripa-lysis-bufferm.htm)

[0156] (2) Add an appropriate amount of RIPA lysate, and lyse on ice for 10-15 minutes. Scrape the cells and transfer them into a new EP tube, and then ultrasonically disrupt the cells (20 times at 40W, 1 s each time, 2 s apart).

[0157] (3) 4°C, 12000g, centrifuge for 15min, take the supernatant and measure the protein concentration by BCA ...

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Abstract

The present invention belongs to the field of biological medicine research and specifically relates to an application of a human PSMD7 gene serving as a target to preparation of a drug for treating pancreatic cancer. Found by extensive and deep research, after the expression of the human PSMD7 gene is reduced by adopting an RNAi method, the proliferation of pancreatic cancer cells may be effectively inhibited, cell apoptosis is promoted, and the growth process of the pancreatic cancer may be effectively controlled. siRNA or a nucleic acid construct containing an siRNA sequence and lentivirus provided by the present invention is capable of specifically inhibiting the proliferation capacity of the pancreatic cancer cells, the cloning of the pancreatic cancer cells, the apoptosis of the pancreatic cancer cells, the metastasis capacity of the pancreatic cancer cells and the growth of the pancreatic cancer, thereby treating pancreatic cancer and opening up a new direction for treating pancreatic cancer.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human PSMD7 gene and related products. Background technique [0002] PSMD7 (Proteasome 26S Subunit, Non-ATPase 7) is a protein-coding gene, which is a subunit of the 19S proteasome, a component of the 26S proteasome. The 26S proteasome is involved in ATP-dependent ubiquitination degradation, and plays a key role in maintaining protein homeostasis by removing misfolded or damaged proteins that may impair cellular function, as well as removing functionally non-essential proteins. The protein encoded by PSMD7 is involved in many cellular processes, including cell cycle progression, cilia / flagella depolymerization, and other processes. Diseases known to be associated with PSMD7 include tracheitis and Huntington's disease. [0003] According to literature reports, PSMD7 is related to the occurrence and development of breast cancer, esophageal cancer and other...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/713A61K31/7105A61K35/768A61K39/395A61P35/00A61P35/04C12N15/113C12N15/867C12N7/01A23L33/13A23L33/00
CPCA23V2002/00A61K31/7105A61K31/713A61K35/768A61K39/39558A61K45/00A23L33/00A23L33/13A61P35/00A61P35/04C12N7/00C12N15/113C12N15/86C12N2310/14C12N2320/30C12N2740/15021C12N2740/15043A23V2200/308
Inventor 金成邬晓敏刘敏丰茆勇陈武强朱茂群
Owner AFFILIATED HOSPITAL OF JIANGNAN UNIV
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