Application of human DEPDC1 gene, and related product

A DEPDC1 gene technology, applied in medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc., can solve problems such as lack of DEPDC1 gene

Pending Publication Date: 2020-06-12
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no relevant report on the use ...

Method used

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  • Application of human DEPDC1 gene, and related product
  • Application of human DEPDC1 gene, and related product
  • Application of human DEPDC1 gene, and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 Preparation of RNAi lentivirus against human DEPDC1 gene

[0103] 1. Screening for effective siRNA targets against the human DEPDC1 gene

[0104] Retrieve DEPDC1 (NM_017779) gene information from Genbank; design effective siRNA targets for DEPDC1 gene. Table 1-1 lists the screened effective siRNA target sequences against DEPDC1 gene.

[0105] Table 1-1 siRNA target sequence targeting human DEPDC1 gene

[0106] SEQ ID NO TargetSeq(5'-3') 1 tatccagtaaggctatcat

[0107] 2. Preparation of lentiviral vector

[0108] 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 Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.

[0109] Table 1-2 Double-stranded DNA Oligo with sticky ends containing...

Embodiment 2

[0128] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0129] Human tongue squamous cell carcinoma CAL27 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 (MOI, CAL27:10), an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of culture, and the cells were collected after the infection time reached 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 the...

Embodiment 3

[0136] Embodiment 3Western Blotting method detects the silencing efficiency of gene

[0137] 1. Extraction of total cell protein

[0138] 1) Infect target cells with control virus and DEPDC1-siRNA lentivirus targeting DEPDC1 interference target according to the multiplicity of infection (MOI, CAL27:10).

[0139] 2) After 5 days of infection, collect the cell samples, take an appropriate amount of RIPA lysate (Beiyuntian, P0013C), and add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM.

[0140] 3) 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).

[0141] 4) Centrifuge at 12000g for 15min at 4°C, take the supernatant and use BCA Protein Assay Kit (manufacturer: Biyuntian, article number: P0010S) to measure the protein concentration.

[0142] 5) Add new lysate to adjust th...

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Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human DEPDC1 gene as a target in preparation of tongue squamous cell carcinoma treatment drugs or tongue squamous cell carcinoma diagnosis drugs. Through wide and deep research, it is found that proliferation of tongue squamous cell carcinoma cells can be effectively inhibited and cell apoptosisis promoted after expression of a human DEPDC1 gene is lowered through an RNAi method, so that the growth process of tongue squamous cell carcinoma 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 tongue squamous cell carcinoma cells, promote apoptosis of the tongue squamous cell carcinoma cells, inhibit cloning of the tongue squamous cell carcinoma cells, influence the period of the tongue squamous cell carcinoma cells and inhibit growth of the tongue squamous cell carcinoma cells, so that the tongue squamous cell carcinoma is treated, and a new direction is opened up for tongue squamous cell carcinoma treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human DEPDC1 gene and related products. Background technique [0002] DEPDC1 (DEP domain containing 1) is an encoding gene that may be involved in transcriptional regulation as a transcriptional co-repressor. Studies have found that DEPDC1 plays an important role in the cell cycle process, and DEPDC1 is highly expressed in the mitotic phase of the cell cycle in synchronized cells. Immunofluorescence detection showed that DEPDC1 was mainly localized in the nucleus during interphase, and redistributed to the whole cell after nuclear envelope rupture in metaphase. In addition, knockdown of DEPDC1 expression caused significant mitotic arrest. In summary, the gene DEPDC1 plays a key role in regulating the process of cell mitosis (Mi Y, Zhang C, Bu Y, Zhang Y, He L, Li H, Zhu H, Li Y, Lei Y, Zhu J. DEPDC1 is novel cell cycle related gene that regulates mitoti...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867A61K45/00A61K31/713A61P35/00
CPCC12N15/113C12N15/86A61K45/00A61K31/713A61P35/00C12N2310/14C12N2740/15043C12N2800/107C12N2310/531
Inventor 郭俊峰谭颖徽徐帅周述作季艳丹
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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