Application of human LSM5 gene and related products

A gene and use technology, applied in the use of human LSM5 gene and related products, can solve the problem that the function of LSM5 has not yet been reported.

Active Publication Date: 2020-03-17
THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, it has been reported that LSM5 is related to the circadian rhythm of humans and plants, but the function of LSM5 in cancer has not been reported yet

Method used

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  • Application of human LSM5 gene and related products
  • Application of human LSM5 gene and related products
  • Application of human LSM5 gene and related products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1 Preparation of RNAi lentivirus against human LSM5 gene

[0109] 1. Screening for effective siRNA targets against human LSM5 gene

[0110] Retrieve LSM5 (NM_012322) gene information from Genbank; design effective siRNA targets for LSM5 gene. Table 1-1 lists the screened effective siRNA target sequences against the LSM5 gene.

[0111] Table 1-1 is targeted at the siRNA target sequence of human LSM5 gene

[0112] SEQ ID NO TargetSeq(5'-3') 1 TGGTACTCTTCTAGGATTT

[0113] 2. Preparation of lentiviral vector

[0114] 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.

[0115] Table 1-2 Double-stranded DNA Oligo with sticky ends containing...

Embodiment 2

[0134] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0135] Human colorectal cancer RKO cells and HCT116 cells in the logarithmic growth phase were digested with trypsin to make a cell suspension (the number of cells was about 2×10 5 / 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, RKO: 10; MOI, HCT116: 10), an appropriate amount of the lentivirus prepared in Example 1 was added, and the culture medium was replaced after 16 hours of culture. After the infection time reached 3 days, the 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 transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 ...

Embodiment 3

[0142] Example 3 Detection of proliferation ability of tumor cells infected with LSM5-siRNA lentivirus

[0143] Human colorectal cancer RKO cells and HCT116 cells in the logarithmic growth phase were digested with trypsin to make a cell suspension (the number of cells was about 2×10 5 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, RKO: 10; MOI, HCT116: 10), add an appropriate amount of virus, replace the medium after 16 hours of culture, and collect cells in each experimental group in the logarithmic growth phase after the infection time reaches 3 days . The complete medium was resuspended into a cell suspension (RKO: 2.5×10 4 / ml, HCT116: 2×10 4 / ml), the cell density is about RKO: 2500 cells / well; HCT116: 2000 cells / well, seeded in 96-well plate. Three replicate wells per group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2 Incubator cultivation. From t...

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Abstract

The invention, which belongs to the field of biomedical research, particularly relates to application of a human LSM5 gene as a target in preparation of a colorectal cancer treatment drug. The wide and deep research discovers that the proliferation of colorectal cancer cells can be effectively inhibited and the apoptosis can be promoted by reducing the expression of the human LSM5 gene based on anRNAi method, thereby effectively controlling the growth process of colorectal cancer. According to the invention, the siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus can specifically inhibit the proliferation rate and proliferation capacity of colorectal cancer cells, promote apoptosis of the colorectal cancer cells, inhibit cloning of the colorectal cancer cells and inhibit growth of the colorectal cancer cells, so that the colorectal cancer is treated and a new direction is opened up for colorectal cancer treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human LSM5 gene and related products. Background technique [0002] LSM5 (LSM5 Homolog, U6 Small Nuclear RNA And MRNA DegradationAssociated; U6 SnRNA-Associated Sm-Like Protein LSm5) is located at 7p14.3, encoding a U6 small nuclear ribonucleic acid-associated Sm-like protein, which can be found in many species , considered to have sequence homology to the Sm protein family (see SNRPD2; MIM 601061). Sm-like proteins contain a 2-domain Sm sequence motif, which consists of 2 domains separated by a variable-length linker that can form a cyclic fold. Sm-like proteins are known to form a stable heteromer of 3-snRNA (U4 / U6-U5), and the heptamer LSM2-8 complex specifically binds to the 3'-terminal U-region of U6 snRNA and participates in the spliceosome Assembly process that plays an important role in the splicing of pre-mRNA. [0003] It has been reported tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61P35/00C12N15/113C12N15/867C12N7/01
CPCA61K45/00A61P35/00C12N15/113C12N15/86C12N7/00C12N2310/14C12N2740/15021C12N2740/15043
Inventor 朱琨李康
Owner THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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