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ShRNA of TMEM2 and application thereof

A drug and sense chain technology, applied in the field of bioengineering, can solve problems such as unclearness, and achieve the effects of inhibiting proliferation, high clinical application value, and promoting chemosensitivity

Active Publication Date: 2022-02-11
NANTONG TUMOR HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of TMEM2 in the development of colorectal cancer is still unclear

Method used

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  • ShRNA of TMEM2 and application thereof
  • ShRNA of TMEM2 and application thereof
  • ShRNA of TMEM2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The expression of TMEM2 in colorectal cancer tissues and paracancerous tissues was detected by western blotting. Fresh cancer (T1-T4) and para-cancerous tissues (N1-N4) of 4 pairs of colorectal cancer patients were collected, and protein was extracted, and the protein content was determined by BCA protein quantification kit (purchased from Beyond Company). After 50 μg of total protein from each group was separated by 12% SDS-PAGE gel electrophoresis, the protein was transferred to PVDF membrane by wet transfer method, and then blocked with blocking solution containing 5% skimmed milk powder at room temperature for 2 hours. Anti-TMEM2 antibody (purchased from Abcam) was used as the primary antibody for 2 h at room temperature, and Horseradish Peroxidase (HRP)-labeled goat anti-rabbit IgG (purchased from Proteintech) as the secondary antibody was incubated at room temperature for 2 h. Finally, chemically enhanced luminescence (ECL) was used to develop bands, and GAPDH was...

Embodiment 2

[0043] Design the siRNA target according to the transcript of the Human TMEM2 gene, and arrange the synthesis of primers. The single-stranded primer is annealed into a double-stranded oligo sequence, which is connected into a double-digested linearized RNA interference vector to replace the original ccdB toxic gene. The transformants were screened by colony PCR, and the screened positive clones were verified by sequencing. Correct clones were verified by sequencing, and high-purity plasmid extraction was carried out. The experiment is divided into the following 8 main steps:

[0044] 1. Interference target design and primer synthesis:

[0045] According to the general principles of shRNA design and the rich experience of Heyuanbio, design siRNA targets and arrange primer synthesis.

[0046] The specific sequence is as follows:

[0047] The TMEM2 siRNA target sequence is as follows:

[0048] Table 1 TMEM2 siRNA target sequences

[0049] Marker Gene Gene ID ...

Embodiment 3

[0065] Example 3: Construction and Identification of Small Interfering Fragments

[0066] According to the above-mentioned three TMEM2 small interfering lentiviruses and the control virus respectively infected the colorectal cancer cell line SW620 cells, the MOI value of the virus was 50, infected for 96 hours, screened with puromycin with a concentration of 2 μg / mL for three days, and extracted and collected respectively. Group cell total protein, BCA protein quantification kit (purchased from Biyuntian Company) was used to determine the protein content. After 50 μg of total protein from each group was separated by 12% SDS-PAGE gel electrophoresis, the protein was transferred to PVDF membrane by wet transfer method, and then blocked with blocking solution containing 5% skimmed milk powder at room temperature for 2 hours. Anti-TMEM2 antibody (purchased from Abcam) was used as the primary antibody for incubation at room temperature for 2 h, and Horseradish Peroxidase (HRP)-labe...

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Abstract

The invention discloses shRNA of TMEM2 and application of the shRNA, and belongs to the technical field of bioengineering. A small interfering RNA fragment of the TMEM2 is invented through an anti-colorectal cancer gene therapy approach based on the difficulty faced by clinical treatment of colorectal cancer, i.e., relapse and metastasis are easy to occur after treatment, and the small interfering RNA fragment can be used as a medicine to be applied to metastasis and relapse of the colorectal cancer. Related cytology experiments show that the small interfering RNA provided by the invention has important functions of inhibiting protein expression of colorectal cancer cells TMEM2 and inhibiting proliferation, migration and invasion of cancer cells. Therefore, a new technical means is provided for developing a new target for tumor treatment, and the shRNA of the TMEM2 has very important application value and prospect in clinical treatment of tumors and development of targeted drugs.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to a shRNA of TMEM2 and its application. Background technique [0002] Colorectal cancer (CRC) is a malignant tumor with the third highest morbidity and mortality worldwide. Although surgery combined with postoperative radiotherapy and chemotherapy can increase the survival rate of patients, the overall curative effect is still not ideal. The reason is because CRC patients are prone to chemotherapy resistance, metastasis and recurrence after chemotherapy. Therefore, elucidating the potential mechanism of chemotherapy resistance of tumor cells and finding potential tumor therapeutic targets can help prevent the spread and invasion of cancer cells, which is of great significance for improving the treatment effect of CRC patients and finding corresponding intervention measures. [0003] Tumor metastasis is a complex process involving changes in multiple stages, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/713A61P1/00A61P35/00A61P35/04
CPCC12N15/1137C12N15/1135C12N15/86A61K31/713A61P1/00A61P35/00A61P35/04C12N2310/14C12N2320/30C12N2740/15043
Inventor 李静杨磊王金明沈爱国
Owner NANTONG TUMOR HOSPITAL
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