Inhibiting NBR1 expression using the AAV6-CRISPR-Cas9 system affects tumor immune regulation in lung cancer mice
10.19405/j.cnki.issn1000-1492.2024.12.006
- Author:
Bokang Wang
1
;
Mingyang Zhu
1
;
Xiusen Zhang
2
;
Jiangtao Sun
2
,
3
,
4
Author Information
1. Clinical Medical College of Henan University of Science and Technology , Luoyang 471000
2. Henan Key Laboratory of Tumor Epigenetics , Luoyang 471000
3. Cancer Institute of Henan University Of Science and Technology , Luoyang 471000
4. Dept of Thoracic Oncology Surgery , Cancer Hospital of The First Afiliated Hospital of Henan University of Science and Technology , Luoyang 471000
- Publication Type:Journal Article
- Keywords:
lung adenocarcinoma;
AAV6;
CRISPR/Cas9;
NBR1;
gene editing;
immunotherapy
- From:
Acta Universitatis Medicinalis Anhui
2024;59(12):2103-2111
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish aNBR1-knockout lung cancer mouse model through CRISPR-Cas9 technology by using adeno-associated virus(AAV) as a vector to specifically inhibitNBR1expression and to investigate the impact ofNBR1knockout on tumor growth and immune cell infiltration and regulation.
Methods:sgRNAs targeting mouseNBR1(Gene ID: 17966) was designed using the online tool CRISPOR(http://crispor.tefor.net/crispor.py). AAV6 was utilized as the vector for sgRNA delivery, and the efficiency of gene knockout was confirmed using PCR and DNA sequencing methods. To determine the best AAV infection approach in mice, 6 C57BL/6J mice were randomly divided into intranasal and endotracheal groups. After 28 days, lung tissue sections were assessed for enhanced green fluorescent protein expression to identify the more efficient infection method for subsequent experiments. Lung tumor growth, as well as immune cell infiltration and activation status in tumor tissues, were detected using methods including HE staining, immunohistochemistry, immunofluorescence, and flow cytometry.
Results:DNA sequencing and immunofluorescence results indicated successful construction of the AAV6-U6-sgNBR1-CAG-Cre-GFP vector with stable knockout efficiency. Fluorescence microscopy showed higher efficiency of lung infection in mice through intratracheal administration(P<0.05). HE staining revealed reduced tumor area in mouse lungs after targetedNBR1knockout compared to the control group(P<0.01). Immunofluorescence and flow cytometry results demonstrated enhanced functional activity of CD8+T lymphocytes in lung cancer tissues of mice with targetedNBR1knockout, characterized by increased effector T lymphocytes and decreased exhausted T lymphocytes(P<0.01).
Conclusion:Using CRISPR/Cas9 technology, we construct a lung cancer mouse model with targetedNBR1knockout. We verify that targeted inhibition of NBR1 expression significantly enhances the functional activity of CD8+T lymphocytes in lung tissues, resulting in suppressed tumor growth, reduced tumor burden, and extended survival in lung cancer mice. This study lays an experimental foundation for investigations into the mechanisms and functions ofNBR1and other genes in lung adenocarcinoma cells.
- Full text:2025022715082182174基于AAV6-CRISPR...肺癌小鼠肿瘤免疫调控的影响_王博康.pdf
