Evaluation of the level of TNFα in the culture medium of MCF7 cells transducted by lenti-CRISPR-HMGB1 vector

Abstract

Introduction: Epidermolysis bollusa is a heterogeneous skin disease that affects 500000 people around the world. The disease is characterized by fragility and chronic blistering of the skin. HMGB1 is a protein that produced and realesed by immune cells such as macrophage, monocytes and dendritic cells. this marker is abundant in the epidermolysis bollusa blisters. During various studies, the CRISPR\CAS9 system has successfully reduced the secretion of various proteins and suppressed selective genes in laboratory and clinical studies. The aim of this study was to investigate the level of TNFα in the culture medium of MCF7 transduced cells with HMGB1 suppressor gene in the laboratory environment.

Methods and materials: For this purpose, MCF7 transduced cells, which HMGB1 gene has been suppressed by the Crisper system, were cultured in Passage 3 with 5,000 pieces in 6 wall plates. The control group included MCF7 cells without gene suppression. The TNFα level in the culture medium of these cells was measured by ELISA after 72 hours of culture and statistical data were obtained by SPSS17 software.

Results: According to the results, the TNFα titer in the case group (MCF7 cancer cells transduced by vector) was 162 pg/mg and the control group (MCF7 cells without manipulation) was 862 pg/ml. Statistical analysis showed a significant difference (almost 5 times) between the case and control group, and the amount of TNFα in the group of suppressed HMGB1 gene was significantly lower than the control group.

Discution: The results of this study showed that the suppression of the HMGB1 gene significantly reduced the expression of TNFα protein in the culture medium. Therefore, the use of gene therapy methods in animal studies is suggested as an effective treatment for epidermolysis bullosa dsease by suppressing the HMGB1 gene.