(A-7) RNA-seq analysis of gene modulation using ultrasound stimulation

Introduction:: A wide range of metabolic diseases including obesity, cancer, and neurodegenerative diseases are closely linked with each other and share genes that control the onset and progress of such critical diseases. Abnormal increase in fat cells causes obesity. Reducing the size and number of fat cells, a procedure known as lipolysis; may be used to prevent obesity as a potential therapy, which also requires a fundamental understanding of the mechanisms of lipolysis at the molecular level upon different types of stimulations. T cells play a crucial role in adaptive immunity. Recent success in immunotherapy uses immune checkpoint blockage therapy. Here, we used low-intensity pulsed ultrasound (LIPUS) stimulation to modulate genes that are related to T cell signature, immune checkpoint blockage (ICB), lipolysis, and autophagy.


Materials and Methods:: We developed an ultrasound biomicroscopy (UBM) to stimulate adipocytes differentiated from 3T3-L1 cells and Jurkat human T cell lines using LIPUS. LIPUS with a center frequency of 2 MHz was applied for 10 minutes per day for three days with the intensity of 370 mW/cm2 and 444 mW/cm2. After LIPUS stimulation, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot were used to determine the regulation of lipolytic factors such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL). Cellular RNA-sequencing (RNA-seq) was performed to study gene regulation after LIPUS stimulation.


Results, Conclusions, and Discussions::

Lipolysis genes of adipocytes and T cell signature and ICB genes of Jurkat cells were modulated after LIPUS stimulation as shown in Figure 1. RT-qPCR and Western blot show the regulation of lipolytic factors such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL). These results demonstrate that LIPUS stimulation can promote the lipolytic capacity of adipocytes in the differentiated state and modulate immune activity of Jurkat cells. The differences between transcriptional genes and metabolites were analyzed by transcript analysis and metabolomic profiling experiments.



All three factors were upregulated at the RNA level while only MGL was upregulated at the protein level in lipolysis, presenting a slightly different lipolysis activation pattern compared to widely used chemical stimulation. Modulation of ICB and T cell signature genes of Jurkat demonstrates a potential usage of LIPUS as remote immune modulator for therapy.



This study may provide an important experimental basis for clinical applications and a fundamental understanding of the mechanisms of gene regulation using LIPUS for cancer treatment as a potential therapeutic approach.   We will further investigate different LIPUS parameters for further applications.










Acknowledgements (Optional): : We thank Melissa Stephens and Joseph Sarro for RNA-sequence analysis at Genomics and Bioinformatics Core Facility (University of Notre Dame). This work was supported in part by the National Institutes of Health (NIH) under Grant No. GM120493, the National Science Foundation (NSF) under Grant No. CBET 1943852, and the Harper Cancer Research Institute, CCV grant.

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