Development Of Anti-tumor Synthetic Genetic Circuit Targeting Cholesterol In Melanoma

Metabolic reprogramming is one of the ten characteristics of tumor.In order to meet the requirements of continuous proliferation and growth,cancer cells must accelerate the metabolic rate,or change the metabolic pathway.As an important component of cell membrane,high synthesis rate of cholesterol is a necessary condition for rapid proliferation of tumor cells.Studies have shown that statins targeting the key enzyme of cholesterol synthesis,HMGCR,can reduce the rate of cholesterol synthesis and inhibit the development of a variety of tumors.However,studies on inhibiting tumor growth by directly converting cholesterol to other metabolites are still rare.Cholesterol and cholesterol synthesis intermediate metabolite 7-dehydrocholesterol(7-DHC)can be transformed each other in multiple species.Previous studies have shown that 7-DHC has a significant inhibitory effect on melanoma,and 7-DHC can significantly inhibit the proliferation of melanoma cells in vitro and induce apoptosis,so the transformation of cholesterol in vivo may be one of potential melanoma treatment strategies.The genetic circuits of synthetic biology,which use the engineering design idea to build standardized components and modules,and synthesizes the new artificial life system to regulate biological behavior,are characterized by precision and flexibility and widely used range can play a huge potential in the treatment of tumor and other diseases.Therefore,the construction of engineered genetic circuits is expected to realize the accurate and controllable reconstruction of the metabolic pathway in vivo,enables homeostasis in the body and provides new treatments for metabolic disorders such as diabetes,cardiovascular disease and tumors.In this study,based on the CRISPR-Cas9 system and cholesterol 7-deasturase,a precise and controlled genetic circuit was constructed in melanoma,which can target the reprogramming of cholesterol metabolism and mediate the transformation of cholesterol into 7-DHC,so as to achieve the inhibition of melanoma.The contents of this study are mainly divided into three parts: the screening of output signal,the screening of input signals,and the construction and verification of gene circuits.In consideration of unsuccessful identification of cholesterol 7-desaturase in higher mammals,this study finally identified the source of silkworm cholesterol 7-desaturase NVD-BM as output signal from five species of eukaryotes by evolutionary conservatism,which can catalyze the conversion of cholesterol to 7-DHC in melanoma cells,so that 7-DHC can accumulate in cells and significantly inhibit the growth of melanoma.Transcription factors are endogenous signaling molecules that bind to promoters to regulate the expression of downstream genes.The expression of transcription factors is strictly regulated in cells,and abnormal expression can lead to multiple tumorigenesis.Therefore,highly expressed transcription factors are ideal input signals for genetic circuits in specific tumors.In this study,ten highly expressed transcription factors were selected in melanoma via analyzing transcriptome data of melanoma and normal skin tissues.According to the dual luciferase assay,pair-wise input signals were optimized that two transcription factors,RELA and STAT1,with the highest efficiency in regulating reporter gene expression,were finally selected as input signals.Then a new type of genetic circuit was constructed in melanoma cells based on CRISPR-Cas9 system and TET-ON switch.Through inducing the up-regulated transcription factors RELA and STAT1 in melanoma,dual-input “AND” gate activated the expression of NVD-BM that catalyzed cholesterol into 7-DHC,which inhibited the proliferation and migration rate of melanoma cells.In this study,for the first time,a genetic circuit was constructed in the tumor with transcription factors as the input signals and heterogenous cholesterol 7-deasturase NVD-BM as the output signal.NVD-BM can significantly inhibit the growth of melanoma,at the same time,we verified that NVD-BM suppressed the activation of Akt1/NF-κB pathway by inhibiting phosphorylation of serine at Akt1-ser473.The results confirmed that genetic circuit can precisely control the expression of cholesterol7-desaturase to realize the reconstruction of metabolic pathway,and thus achieve the effect of killing tumors.Meanwhile,the construction idea of genetic circuit is expected to be applied to other tumor types with abnormal metabolism,providing a new idea for targeted therapy of tumors.