Preliminary Study On Influence Of The Interaction Between DHCR24 And HRAS In The Pathogenesis Of Cancer

Cancer is a disease caused by the disorder of cell growth,proliferation and apoptosis.Recent studies have found that the development of cancer is related to the level of cholesterol in cells,and many cancers can be treated with drugs that lower cholesterol and inhibit lipid droplets.3β-hydroxysterol Δ24-reductase(DHCR24)is an enzyme in the final step of cholesterol biosynthetic process,which catalyzes the reduction of chain sterol into cholesterol.However,the molecular mechanism of DHCR24 in cancer pathogenesis has not been studied.Based on the previous studies,this study aimed to study the interaction group of DHCR24 and used Duolink demonstrate the possible interaction of DHCR24 and HRAS in cancer,and speculated the important role of DHCR24 and its interaction proteins in the occurrence and development of cancer through cell experiments.First,using the identification data obtained from immune coprecipitation mass spectrometry in the laboratory and four public protein interaction databases we constructed the DHCR24 interaction proteome.Through the pathway analysis,we found that DHCR24 interaction group enriched in the related pathways in cancer development,and the data obtained from the PPI network analysis showed that DHCR24 might affect cancer development through the interaction with HRAS.GEPIA online analysis showed that DHCR24 was significantly up-regulated in patients with a variety of cancers,and the expression of DHCR24 was correlated with the expression of HRAS in cancer.Moreover the expression level of DHCR24 was negatively correlated with the survival rate of patients.These result suggested that DHCR24 and its interacting proteins may play an important role in the occur and development of cancer.Secondly,the multi-template model of DHCR24 was constructed by protein homologous modeling,and the three-dimensional structure of activated HRAS was downloaded.The direct interaction between DHCR24 and HRAS was verified by using the protein docking software ZDOCK.The interaction between DHCR24 and HRAS was further studied by using the molecular dynamics simulation software NAMD.It was found that the structures of DHCR24,such as I45-K59,L354-P361 and Y440-R444,are located at the interface between DHCR24 and HRAS,suggesting that the interaction between DHCR24 and activated HRAS may be related to its catalytic function of cholesterol synthesis.Subsequently,the Proximity ligation assay(PLA)Duolink technique was used to demonstrate that in 5637 cells of bladder cancer cell lines.The result revealed that DHCR24 had a direct interaction with HRAS,and this interaction occurred outside the nucleus.Further studies showed that the interaction between DHCR24 and HRAS was significantly enhanced after 5637 cells were stimulated by Epidermal Growth Factor(EGF).These studies strongly demonstrated there is a direct interaction between DHCR24 and HRAS in 5637 cells,and the interaction was enhanced by EGF,suggesting that the interaction between DHCR24 and HRAS may be involved in EGF mediated cell proliferation in cancer cells.Finally,the data obtained from filipin’s fluorescence staining and cell proliferation and survival assay showed that both EGF-induced cell proliferation and none EGF-induced proliferation of 5637 cells could be inhibited by HRAS inhibitor lonafarnib as well as DHCR24 inhibitor U18666 A.At the same time,EGF can promote cholesterol synthesis,and this effect could be blocked by lonafarnib or U18666 A.These results demonstrate that EGF can promote cell proliferation and cholesterol synthesis,and this effect requires the participation of DHCR24 and HRAS,suggesting that the interaction between DHCR24 and HRAS may be involved in the proliferation of cancer cells by promoting cholesterol synthesis.In summary,through the study on the DHCR24 interaction protein group,this study identified that the DHCR24 interaction group was mainly enriched in the cancer-related signaling pathway,and DHCR24 might influence the development of cancer through HRAS.Then,computer simulation and Duolink anaalysis,proved that DHCR24 has a direct interaction with HRAS,and EGF can promote the occurrence of this effect,which occurs outside the nucleus.Further studies showed that in 5637 cells,the role of DHCR24 and HRAS in EGF promoting cell proliferation and cholesterol synthesis was required.The preliminary results of this study on the interaction between DHCR24 and HRAS laid a foundation for the deep understanding of DHCR24 protein function in the molecular mechanisms of cancer development,and provide a basis for the development of new anti-cancer drugs through lowering cholesterol through targeting at DHCR24.