HCAR1 Promotes The Proliferation Of Osteosarcoma Cells By Mediating Glucose Metabolism Through AKT/mTOR/HIF-1a Signaling Pathway

Malignant tumor is regarded as a major public health problem in the world.It has been reported that the rate of glycolysis in cancer cells is much higher than in non-tumor cells(e.g.,normal cells,immune cells,fat cells,etc.).In cancer cells,glucose is preferentially provided for energy by glycolysis,even in the presence of oxygen.Therefore,reducing glucose uptake by tumor cells is an effective way to inhibit tumor.GPR81 belongs to a subfamily of G Protein-coupled receptor(GPCRs),called Hydroxyl carboxylic acid receptor(HCAR),and consists of three members: HCAR1(GPR81),HCAR2(GPR109A)and HCAR3(GPR109B).This unique GPCR family is sequentially located on human chromosome 12 Q.In rodents,only HCAR1 and 2 are expressed,both of which are located on chromosome 5 of mice.HCAR3 is not present in rodents.HCAR1 is considered to be the most evolutionarily conserved of the three receptors,as GPR81 is present in all mammals and fish,while GPR109 A is only present in higher mammals.The HCAR gene family is unique in that they consist of a single exon without introns,recognize metabolic intermediates as their endogenous agonists,and have a relatively low affinity for their agonists(from low to high in the milimolar range)compared to other members of the GPCR family.Among them,L-lactic acid(EC50= ～ 5 mM),a ubiquitous metabolite,acts as a signal molecule as a natural ligand and agonist of GPR81.As a cell-surface receptor for lactic acid,GPR81 may play an important role in tumor growth based on the fact that cancer cells produce lactic acid as the end product of glycolysis and export it to the extracellular environment.This effect may be autocrine,in which extracellular lactic acid acts on GPR81 expressed by tumor cells themselves.Alternatively,it may be paracrine,in which extracellular lactic acid produced by cancer cells acts on GPR81 expressed on non-cancer cells in the tumor microenvironment.Both autocrine and paracrine are involved in promoting tumor growth and/or metastasis.To sum up,HCAR1 is a tumor promoting factor and is expected to become a therapeutic target for osteosarcoma.However,its specific expression pattern and clinical value in osteosarcoma are still unknown.In this study,we will systematically evaluate the expression of HCAR1 in osteosarcoma and its clinical value.Secondly,explore the biological function of HCAR1 in osteosarcoma cells.Finally,the potential mechanism of HCAR1 carcinogenesis in osteosarcoma cells was explored.The purpose of this study is to provide a theoretical basis for the clinical treatment of tumors,especially osteosarcoma.In this experiment,we first from cbiopotal TCGA database search(https://www.cbioportal.org/)database provides 32 kinds of human tumor samples of exon sequenced data set of 10967 samples retrieved HCAR1 mutation frequency and mutation model.Based on TCGA again(https://portal.gdc.cancer.gov/)database and CCLE database(https://portals.broadinstitute.org/ccle/about),analysis HCAR1 in generic carcinoma in expression pattern and clinical value;Then,in osteosarcoma cells,CCK-8,tranwell,scratch and glucose uptake experiments were used to explore whether HCAR1 affected the biological function of osteosarcoma cells;Finally,osteosarcoma cells were co cultured with bone marrow cells to test whether HCAR1 promoted the glucose competition of osteosarcoma cells.In this study,based on 10,967 samples from 32 human tumors provided by the Cbiopotal database,we found that the mutation frequency of HCAR1 was only 0.9%;Based on RNA sequencing and clinical information provided by TCGA and CCLE databases,we found that HCAR1 was up-regulated in the solid texture and predicted a poor prognosis in generalized carcinoma.Based on cell function experiments,we found that silencing HCAR1 inhibits while overexpression of HCAR1 promotes carcinogenic properties of four malignant tumor cells.Based on THE RNA sequencing provided by TCGA,we found that DNA methyltransferase was positively correlated with the expressions of DNMT1,DNMT2,DNMT3 A,DNMT3B and HCAR1 in generalized carcinoma.Real-time quantitative PCR and WB results showed that overexpression of DNMT3 A promoted HCAR1 expression,while silencing DNMT3 A inhibited HCAR1 expression.The results of CCK-8,Tranwell,scratch and glucose uptake assay showed that DNMT3 A promoted the malignant characteristics of osteosarcoma cells by promoting the expression of HCAR1.Hcar1-silenced osteosarcoma cells co-cultured with bone marrow cells increased glucose uptake and decreased apoptosis of myeloid cells.The osteosarcoma cells with HCAR1 overexpression were co-cultured with myeloid cells,and the glucose uptake of myeloid cells decreased and apoptosis increased.The results were as follows: Based on 10,967 exon sequencing samples processed by cbiopotal database,we found that the total mutation frequency of HCAR1 was only0.9%.Therefore,point mutation of HCAR1 may not be the main factor of its carcinogenic effect.The biological functions of HCAR1 in lung cancer,colorectal cancer and osteosarcoma have not been reported.We found that HCAR1 promotes proliferation,invasion,and migration of breast,lung,colorectal,and osteosarcoma.Conclusion: HCAR1 is up-regulated in pancarcinomas(except CESC,UCEC,HNSC,KIRC,KIRP)and plays a carcinogenic role.Glucose is completely broken down to produce 36 molecules of ATP from one molecule of glucose.Hypoxia is a hallmark of cancer,and as a result,cancer cells require more molecules of glucose than non-malignant cells,thus facilitating the carcinogenesis process and increasing the aggressiveness and aggressiveness of the tumor.In this chapter,we found that overexpression of HCAR1 promotes the absorption of glucose and lactic acid by osteosarcoma cells;In addition,2-deoxy-d-glucose(2-DG),as an inhibitor of glucose metabolism,under its intervention,we found that HCAR1 promotes the proliferation of osteosarcoma cells by promoting glucose metabolism.In addition,we found that HCAR1 promotes the malignant characteristics of osteosarcoma cells,and its carcinogenic characteristics depend partly on AKT/mTOR/HIF-1a signaling pathway;HCAR1 can promote the competition between osteosarcoma cells and bone marrow cells for glucose.This result suggests that HCAR1 regulates the metabolic reprogramming between tumor cells and bone marrow cells.Small molecules,monoclonal antibodies or gene editing targeting HCAR1 may become a new idea for tumor treatment.