Methionine Enkephalin (MENK) Inhibits Human Osteosarcoma Through Opioid Receptor (OGFR) And Its Related Mechanism

Objective : Osteosarcoma,the most common primary malignant bone tumor in children and adolescents,is characterized by the direct production of bone or osteoid tissue by tumor cells.Before the 1970 s,limb osteosarcoma was generally amputated,and the 5-year survival rate was less than 20%.At present,the treatment mode of osteosarcoma has developed into a comprehensive treatment mode based on multi-drug combined chemotherapy and limb salvage surgery,The tumor-free survival rate of patients can reach 60%-70%,the overall survival rate can reach60%-80%,and more than 90% patients can implement limb salvage therapy.Methionine enkephalin(MENK)is an endogenous opioid peptide encoded by the proenkephalin(PENK)gene,which mediates the regulation of the nervous and immune systems.MENK is also known as opioid growth factor(OGF)due to its growth-regulating properties.Studies have shown that MENK interacts with opioid growth factor receptor(OGFr)to upregulate p16 and/or p21 to inhibit DNA synthesis,thereby delaying the transition of the cell cycle from G0/G1 phase to S phase and inhibiting tumor cell proliferation.Moreover,through the interaction of MENK with OGFr,immune activity and function in vivo are also regulated.MENK has been shown to act as an immunomodulator for a variety of autoimmune diseases,including multiple sclerosis,inflammatory bowel disease,diabetes,and viral infections,and has been shown to alleviate symptoms of certain diseases in in vitro and in vivo experiments.Preliminary studies of our research group have confirmed that MENK can induce apoptosis and block cell cycle in G0/G1 phase,indicating that it plays an inhibitory role in melanoma,lung cancer,epidermal squamous cell carcinoma and gastric cancer.So does MENK also have an inhibitory effect on osteosarcoma cells?What is its possible mechanism of action?This study,in vitro the effects of methionine enkephalin(MENK)on the proliferation,invasion,migration and apoptosis of osteosarcoma cells,as well as on the expression of related signaling pathway proteins were detected,in vivo the effectof methionine enkephalin(MENK)on tumor microenvironment was detected,to investigate the role and molecular mechanism of MENK in the regulation of osteosarcoma cells,so as to provide new ideas for its clinical treatment.Methods:1.In vitro,use 2mg/ml,4mg/ml,5mg/ml,6mg/ml,8mg/ml,10mg/ml of MENK to stimulate human osteosarcoma cells MG-63 and Saos-2 respectively,and CCK-8 assay was used to detect the proliferation of two kinds of human osteosarcoma cells at 24 hours,48 hours and 72 hours respectively;the optimal dose and duration of action were selected for subsequent cell function experiments.Colony formation assay was used to detect the number of clones after 2mg/ml,5mg/ml and 10mg/ml of MENK were incubated with human osteosarcoma MG-63 and Saos-2 cells for 15days;MG-63 and Saos-2 were stimulated at 5mg/ml MENK after 48 hours,flow cytometry was used to detect the distribution of MG-63 and Saos-2 cells in G0/G1,S,G2/M phases;Transwell technology was used to detect the effect of MENK on the migration and invasion of osteosarcoma cells;Cell scratch assay was used to detect the changes of migration ability of osteosarcoma cells,and flow cytometry was used to detect the changes of apoptosis;q RT-PCR,WB and WES technology were used to detect 5mg/ml MENK for 48 hours,expression of m RNA and protein levels of OGFr,Bax,Bcl-2,caspase-3,caspase-9,and PARP in MG-63 and Saos-2 cells.2.To further study the regulation of MENK on osteosarcoma in vivo.A nude mice tumor model was constructed using human osteosarcoma cell MG-63.After subcutaneous tumor formation in nude mice,nude mice were randomly divided into two groups: MENK administration group(10 mg/kg)and negative control group(NS).The vital signs of nude mice were observed daily,and the changes of body weight and subcutaneous tumor volume of nude mice were dynamically monitored.After 21 days of treatment,the nude mice were sacrificed by cervical dislocation,and the tumors were stripped and weighed;HE staining was used to detect the pathology of the heart,liver,kidney and tumor tissue in the mice;OGFr,Ki67,Bax,Bcl-2 were detected by immunohistochemistry in tumor tissue;TUNEL was used to detect Apoptosis of tumor tissue,flow cytometry to detect the proportion of myeloid-derived suppressor cells(MDSCs)in spleen,bone marrow and tumor tissue in mice.At the same time,detect the expression levels of tumor related macrophages(TAMs)surface markers F4/80,CD86,CD206 and secretory cytokines TNF-α and IL-10 in tumors by immunohistochemistry.3.In order to explore the molecular mechanism of MENK regulating osteosarcoma cells in vitro,next,MG-63 and Saos-2 stable OGFr silenced cell lines were constructed by lentiviral vector,and the m RNA level and protein of OGFr were detected by q RT-PCR and WES to detect knockdown efficiency.After that,the cells were divided into three groups,including si-NC group,si-NC+MENK group,and si-OGFr+MENK group.Flow cytometry was used to detect the apoptosis of osteosarcoma cells,and Transwell technology was used to detect changes in migration and invasion of osteosarcoma cells.The m RNA levels of Bax,Bcl-2,caspase-3,caspase-9 and PARP were detected by q RT-PCR;the protein expression changes of Bax and Bcl-2 were detected by WB,the protein expression changes of caspase-3,caspase-9,PARP,PI3 K,p AKT,AKT were detected by WES.Results:1.The results of in vitro showed that the proliferation inhibitory effect of MENK by MG-63 and Saos-2 was positively correlated with the concentration and time of stimulation.The higher the concentration of MENK or the longer the duration of action,the more obvious inhibitory effect on osteosarcoma cells.Subsequent experiments were performed with 5mg/ml MENK for 48 h.2mg/ml,5mg/ml and10mg/ml of MENK all inhibited the formation of MG-63 and Saos-2 cell clones,and the higher concentration,the less clones formed.Compared with the control group,the proportion of G0/G1 phase cells in human osteosarcoma MG-63 and Saos-2 cells was increased,while the proportion of S phase cells was decreased after MENK stimulation.MENK can significantly inhibit the migration and invasion of osteosarcoma cells and promote the apoptosis of osteosarcoma cells.Stimulation of MENK resulted in up-regulation of OGFr m RNA and protein expression in MG-63 and Saos-2 cells.The m RNA and protein expressions of apoptosis-related genes Bax,caspase-3,caspase-9,and PARP were increased,while the m RNA and protein levels of Bcl-2 decreased.2.In vivo results showed that MENK could inhibit the growth of humanosteosarcoma subcutaneously transplanted in nude mice.After MENK treatment,the volume of the subcutaneously transplanted tumor was significantly reduced,the tumor weight was significantly reduced,and the body weight of the nude mice did not change significantly.Histopathological tests showed that MENK did not affect the heart,liver,and kidney tissues,but a large amount of necrosis occurred in the tumors in the MENK treatment group.In contrast,the tumor cells in the blank group were neat and tightly arranged,indicating that MENK can inhibit tumor cells grow in vivo;the expression levels of OGFr and Bax in the tumor tissue of MENK treatment group were up-regulated,and the expression level of Bcl-2 and Ki67 were down-regulated;TUNEL analysis further confirmed that MENK can promote the apoptosis of tumor cells,the results of flow cytometry showed that the proportion of MDSCs in the spleen,bone marrow and tumor tissues was significantly decreased,the results of immunohistochemistry showed that the proportion of tumor associated macrophages expressing CD86 was increased,the proportion of tumor associated macrophages expressing CD206 was decreased,the expression of M1 related cytokine TNF-α was increased,and the expression of M2 related cytokine IL-10 was decreased.3.The effective sequence of OGFr-si RNA was used to encapsulate the lentivirus,to construct stable OGFr-knockdown human osteosarcoma MG-63 and SAOS-2 cell lines.The results of q RT-PCR and WES showed that the m RNA and protein expressions of OGFr in OGFr knockdown cell lines MG-63 and Saos-2 were significantly inhibited.The results of flow cytometry showed that the proportion of early apoptotic cells and late apoptotic cells in si-NC+MENK group increased significantly,the proportion of early apoptotic and late apoptotic cells in si-OGFr+MENK group was lower than that in si-NC+MENK group.Further cell invasion results showed that the number of invasive cells in si-NC+MENK group was significantly decreased compared with si-NC group,while the number of invasive cells in si-OGFR+MENK group was increased compared with si-NC+MENK group.The results of cell migration showed that the number of migrating cells in si-NC+MENK group was significantly reduced,and the number of migrating cells in si-OGFR+MENK group was increased compared with si-NC+MENK group.Compared with si-NC group,the m RNA and protein expressions of Bax,caspase-3,caspase-9 and PARP in si-NC+MENK group were increased,while the m RNA and protein expressions of Bcl-2 were decreased,however,the m RNA and protein expression changes of Bax,Bcl-2,caspase-3,caspase-9 and PARP in Si-OGFr+MENK group were partially reversed compared with si-NC+MENK group.MENK could down-regulate the expression of PI3 K,p-AKT and AKT proteins in the PI3K/AKT pathway,and these effects were partially reversed after OGFr knockdown.Conclusion:1.Both in vitro and in vivo experiments have confirmed that MENK has a significant inhibitory effect on osteosarcoma at an appropriate dose.2.MENK can inhibit osteosarcoma by blocking cell cycle,inducing apoptosis,inhibiting cell migration and invasion.3.MENK may inhibit osteosarcoma by inhibiting myeloid derived suppressor cells(MDSCs)and inducing the transformation of tumor-associated macrophages(TAMs)from M2 to M1 phenotype.4.MENK can up-regulate the expression level of OGFr in osteosarcoma cells and exert anti-tumor effect by binding with OGFr.MENK may regulate PI3K/AKT signaling pathway after binding OGFr to inhibit osteosarcoma.