Effects And Mechanisms Of KiSS1/GPR54 In Metastases And Proliferation Of Osteosarcoma

BackgroundOsteosarcoma is the most common primary malignant bone tumor in adolescents,which is prone to metastasis and recurrence.Distant metastatic lesions could be found in nearly 15% to 20% of patients at first visit.Once metastasis occurred,the survival time of patients was significantly shortened.There has been a lack of effective targeted therapeutic drugs for osteosarcoma.G protein coupled receptors(GPCRs)are the largest superfamily of cell surface receptors in human,and are the most successful drug targets at present.It is of great significance to search for potential diagnostic and therapeutic targets in GPCRs for osteosarcoma.KiSS1/GPR54 system is a pair of ligand and receptor proteins encoded by KiSS1 gene and GPR54 gene.It has been widely studied in adolescent development and tumor,serving as an important regulator of adolescent development genes and playing an important role of "on and off" in puberty.It also has the function of inhibiting tumor metastasis in multiple tumor progression,known as " tumor metastasis suppressor genes".Since osteosarcoma is a common and highly metastatic tumor in adolescence and KiSS1/GPR54,as an "on-off" of adolescence,can inhibit the metastasis of tumors in many tumors,this suggests that KiSS1/GPR54 may play an important role in the development of osteosarcoma.Objective:(1)To detect the expression of KiSS1 and GPR54 in osteosarcoma tissues and explore their potential clinical significance.(2)To study the effects of KiSS1 gene coding product KP-10 and receptor GPR54 on the biological characteristics of osteosarcoma cell metastasis and proliferation in vitro and in vivo experiments.(3)To explore the possible molecular mechanism of KiSS1/GPR54 in inhibiting osteosarcoma metastasis and proliferation.Methods:(1)Immunohistochemistry(IHC)was used to detect the expression of KiSS1 and GPR54 in clinical specimens of osteosarcoma and to analyze the relationship between the expression of KiSS1 and GPR54 and the patients’ age and sex.Kaplan-Meier method was used to analyze the relationship between the expression of KiSS1/GPR54 and the survival time of patients.qRT-PCR and Western Blot were used to detect the expression of KiSS1 and GPR54 and their differential expression in 5 clinical specimens of osteosarcoma and corresponding adjacent tissues.qRT-PCR and Western Blot were used to detect the expression of KiSS1/GPR54 in osteosarcoma cell line and osteoblastic cell line hFOB1.19 cells.(2)shRNA interference sequences of GPR54 were constructed,and stable cell lines with low-expressed GPR54(shGPR54)were constructed on MNNG/HOS and ZOS cell lines with relatively high expression of GPR54.GPR54 overexpressed plasmid was constructed,and stable cell lines with overexpressed GPR54(gpr54-oe)were constructed on 143B(from the same cell line as MNNG/HOS)and ZOSM(from the same patient source as ZOS)cell lines with relatively low expression of GPR54.qRT-PCT and Western Blot were used to detect the interference and overexpression efficiency of GPR54 in osteosarcoma cells.KiSS1 gene coding product KP-10 was synthesized in vitro.Wound healing and Transwell assay were used to detect the effects of KP-10 or low-and over-expressed GPR54 on the migration ability of osteosarcoma cells.MTS assay was used to detect the effects of KP-10 or low-expressed GPR54 on the viability of osteosarcoma cells.Colony formation assay was performed to detect the effects of KP-10 or low-expressed GPR54 on the colony formation rate and proliferation of osteosarcoma cells.MNNG/HOS cells labeled with shGPR54 were injected into the tail vein of nude mice,and IVIS was used to detect the effect on the ability of lung metastasis of osteosarcoma cells with interference of GPR54.143 B cells labeled with luciferase were injected into the tibia of nude mice in situ while the treatment group was given KP-10 drugs,and IVIS was used to detect the effect of KP-10 on in situ growth and distant metastasis of osteosarcoma cells injected into the tibia of nude mice.(3)RNA-seq high-throughput sequencing method was used to detect and analyze the gene expression changes of 143 B cells after KP-10 was given.KEGG pathway was used to analyze the signal pathway enriched with differentially expressed genes.Based on the results of RNA-seq sequencing,qRT-PCR was used to verify the ETV4 expression of osteosarcoma cells after KP-10/GPR54 intervention.Western Blot was further used to detect the expression of ERK,MMP2,MMP9,E-cadherin and N-cadherin in the upstream and downstream of ETV4,and to analyze the molecular mechanism of KiSS1/GPR54 in inhibiting osteosarcoma metastasis.Results:(1)There was no significant correlation between the expression of KiSS1 and GPR54 and the osteosarcoma patients’ age and sex.Kaplan-Meier analysis found that patients with lower expression of KiSS1 or GPR54 in osteosarcoma had a shorter survival time.Western Blot and qRT-PCR results showed that the expression of KiSS1 and GPR54 in tumor tissues was lower than that in adjacent tissues.Western Blot showed that the expression of KiSS1 and GPR54 in osteosarcoma cell line was lower than that in hFOB1.19.The expression of KiSS1 and GPR54 in 143 B and ZOSM cells with strong metastasis ability was the lowest.(2)Wound healing and Transwell experiments showed that KP-10 or overexpressed GPR54 could inhibit the migration of osteosarcoma cells,while low-expressed GPR54 could promote the migration of osteosarcoma cells.MTS assay and colony formation assay showed that interfering with GPR54 could promote cell viability and colony formation of osteosarcoma cells.KP-10 drug could inhibit the proliferation activity and colony formation ability of osteosarcoma cells.Experiments of MNNG/HOS osteosarcoma cells injecting into tail vein of nude mice showed that the low expression of GPR54 could promote the metastasis of osteosarcoma cells in the lungs of mice.In situ tibia injection of 143 B cells into nude mice,KP-10 inhibited lung metastasis of osteosarcoma cells in situ tibia.(3)RNA-seq high-throughput sequencing analysis showed that 440 genes were up-regulated and 420 genes were down-regulated after KP-10 was given to 143 B cells.KEGG pathway analysis indicated that differentially expressed genes were mainly enriched in 16 signaling pathways after KP-10 treatment.The Transcriptional misregulation in cancer signaling pathway with the highest concentration of differentially expressed genes was focused,and the ETV4 gene whose function was most consistent with the change trend was selected for further exploration.qRT-PCR confirmed that the expression of ETV4 in osteosarcoma cells was down-regulated after KP-10 treatment or GPR54 overexpression,and up-regulated after GPR54 interference.Western Blot showed that the phosphorylation levels of ERK in the upstream of ETV4 and the expression of MMP-2,MMP-9,N-cadherin in the downstream of ETV4 were down-regulated after KP-10 treatment or GPR54 overexpression in osteosarcoma cells,while the expressions of E-cadherin was up-regulated.What’s more,the expression of P-ERK,MMP-2,MMP-9,N-cadherin were up-regylated after GPR54 interference.Conclusions: KiSS1 and GPR54 are down-regulated in osteosarcoma tissues,and the expression of KiSS1 and GPR54 in osteosarcoma tissues is closely related to the survival time of patients Also,KiSS1 gene encoding products KP-10 and GPR54 are involved in the regulation of migration and proliferation of osteosarcoma.KiSS1/GPR54 may regulate ERK phosphorylation thereby regulating the ETV4-MMP&EMT signaling to play a biological role in inhibiting osteosarcoma cells.