| Objectives:Osteosarcoma(OS)is the most common primary malignant bone tumor,which has high local infiltration ability and the tendency of lung parenchyma preferentially metastasis,and the metastasis and recurrence rate are very high.Over the past decades,the prognosis of metastatic osteosarcoma has been stagnant.Therefore,it is the key to reduce the death rate and improve the prognosis of osteosarcoma by studying the growth and metastasis of osteosarcoma,exploring and understanding the mechanism of osteosarcoma formation and lung metastasis,then developing effective anti-metastasis drugs.Recently,Interferon-stimulated Gene 15(ISG15)has been found to be abnormally expressed in a variety of malignant tumors and is involved in the pathophysiological processes of tumor formation and metastasis.In the preliminary study,the research team carried out high throughput RNA-sequencing to analysis on the tissue samples of patients with primary osteosarcoma and metastatic osteosarcoma and drew the protein interaction network of differentially expressed genes.It was found that ISG15 was the most significantly up-regulated hub protein in the protein interaction network of metastatic osteosarcoma,suggesting that over-expression of ISG15 might promote the invasion and metastasis of osteosarcoma.This result was also confirmed by cell function experiments,but the mechanism was not clearly.Subsequently,gene annotation revealed that Long Noncoding RNA(Lnc RNA)RP11-54O7.3 was within the range of 100 kb before and after ISG15,and analysis of TCGA sarcoma data revealed that lncRNA RP11-54O7.3 was one of ISG15-related differentially expressed genes,which was negatively correlated with expression of ISG15.Several studies have shown that ISG15 and lncRNA are the key targets for regulating autophagy of tumor cells.Previous studies also confirmed that after inhibiting the autophagy level of osteosarcoma cells with the autophagy inhibitor3-methyladenine,the effect of over-expression of ISG15 on promoting proliferation,migration and invasion of osteosarcoma cells was weakened.This study aimed to investigate whether there were differences in the expression of ISG15 between the primary tissue of osteosarcoma and the normal/adjacent tissue samples,and whether the expression level was related to the the occurrence of lung metastasis and clinical prognosis.Besides,the study clarified the correlation between the expression of ISG15 and lncRNA RP11-54O7.3 and determine the role of lncRNA RP11-54O7.3 in the proliferation,migration and invasion of osteosarcoma cells.In addition,rescue experiments were conducted to further clarify whether the oncogenic effect of ISG15 on osteosarcoma was realized by influencing the expression of lncRNA RP11-54O7.3.At last,the role of autophagy in the influence of ISG15/lncRNA RP11-54O7.3 axis on osteosarcoma formation and metastasis was explored.Through the above research,we revealed an ISG15/lncRNA RP11-54O7.3axis that regulates cell autophagy and affects the signaling pathway of osteosarcoma invasion and metastasis.We have also gained a deeper understanding of the molecular mechanisms underlying the biological processes of invasion and metastasis of osteosarcoma.This study provided a theoretical reference for exploring new targets for lung metastasis of osteosarcoma and treating this disease.Methods:Part 1: The samples of primary osteosarcoma and normal/adjacent tissues were collected,and the levels of ISG15 in the gene and protein were detected by RT-q PCR and immunohistochemistry,respectively.The relevant clinical information of the patients included in the study was collected,and the relationship between the expression of ISG15 and the occurrence,development and prognosis of osteosarcoma were discussed.Furthermore,the effect of ISG15 on osteosarcoma formation in vivo was further studied by constructing nude mice subcutaneous tumorigenesis and tibia in situ tumorigenesis models.Part 2: The subcellular localization of lncRNA RP11-54O7.3 in osteosarcoma cells was determined by RNA FISH.And lncRNA RP11-54O7.3 was knocked down by ASO technique.Using lentivirus to infect target cells to construct a stable transgenic over-expression of lncRNA RP11-54O7.3.RT-q PCR was used to verify the expression level correlation between ISG15 and lncRNA RP11-54O7.3.The effects of lncRNA RP11-54O7.3 on the proliferation,migration and invasion of osteosarcoma cells were determined by CCK-8,cloning,transwell migration/invasion and other methods,and the rescue experiment was designed to determine whether the effects of knockdown of ISG15 on the proliferation,migration and invasion of osteosarcoma cells could be partially reversed by knockdown of lncRNA RP11-54O7.3.The subcutaneous tumorigenesis,tibia in situ tumorigenesis and lung metastasis nude mice models of osteosarcoma were constructed using 143 B cells stably transfected with luciferase to determine whether the promotion effect of over-expression of ISG15 on osteosarcoma formation,invasion and lung metastasis in vivo depends on lncRNA RP11-54O7.3.Part 3: Autophagy was detected by a series of gold standard methods such as electron microscopy,Western blot,m RFP-GFP-LC3,to determine the effect of ISG15/lncRNA RP11-54O7.3 axis on autophagy of osteosarcoma cells,and to determine whether the proliferation,migration and invasion of ISG15/lncRNA RP11-54O7.3 axis on osteosarcoma cells depended on the autophagy process regulated by ISG15.Results:Part 1: RT-q PCR was used to detect the m RNA level of ISG15 in clinical samples of osteosarcoma.It was found that the overall expression level of ISG15 in primary tissues of osteosarcoma was significantly higher than that in normal/adjacent tissues(P=0.004)in 19 paired samples.In the unmatched samples containing 28 tumor tissues and 19 normal/adjacent tissues,the overall expression level of ISG15 in the primary tissues of osteosarcoma was significantly higher than that in the normal/adjacent tissues(P=0.032).Correlation analysis showed that the expression of ISG15 was associated with lung metastasis.The incidence of lung metastasis was higher in the high expression group of ISG15(P=0.021),but there was no significant correlation with sex,age,T stage,and primary tumor location.Next,we expanded the collection of 71 primary tumor tissue samples of osteosarcoma,and detected the expression level of ISG15 protein by immunohistochemistry,and analysed the correlation between the expression of ISG15 and the prognosis of osteosarcoma.It was found that the high expression of ISG15 is a risk factor for OS in osteosarcoma patients(P<0.001).Correlation analysis also found that the expression of ISG15 protein level was associated with lung metastasis.The incidence of lung metastasis was higher in the high expression group of ISG15(P=0.003),but there was no significant correlation with sex,age,T stage,and primary tumor site.The nude mice models of osteosarcoma subcutaneous tumorigenesis and tibia in situ tumorigenesis were successfully constructed.The growth rate of subcutaneous tumor in OE-ISG15 group was significantly faster than that in NC group,and the growth rate of subcutaneous tumor in sh-ISG15 group was significantly slower than that in NC group.The degree of osteogenic destruction of tibia in situ in OE-ISG15 group was significantly higher than that in NC group(P<0.05),and the degree of osteogenic destruction of tibia in situ in sh-ISG15 group was significantly lower than that in NC group(P<0.05).Part 2: RNA FISH showed that lncRNA RP11-54O7.3 was distributed in the nucleus and cytoplasm of 143 B and U2 OS,mainly located in the nucleus.The number 181 sequence was knocked down by ASO technology.At 48 hours after transfection,the lncRNA RP11-54O7.3 of U2 OS and 143 B were significantly reduced compared with the control group,with a statistically significant difference(P=0.0098;P=0.0003).The number 181 sequence was knocked down for subsequent experiments.The stable transgenic cells of 143 B and U2 OS which over-expression of lncRNA RP11-54O7.3 were successfully constructed.The lncRNA RP11-54O7.3 in the over-expression group was significantly higher than that in the control group,with a statistically significant difference(P<0.0001;P<0.0001).RT-q PCR detection finding revealed that in the cells of 143 B and U2 OS,ISG15 could negatively affect the expression of lncRNA RP11-54O7.3(P=0.0012;P=0.0004),while lncRNA RP11-54O7.3 did not affect the expression of ISG15.The experiments of CCK-8,cloning,scratch and transwell migration/invasion proved that knockdown of lncRNA RP11-54O7.3 promoted the proliferation,migration and invasion of osteosarcoma cells(P<0.05),and over-expression of lncRNA RP11-54O7.3 inhibited the proliferation,migration and invasion of osteosarcoma cells(P<0.05).The rescue experiments included CCK-8 and transwell migration/invasion were designed.The results showed that the inhibitory effect of knocking down ISG15 on the proliferation,migration and invasion of osteosarcoma could be partially reversed by knocking down lncRNA RP11-54O7.3,with significant difference(P<0.05).The nude mice models of subcutaneous tumorigenesis,tibia in situ tumorigenesis and lung metastasis of osteosarcoma with Luc bioluminescence was successfully constructed.The results showed that the incidence of lung metastasis in OE-ISG15+OE-RP11-54O7.3 group was significantly lower than that in OE-ISG15 group,and the growth rate of subcutaneous tumor and the degree of bone destruction were significantly reduced,with statistically significant differences between groups(P<0.05).Part 3: Through transmission electron microscopy,it was observed that compared with the control group,143 B and U2 OS cells which over-expression of ISG15 had more autophagosomes and autolysosomes,and autophagy was more active.Western blot was used to detect the changes in the expression level of autophagy-related protein LC3,and m RFP-GFP-LC3 was used to observe the autophagy flow.It was found that the over-expression of ISG15 increased the level of autophagy,and the over-expression of lncRNA RP11-54O7.3 could reverse the effect of over-expression of ISG15 on enhancing autophagy.The results of CCK-8 and transwell migration/invasion experiments showed that the proliferation,migration and invasion ability of 143 B and U2 OS cells knocked down lncRNA RP11-54O7.3 were significantly reduced(P<0.05)after adding autophagy inhibitor 3-MA.Conclusions:1.Compared with normal/adjacent tissues,ISG15 is significantly higher in the primary tissues of osteosarcoma,and the higher expression of ISG15 promotes lung metastasis of osteosarcoma,which is related to the poor clinical prognosis.2.In vivo experiments have confirmed that the high expression of ISG15 can promote the occurrence and development of osteosarcoma.3.There is a negative correlation between the expression of ISG15 and lncRNA RP11-54O7.3,and it have confirmed that the promotion effect of over-expression of ISG15 on the occurrence,development and lung metastasis of osteosarcoma depends on down-regulating the expression of lncRNA RP11-54O7.3 both in vitro and in vivo experiments.4.The effect of ISG15/lncRNA RP11-54O7.3 axis on the proliferation,migration and invasion of osteosarcoma cells depends on the autophagy process regulated by ISG15. |
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