Circular RNA ROCK1-E3/E4 Suppresses Proliferation And Migration In Osteosarcoma Cells

IntroductionOsteosarcoma（OS）is the most common primary malignant tumor of bone tissue in the epiphysis of the long shaft of adolescents.Osteosarcoma is believed to originate from primitive mesenchymal cells,which is characterized by the transformation of osteoblasts to produce bone like matrix.Because of its high degree of malignancy,rapid growth and early lung metastasis,the prognosis of osteosarcoma is often poor.Because osteosarcoma itself has the characteristics of strong malignant biological behavior,about 20%of clinical patients have lung metastasis at the first visit.Therefore,targeted research on the key factors and targets of reversing osteosarcoma metastasis and clarifying its regulation mechanism has important theoretical significance and practical value.Circular RNA（circRNA）is a kind of noncoding RNA with covalently closed structure without 5 ’end cap and 3’ end polyadenylate tail.It has the characteristics of endogenous,abundant,conservative and stable.CircRNA is transcribed by RNA polymerase Ⅱ,which has the same transcription efficiency as linear RNA.CircRNA can act as a miRNA sponge,interact with proteins,translate into proteins and regulate transcription.CircRNA is widely involved in the occurrence and development of pathological processes such as inflammation,tumor,vascular disease and nervous system disease.More and more evidence shows that circRNA plays an important regulatory role in a variety of tumor diseases,including osteosarcoma.Rho associated coiled coil forming protein kinase 1（ROCK1）belongs to Rho family.It is a small molecule G protein.It can regulate cell division and proliferation through its downstream target effector molecules,participate in actin recombination and regulate the activities of cytoskeleton,and then affect the invasion and metastasis of many cells,including tumor cells.In recent years,our research group has been engaged in basic research on Rock1 and the occurrence and development of osteosarcoma.Our previous study found that the expression of ROCK1 was increased in osteosarcoma.The overall survival（OS）and disease-free survival（DFS）of osteosarcoma patients with elevated Rock1 were significantly shortened,and the increased Rock1 was closely related to the distant metastasis and higher clinical stage of osteosarcoma.At the same time,we found that multiple long-chain noncoding RNAs,including TUG1 and MALAT1,can up regulate the expression of Rock1 by means of competitive endogenous RNA（CeRNA）and promote the invasion,metastasis and proliferation of osteosarcoma cells.It can be seen that ROCK1 is a key molecule in the invasion and metastasis of osteosarcoma.If a new endogenous molecule that can inhibit its expression can be found upstream,it will provide an important new target for inhibiting the invasion and metastasis of osteosarcoma at the molecular level.On the basis of previous researches,this research intends to focus on the expression and function of circRNAs that may be cut by ROCK1 precursor RNA in osteosarcoma,and verify the specific mechanism of its possible role.This study is intended to provide a new target for the development of molecular targeted therapy of osteosarcoma.ObjectiveThe purpose of this study was to investigate the expression of circular RNA（circROCK1-E3/E4）cut by ROCK1 in osteosarcoma and its effect on the proliferation,invasion and migration of osteosarcoma cells;At the same time,this study intends to explore whether circROCK1-E3/E4 can indirectly regulate PTEN mediated osteosarcoma cell proliferation,invasion and metastasis by adsorbing miR-532-5p.Methods1.CircROCK1 in osteosarcoma,expression,identification,biogenesis and its correlation with poor clinicopathological features in patients with osteosarcoma.（1）Bioinformatics analysis and RT-qPCR were used to verify the expression of circRNAs that may be formed by the cleavage of ROCK1 precursor RNA in osteosarcoma.（2）RT-qPCR and fish were used to detect the differential expression of circROCK1-E3/E4 in osteosarcoma tissues and cell lines;FISH verified the localization of circROCK1-E3/E4 in the subcellular organelles of osteosarcoma cells.（3）Divergent primers and polymerization primers based on cyclization sites,random primers and oligo DT,nucleic acid electrophoresis and RTqPCR were designed to verify the circular structure of circROCK1-E3/E4.（4）RNase R and actinomycin D verified the stability of circROCK1E3/E4.（5）Statistical analysis confirmed the correlation between different circROCK1-E3/E4 expression levels and adverse pathological parameters of osteosarcoma patients.（6）The overexpression vectors containing different ROCK1 flanking intron sequences were constructed,and the effects of different overexpression vectors on the expression level of circROCK1-E3/E4 were detected by RT-qPCR.（7）Osteosarcoma cell models with different QKI expression levels were constructed and verified.The effects of different QKI expression levels on the expression of circROCK1-E3/E4 were detected by RT-qPCR;RNA immunoprecipitation（RIP）experiment verified the binding effect of ROCK1 flanking intron fragment with QKI.2.Mechanism of CircROCK1-E3/E4 inhibiting osteosarcoma proliferation and migration through miR532-5p/PTEN pathway.（1）Osteosarcoma cell models with different circROCK 1-E3/E4 expression levels were constructed and verified.（2）CCK8 and edu experiments verified the effects of different circrock1-e3/E4 expression levels on the proliferation of osteosarcoma cells;Transwell experiment verified the effects of different circrockl-e3/E4 expression levels on the invasion and migration of osteosarcoma cells.（3）RIP experiment verified the binding effect of circROCK1-E3/E4 and AGO2.（4）Bioinformatics screening miRNAs that may interact with circROCK1-E3/E4.（5）Bioinformatics analysis and ISH were used to detect the expression of miR-532-5p in osteosarcoma;Spearman correlation analysis was used to investigate the correlation between miR-532-5p and circROCK1-E3/E4 expression;The expression of miR-532-5p in osteosarcoma cell line was detected by RT-qPCR;Fish verified the co localization of miR-532-5p and circROCK1-E3/E4 in the subcellular organelles of osteosarcoma cells.（6）The reporter gene vector plasmid containing wild and mutant circROCK1-E3/E4 fragments was constructed.The double luciferase reporter gene was used to investigate the targeted binding effect of miR-5325p with circROCK1-E3/E4.（7）RT-qPCR was used to verify whether there was mutual inhibition between circROCK1-E3/E4 and miR-532-5p.（8）Osteosarcoma cell models with different miR-532-5p expression levels were constructed and verified;Western blot was used to detect the effect of different miR-532-5p expression levels on PTEN expression.（9）PTEN overexpression vector plasmid was transferred to osteosarcoma cell model with miR-532-5p overexpression,and PTEN specific small interfering RNA was transferred to osteosarcoma cell model with miR-532-5p silencing expression;CCK8 experiment,EdU experiment and Transwell experiment verified the role of PTEN in the proliferation,invasion and migration of osteosarcoma cells mediated by miR-532-5p.（10）The reporter gene vector plasmid containing wild and mutant PTEN fragments was constructed.The double luciferase reporter gene was used to investigate the targeted binding of miR-532-5p to PTEN.（11）The effects of different circROCK1-E3/E4 expression levels on PTEN expression were investigated by Western blot（12）The effects of different circROCK1-E3/E4 expression levels on PTEN expression were investigated by Western blot;Western blot was used to investigate the effect of circROCK1-E3/E4 and miR-532-5p on PTEN expression;Effects of CCK8 experiment,EdU experiment,Transwell experiment,circROCK1-E3/E4 and miR-532-5p on proliferation,invasion and migration of osteosarcoma cells.Results1.The expression of circROCK1-E3/E4 decreased in osteosarcoma,and its expression was regulated by QKI.The downregulated of circROCK1-E3/E4 was closely related to the adverse clinicopathological parameters of osteosarcoma patients.（1）There are 53 circRNAs that may be cut by Rock1 in the human genome,including 20 circRNAs with a length of less than 500 bp.（2）Compared with the other 19 circRNAs,circROCK1-E3/E4（hsa_circ₀108024）was stably down regulated in 5 osteosarcoma tissue samples;Compared with hFOB1 19.The expression of circROCK1-E3/E4 decreased significantly in four osteosarcoma cell lines MG63,U2OS,HOS and 143B cells.（3）Compared with the polymerization primer,circROCK1-E3/E4 was expressed in the cDNA product of divergent primer;Compared with random primers,the expression of circROCK1-E3/E4 in oligo DT primer group was significantly reduced,which confirmed that circROCK 1-E3/E4 had no polyadenylate tail structure.（4）Compared with linear ROCK1 mRNA,circROCK1-E3/E4 has better stability and is more resistant to the digestion of RNase R and actinomycin D（5）CircROCK1-E3/E4 is localized in the cytoplasm of HOS and U2OS cells.（6）The expression level of circROCK1-E3/E4 in osteosarcoma patients with lymph node metastasis was lower than that in osteosarcoma patients without lymph node metastasis;The expression level of circROCK1-E3/E4 in osteosarcoma patients with distant metastasis was lower than that in osteosarcoma patients without distant metastasis;Through statistical analysis,it was verified that the low expression level of circROCK1-E3/E4 was closely related to the low survival rate of osteosarcoma patients.（7）Western blot was used to detect the downregulated of circROCK1E3/E4 expression of QKI knockout by RNAi technology.Compared with the negative control group,the downregulated of circROCK1-E3/E4 expression of QKI knockout was detected by RT qPCR,while the ROCK1 mRNA of QKI knockout was almost unchanged.（8）The targeted binding effect between QKI and intron 2 and intron 4 sequences was confirmed by RIP analysis.2.CircRNA ROCK1-E3/E4 upregulates PTEN by adsorbing mir-532-5p,thereby suppressed the proliferation and migration of osteosarcoma cells.（1）Osteosarcoma cell models with different circROCK1-E3/E4 expression levels were successfully constructed.（2）CCK8,EdU and Transwell experiments showed that upregulating the expression of circROCK1-E3/E4 could significantly inhibit the proliferation,invasion and migration of osteosarcoma cells;Downregulation of circROCK1-E3/E4 expression significantly promoted the proliferation,invasion and migration of osteosarcoma cells.（3）The results of RIP experiment showed that with IgG as the internal reference,circROCK1-E3/E4 was widely enriched in AGO2 compared with circANRIL,that is the existence of circROCK1-E3/E4 was the biological basis of ceRNA.（4）By using circBank,miRDB and TargetScan as bioinformatics prediction tools,miR-532-5p was screened as a potential miRNA that can interact with circROCK1-E3/E4.（5）In the osteosarcoma related data set GSE65071,the miR-532-5p in osteosarcoma is overexpression compared to which in healthy people;ISH results showed that the miR-532-5p in osteosarcoma tissues is overexpression compared to adjacent tissues;Compared with osteoblast line hFOB1 19,the overexpression of miR-532-5p was in osteosarcoma cell lines MG63,U2OS,HOS and 143B;There was a significant negative correlation between circROCK1-E3/E4 and miR-532-5p expression in osteosarcoma tissue samples;FISH results confirmed that circROCK1-E3/E4 and miR-5325p were colocated in the cytoplasm of HOS and U2OS.（6）The results of double luciferase reporter gene showed that compared with the control simulant（miR NC）,the luciferase activity decreased significantly after cotransfection of miR-532-5p simulant（miR-532-5p mimic）and wild-type circROCK1-E3/E4（circROCK1 wt）reporter plasmid,while the luciferase activity did not change significantly after cotransfection of miR-532-5p mimic and mutant circROCK1-E3/E4（circROCK1 mut）reporter plasmid,That is,miR-532-5p can be targeted and combined with circROCK1-E3/E4 through specific microRNA response elements（MREs）.（7）RT-qPCR results showed that upregulation or downregulation of circROCK1-E3/E4 expression did not affect the expression level of miR532-5p.On the contrary,upregulation or downregulation of miR-532-5p did not affect the expression of circROCK1-E3/E4.（8）Osteosarcoma cell models with different miR-532-5p expression levels were successfully constructed;Western blot and RT-qPCR showed that miR-532-5p could negatively regulate the expression of PTEN.（9）MiR-532-5p overexpression could promote the proliferation,invasion and migration of HOS and U2OS cells,which could be reversed by PTEN overexpression vector plasmid;miR-532-5p silencing expression could inhibit the proliferation,invasion and migration of HOS and U2OS cells,and this inhibition was weakened after transfection of PTEN specific small interfering RNA;That is,mir-532-5p could promote the proliferation,invasion and migration of HOS and U2OS cells by regulating PTEN.（10）The results of double luciferase reporter gene showed that compared with the control simulant（miR NC）,the luciferase activity decreased significantly after cotransfection of mir-532-5p mimic and wildtype PTEN（PTEN wt）reporter plasmid,while the luciferase activity did not change significantly after cotransfection of miR-532-5p mimic and mutant PTEN（PTEN mut）reporter plasmid,That is,miR-532-5p could be targeted and combined with PTEN 3 ’untranslated region（3’ UTR）through specific MREs.（11）Western blot showed that circROCK1-E3/E4 could positively regulate the expression of PTEN protein.（12）Western blot showed that circROCK1-E3/E4 could promote the expression of PTEN protein,which was significantly inhibited after further up regulating miR-532-5p;The results of CCK8 experiment,EdU experiment and Transwell experiment showed that circROCK1-E3/E4 could inhibit the proliferation,invasion and migration of HOS and U2OS cells,and this inhibition was significantly weakened after further up regulating miR-5325p.Conclusion1.CircRNA ROCK1-E3/E4 is downregulated in osteosarcoma patients and cell lines.The low expression of circROCK1-E3/E4 was closely related to the adverse pathological parameters of osteosarcoma patients.2.QKI can regulate the formation of circROCK1-E3/E4 by binding to the flanking intron fragment of ROCK1.3.CircROCK1-E3/E4 can upregulate the expression of PTEN by adsorbing miR-532-5p,so as to inhibit the proliferation,invasion and migration of osteosarcoma cells.