| Background and purpose:Renal cell carcinoma(RCC)is a common malignancy in the genitourinary system originating from renal tubular epithelial cells,accounting for approximately 80-85% of renal malignancies,and its incidence has been on the rise over the past decade.According to the WHO database,renal cell carcinoma is considered one of the most common epithelial cancers worldwide,with more than 430,000 new cases and nearly 180,000 deaths worldwide in 2020.The mortality and severity of the disease is strongly associated with late diagnosis,as only 10%of patients present with typical symptoms,such as hematuria,back pain or abdominal masses.Due to the heterogeneous nature of renal cell carcinoma,approximately 20-30% of patients are prone to local recurrence and distant metastases after surgery,with 25% of patients diagnosed with limited renal cell carcinoma developing distant metastases.Therefore,deciphering the molecular mechanisms of renal cell carcinoma progression and metastasis is of great clinical importance.Circular RNAs(circ RNAs)are a newly discovered type of non-coding RNAs(nc RNAs)that are prevalent in many species.Unlike typical linear RNAs,circ RNAs form a covalently closed continuous loop from exons or introns through specific selective shearing,with neither a 5’ cap nor a 3’ polyadenylate tail,which makes them more stable than linear RNAs.There is growing evidence that circ RNAs play a key role in the pathogenesis of a variety of malignancies including renal cell carcinoma,bladder cancer,breast cancer and hepatocellular carcinoma,and are expected to be biomarkers for tumor diagnosis,prognostic assessment,and effective targets for drug therapy.Metastatic renal cell carcinoma is insensitive to both radiotherapy and chemotherapy,with poor prognosis and limited effect of traditional cytokine therapy page.In recent years,with the increasing use of immune checkpoint inhibitors and targeted agents in the clinic,the survival of patients with metastatic renal cell carcinoma has been significantly improved.The current study shows that although targeted therapy has certain efficacy on metastatic renal cell carcinoma,the effect of targeted therapy is still limited: on the one hand,targeted drugs have obvious toxic side effects on patients;on the other hand,drug resistance to targeted therapy arises.Therefore,there is an urgent clinical need to study the biological mechanism of renal cell carcinoma,and early identification of the progression and metastasis mechanism of renal cell carcinoma can help to develop targeted drugs more accurately to improve the survival rate of renal cell carcinoma patients.Poly(β-amino)esters(PBAEs)class of polymer molecules is a newly synthesized class of efficient polymeric gene vector materials.It contains primary,secondary and tertiary amines as well as hydrolyzable ester bonds,which allow higher cell affinity and better transfection efficiency.In addition,PBAE can have specific affinity for tumor cells by changing the position of its subunits,thus avoiding damage to surrounding normal tissues.All these advantages make this class of polymers ideal for the construction of polymeric gene delivery nanoparticles for tumor targeting therapy.In this work,we propose to search for circ RNAs related to renal cell carcinoma progression and metastasis by sequencing data from public databases;to detect the expression of screened circ RNAs in renal cell carcinoma tissue samples and renal cell lines,and to evaluate the relationship between screened circ RNAs and clinicopathological variables and prognosis;to detect the effects of screened circ RNAs on renal cell carcinoma cell line proliferation,migration,and proliferation in ex vivo functional assays.In addition,we will examine the effects of circ RNAs on proliferation,migration and invasion,tumor growth and metastasis of renal cell lines;investigate the specific molecular mechanisms and possible signaling pathways of circ RNAs;construct relevant nanocomplexes and explore the therapeutic effects of nanocomplexes in vivo,and provide new directions and theoretical support for the search of molecular targets for diagnosis,prognosis and treatment of renal cells.Method:(1)Search for circ RNAs associated with renal cell carcinoma progression and metastasis:renal cell carcinoma-associated circ RNA datasets were obtained from the Gene Expression Omnibus(GEO)database,and differential expression analysis was performed on the normalized data using limma software in R.The differentially expressed circ RNAs were selected using lg FC>=2 and P value <0.05 as screening criteria(hsa＿circ＿0001946(ciRS-7)).(2)Detection of ciRS-7 expression and the relationship with clinicopathology and prognosis: firstly,agarose gel electrophoresis,Sanger sequencing and treatment of cells with RNase R enzyme and actinomycin D(Act D)were performed to verify the presence as well as stability and closed loop structure of ciRS-7,followed by fluorescence in situ hybridization(FISH)experiments,combined with real-time fluorescent quantitative polymerase The sublocalization of ciRS-7 in renal cell carcinoma cell lines was then clarified by FISH,nucleoplasmic separation assay combined with quantitative real-time fluorescence polymerase reaction(q RT-PCR).Normal renal tubular epithelial cells and renal cell carcinoma cell lines,85 pairs of nephrectomized renal cell carcinoma tumor tissues and corresponding adjacent normal kidney tissues were collected,and the expression of ciRS-7 in renal cell carcinoma cell lines,renal cell carcinoma tissues and adjacent normal kidney tissues specimens was detected by q RT-PCR.All patients were divided into two groups(high ciRS-7 group and low ciRS-7group)according to the mean of ciRS-7 expression levels,and the correlation between ciRS-7expression levels and clinicopathological variables in renal cell carcinoma patients was examined.Kaplan-Meier survival curves were used to detect the relationship between patients in different ciRS-7 expression level groups and the overall survival rate(OS).(3)Functional assays to detect the effects of ciRS-7 on the biological behavior of renal cell carcinoma cell lines in terms of proliferation,migration and invasion,tumor growth,and lung metastasis: three ciRS-7-specific small interfering RNAs(si-ciRS-7#1,si-ciRS-7#2,and si-ciRS-7#3)and ciRS-7 overexpression lentivirus were designed and infected by transfection reagents with renal cell carcinoma 786-O and ACHN cell lines.The transfection efficiency of small interfering RNA and overexpressing lentivirus was examined by q RT-PCR and FISH assay;the effect of ciRS-7 on the proliferation ability of renal cell carcinoma cells was examined by Ed U cell proliferation assay,CCK-8 and colony formation assay;the effect of ciRS-7 on the migration and invasion ability of renal cell carcinoma cells was examined by wound healing assay,Transwell migration and invasion assay;finally,the effect of ciRS-7 on the migration and invasion ability of renal cell carcinoma cells was examined by transfection.Finally,786-O cell lines were transfected with a stable lentivirus(sh-ciRS-7)containing a ciRS-7 interference sequence(si-ciRS-7#2)and a lentivirus overexpressing ciRS-7(OE-ciRS-7)and inoculated into the subcutaneous and tail vein of immunodeficient mice to observe the subcutaneous tumorigenic ability and metastatic foci in the experimental group compared with the control group.The changes of tumorigenic ability and metastatic foci in lung tissue were observed in the experimental group compared with the control group,and the effects of ciRS-7on tumor growth and metastatic ability of renal cell carcinoma were verified by in vivo experiments.(4)To investigate the specific molecular mechanisms and possible signaling pathways through which ciRS-7 acts.1.search for ciRS-7 downstream miRNA(miR-139-3p): firstly,we predicted the possible miRNAs and binding sites related to ciRS-7 by bioinformatics software,and screened the target miRNA(miR-139-3p)according to the expression level in the database.The direct target binding of ciRS-7 to miR-139-3p was verified by dual luciferase reporter gene assay,RNA pulldown assay and FISH assay,and the expression status and OS relationship of miR-139-3p and the correlation between ciRS-7 and miR-139-3p were examined in tissue samples as well as TCGA database.2.Functional assays to detect the effects of miR-139-3p on biological behaviors such as proliferation,migration and invasion of renal cell carcinoma cell lines: q RT-PCR to verify the effects of overexpression and knockdown of ciRS-7 in 786-O and ACHN cell lines on miR-139-3p expression levels;Ed U cell proliferation assay,CCK-8 and colony formation assay to detect The effect of miR-139-3p on the proliferation ability of renal cell carcinoma cells;the effect of miR-139-3p on the migration and invasion ability of renal cell carcinoma cells was detected by wound healing assay,Transwell migration and invasion assay.3.Search for ciRS-7 downstream target genes(TAGLN)and signaling pathway(PI3K/AKT signaling pathway): sh-ciRS-7 and sh-NC cell lines were first sequenced at transcriptional level and validated at protein level to screen ciRS-7 target genes(TAGLN).Bioinformatics software predicted the binding site of miR-139-3p to TAGLN,and dual luciferase reporter gene experiments verified the direct target binding of miR-139-3p to TAGLN.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis was performed to screen for possible downstream signaling pathways(PI3K/AKT signaling pathway),and Western-blot method was further applied to detect changes in the expression of PI3K/AKT signaling pathway-related proteins.The effect of elevated miR-139-3p expression after overexpression of ciRS-7 on the proliferation,migration and invasion of renal cell carcinoma cell lines and the expression changes of downstream TAGLN and PI3K/AKT signaling pathway related proteins were examined in the salvage assay;the effect of decreased miR-139-3p expression after knockdown of ciRS-7 on the proliferation,migration and invasion of renal cell carcinoma cell lines and the expression changes of downstream TAGLN and PI3K/AKT signaling pathway related proteins were examined.The effects of reducing the expression of miR-139-3p after knocking down ciRS-7 on the proliferation,migration and invasion of renal cell carcinoma cell lines,and the changes in the expression of downstream TAGLN and PI3K/AKT signaling pathway-related proteins.(5)Construct related nanocomplexes(PBAE/si-ciRS-7)and explore the therapeutic effects of nanocomplexes in vivo: different ratios of nanocomplexes were prepared according to the weight ratio of PBAE and si-ciRS-7.Particle size,polymer dispersibility index(PDI)assay and agarose gel electrophoresis were used to detect the optimal weight ratio of PBAE and si-ciRS-7 as well as the si RNA encapsulation rate.Transmission electron microscopy(TEM)and particle size potentiometry were used to detect the particle size of PBAE/si-ciRS-7nanocomplexes.CCK-8 assay was performed to detect the effect of PBAE/si-ciRS-7nanocomplexes on the proliferation ability of renal cell carcinoma cells;786-O cell lines overexpressing ciRS-7 lentivirus(OE-ciRS-7)were inoculated into immunodeficient mice by subcutaneous and tail vein injection,and observed the changes of subcutaneous tumorigenic ability and metastatic foci in lung tissue in different treatment groups(saline,si-ciRS-7 and PBAE/si-ciRS-7 nanocomplex),and in vivo experiments to verify the effects of PBAE/si-ciRS-7 nanocomplex on tumor growth as well as metastatic ability of renal cell carcinoma.Results:(1)ciRS-7 is associated with renal cell carcinoma progression and metastasis: according to the screening criteria,743,110 and 210 upregulated circ RNAs were identified in the GSE100186,GSE108735 and GSE137836 datasets,respectively,and the Venn diagram showed only 2 circ RNAs associated with renal cell carcinoma progression and metastasis(hsa＿circ＿0001946 and hsa＿circ＿0002484),and hsa＿circ＿0001946(ciRS-7)was finally identified as the subject of this study.(2)High expression of ciRS-7 in renal cell carcinoma tissues and cell lines correlated with poor prognosis: agarose gel electrophoresis verified the presence of ciRS-7 in 786-O,Caki-1,769-P and ACHN cell lines;Sanger sequencing confirmed that ciRS-7 is a covalently closed continuous loop formed by reverse shearing of CDR1;RNase Rase and Act FISH and nucleoplasmic separation experiments confirmed that ciRS-7 was mainly distributed in the cytoplasm of renal cell carcinoma cells.The high expression of ciRS-7 in renal cell carcinoma cell lines and tissues relative to normal tubular epithelial cells and normal renal tissues was confirmed by q RT-PCR assay,and the high expression of ciRS-7 correlated with tumor size and Fuhrman grade.Kaplan-Meier survival analysis showed that high ciRS-7 expression was associated with poor survival,and multivariate Cox regression analysis showed that high ciRS-7 expression was a poor prognostic risk factor for patients with renal cell carcinoma.(3)ciRS-7 promotes proliferation,migration and invasion,tumor growth,and lung metastasis of renal cell carcinoma cells in vitro and in vivo: q RT-PCR and FISH assays revealed that si-ciRS-7#2 significantly decreased ciRS-7 expression and OE-ciRS-7 significantly increased ciRS-7 expression in 786-O and ACHN cell lines;Ed U cell proliferation assay,CCK-8,and colony formation assay confirmed that compared with the control,the proliferation ability of 786-O and ACHN cell lines transfected with si-ciRS-7#2 was significantly decreased,and the proliferation ability of 786-O and ACHN cell lines transfected with OE-ciRS-7 was significantly enhanced;wound healing assay,Transwell migration,and invasion assay showed that the proliferation ability of 786-O and ACHN cell lines transfected with si ciRS-7#2 showed a significant decrease in the migration and invasion ability of 786-O and ACHN cell lines transfected with OE-ciRS-7,and a significant increase in the migration and invasion ability of786-O and ACHN cell lines transfected with OE-ciRS-7.Subcutaneous tumorigenesis models as well as lung metastasis models showed that decreasing ciRS-7 expression significantly inhibited the growth and lung metastatic ability of subcutaneous tumors,and increasing ciRS-7 expression significantly enhanced the growth and lung metastatic ability of subcutaneous tumors.(4)1.miR-139-3p is a downstream miRNA of ciRS-7: two miRNAs(miR-7-5p and miR-139-3p)potentially related to ciRS-7 were predicted by circ Bank,circ Atlas,miRanda and RNAhybrid software in the TCGA database.miR-7-5p was highly expressed in renal cell carcinoma tissues,while miR-139-3p was lowly expressed,identifying miR-139-3p as the target miRNA.bioinformatics software predicted three possible binding sites for ciRS-7 and miR-139-3p,and dual luciferase reporter gene assays showed that miR-139-3p mimics could bind to wild-type ciRS-7 The third binding site directly binds to repress the fluorescence activity of the reporter gene but does not reduce the fluorescence activity of the mutant ciRS-7 reporter gene.RNA pull-down assays revealed that miR-139-3p could pull down more ciRS-7 compared to the control,and FISH assays revealed that ciRS-7 and miR-139-3p were co-localized in the cytoplasm.In addition,q RT-PCR assay found that ciRS-7 was negatively correlated with miR-139-3p expression in 85 pairs of tissue samples.The above experiments suggest that ciRS-7can directly bind miR-139-3p.2.miR-139-3p inhibited the proliferation,migration and invasion of renal cell carcinoma cells in vitro: Ed U cell proliferation assay,CCK-8 and colony formation assay confirmed that the proliferation ability of 786-O and ACHN cell lines was significantly decreased after overexpression of miR-139-3p compared with the control group,and the proliferation ability of 786-O and ACHN cell lines was significantly enhanced after knockdown of miR-139-3p expression.Wound healing assay,Transwell migration and invasion assay showed that the migration and invasion ability of 786-O and ACHN cell lines were significantly decreased after overexpression of miR-139-3p,and the migration and invasion ability of 786-O and ACHN cell lines were significantly increased after knockdown of miR-139-3p expression.3.TAGLN is a target gene of ciRS-7,which activates PI3K/AKT signaling pathway:transcriptional level sequencing of sh-ciRS-7 and sh-NC cell lines revealed 41 up-regulated differentially expressed genes(DEGs)and 36 down-regulated DEGs.in addition,protein level validation of sh-ciRS-7 and sh-NC cell lines,492 proteins were found to be highly expressed and 555 proteins were found to be lowly expressed.Μltimately,TAGLN was found to be downregulated at both the transcriptional and protein levels.Dual luciferase reporter gene assays showed that miR-139-3p could directly bind TAGLN.in addition,further KEGG pathway partitioning showed that the PI3K/AKT signaling pathway was significantly enriched.Subsequent,western-blot assays revealed that transfection with sh-ciRS-7 or miR-139-3pmimic resulted in decreased protein levels of TAGLN,p-AKT and p-PI3 K in 786-O and ACHN cells,and transfection with OE-ciRS-7 or miR-139-3p-inhibitor resulted in decreased protein levels of TAGLN,p-AKT and p-PI3 K protein levels increased after transfection with OE-ciRS-7 or miR-139-3p-inhibitor.western-blot assay revealed that miR-139-3p-mimic could partially counteract the increased expression of TAGLN,p-AKT and p-PI3 K brought about by OE-ciRS-7,while miR-139-3p-inhibitor could partially rescue the sh-ciRS-7 inhibited the reduced expression of TAGLN,p-AKT and p-PI3 K.In addition,the results of Ed U cell proliferation assay,CCK-8,colony formation assay,wound healing assay,and Transwell migration and invasion assay also revealed that miR-139-3p-mimic could partially counteract the enhanced cell proliferation,migration and invasion ability brought about by OE-ciRS-7,while miR-139-3p-inhibitor could partially rescue the inhibition of cell proliferation,migration and invasive ability brought about by sh-ciRS-7.(5)Construction of PBAE/si-ciRS-7 nanocomplexes and therapeutic effects in vivo: Nine different ratios of nanocomplexes were prepared based on the weight ratio of PBAE and siciRS-7.It was found that relatively uniform nanoparticles were formed when the weight ratio of PBAE and si-ciRS-7 exceeded 40,while excellent performance was exhibited when PBAE/si-ciRS-7 was 80.Agarose gel electrophoresis analysis showed that when the weight ratio of PBAE to si-ciRS-7 exceeded 80,PBAE and si-ciRS-7 could effectively coalesce together and the loading efficiency of PBAE could reach 98%.Further TEM as well as particle size potentiometry showed that the particle size of the PBAE/si-ciRS-7 nanocomplex was about160 nm.In addition,CCK-8 experiments showed that the PBAE/si-ciRS-7 nanocomplex had a stronger ability to inhibit the proliferation of 786-O and ACHN cells than PBAE and si-ciRS-7.Results from in vivo subcutaneous tumorigenesis models as well as lung metastasis models showed that PBAE/si-ciRS-7 nanocomplexes better inhibited renal cell carcinoma tumor growth and metastasis compared to animal-grade si-ciRS-7.Conclusion:We first screened circ RNAs associated with renal cell carcinoma progression and metastasis by microarray and focused on the intrinsic mechanism of ciRS-7 in renal cell carcinoma.We found that ciRS-7 was highly expressed in renal cell carcinoma tumor tissues and cell lines,and correlated with tumor size,high Fuhrman grade,and poor prognosis.In vitro and in vivo experiments revealed that depletion of ciRS-7 significantly inhibited proliferation,invasion,tumor growth and metastasis of renal cell carcinoma cells in vivo,while overexpression of ciRS-7 had the opposite effect.Mechanistically,ciRS-7 acts as a "ce RNA" for miR-139-3p by blocking the degradation of TAGLN and through the PI3K/AKT signaling pathway promotes the progression and metastasis of renal cell carcinoma.In addition,miR-139-3p mimics or inhibitors can reverse the altered malignant tumor behavior caused by ciRS-7 overexpression or silencing.Furthermore,PBAE/si-ciRS-7 nanocomplex could significantly inhibit tumor progression and metastasis in RCC in vivo,which could provide new ideas for related drug development. |
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