The Role And Mechanism Of Circular RNAs(cSLC8A1) In Sunitinib Resistance Of Renal Cell Carcinoma

ObjectiveKidney cancer,also known as renal cell carcinoma(RCC),is one of the most common malignant tumors in the urinary system.Its incidence rate has increased rapidly at 2% per year in the past two decades.However,most of the early stage RCC have no obvious clinical symptoms,and about 20% of patients have metastasis at initial diagnosis.Even after early surgical treatment,30% of the patients with RCC in situ still undergo long-term recurrence and metastasis.As the tumor cells are insensitive to radiotherapy and chemotherapy,which greatly limits the effective treatment of metastatic RCC.The first targeted drug has been used in the treatment of metastatic RCC since 2005 and has achieved good results.Subsequently,the drugs of TKI represented by Sunitinib,Pazopanib,Sorafenib were widely used in the clinical treatment of metastatic RCC.Sunitinib,as a multitargeted tyrosine kinase inhibitor that has potent anti-angiogenic effects and direct anti-tumor activities.Evidence-based clinical trials had confirmed that sunitinib can effectively prolong progression-free survival(PFS),overall survival(OS),and improve objective response rate(ORR)and disease control rate(DCR).Sunitinib has been recommended by major urology guidelines as first-line treatment for metastatic RCC.However,clinical practice found that about one-third of the patients became resistant to sunitinib during the initial medication,and the major patients who were initially sensitive also had secondary drug resistance after one year of treatment,which resulted in a failure to achieve a substantial breakthrough in targeted drug therapy for metastatic RCC.At present,the molecular mechanism of targeted drug resistance for RCC is unclear,and there is no effective means to reverse this resistance.Therefore,it is the focus of research to explore the molecular basis and mechanism of drug resistance for RCC targeted therapy,and use effective targets to break and reverse drug resistance to achieve prolonged patient survival.Circular RNAs(circ RNAs) were a class of small RNAs with closed loop structures.They were formed by exon skipping or back splicing of pre-m RNAs.Owing to their circular structure,circ RNAs have higher stability to exist in the human nuclei and avoid degradation by RNases.Circ RNAs were previously considered to be a non-functional substance.With the frequent use of high throughput sequencing and in-depth mechanistic research,circ RNAs have been found to be widely present in various organs and tissues and plays an important biological function.Many studies have proved that circ RNAs were involved in important physiological processes of tumorigenesis and development,and were closely related to tumor proliferation,invasion,and metastasis.This study expects to explore the new function of circ RNAs in the resistance of sunitinib to RCC,and to further research the molecular mechanism of drug resistance.At the same time,new targets for reversing the targeted resistance of RCC and new biomolecular markers to predict drug resistance were provided to clinical application through our research.Methods1.The RCC cell lines were sequentially treated with sunitinib in vitro to construct RCC sunitinib-resistant cell lines.In order to verify the cell lines sunitinib resistance,CCK-8 experiments and plate clone experiments were performed.2.After confirming the successful construction of drug-resistant cell lines,high throughput RNA sequencing(RNA-seq)of the whole transcriptome was performed to analyze the different expressions and distribution characteristics between the two groups of drug-resistant and sensitive cells(3: 3).3.Design circ RNAs "back-to-back" primers and verify primers specificity by Sanger sequencing and agarose gel electrophoresis.Six up-regulation and six down-regulation circular RNAs were selected,and they were verified by real-time PCR(qRT-PCR).4.Expand the verification samples to determine the circ RNA of interest.qRT-PCR was used to verify the correlation between the circ RNAs expression level of different RCC cell lines and the half inhibitory concentration(IC50)of the corresponding sunitinib.qRT-PCR was used to verify the correlation between target circ RNA and clinical prognosis in 46 renal cell carcinoma tissue samples.5.To verify the circularity characteristics and cell sub-localization of cSLC8A1,the actinomycin D experiment,RNase R digestion experiment,and fluorescence in situ hybridization(FISH)experiment were performed.6.Knockdown lentivirus of cSLC8A1 sh RNA(short hairpin RNA)and over-expressed lentivirus were constructed stable transfected to RCC cell lines.After RCC cells were stably transfected successfully,qRT-PCR was used to verify knockdown and overexpression efficiency.CCK-8 experiment and plate clone experiment were performed to verify the reactivity of RCC cells to sunitinib after intervention.786-O RCC cells and control cells whose knockdown of cSLC8A1 were implanted subcutaneously in mice,and sunitinib was administered at a dose of 40 mg / kg / day after tumor formation,and tumor size was measured.7.Perform identification of Pull-down target protein by mass spectrometry and combine with the database to predict that RNA-binding proteins(RBP)of cSLC8A1,and further verify that cSLC8A1 can be combined with the target protein FUS by RNA immunoprecipitation experiments.8.Through qRT-PCR experiment,Western-blot experiment,and immunohistochemical experiment,analyze the protein expression level of FUS and m RNA transcription expression level of the RBP in drug-resistant and sensitive cells and tissues,and analyze its correlation with clinical prognosis.By constructing knockdown and over-expressing FUS with RCC cell lines,through CCK-8 experiment and plate clone experiment to further verify the functional role of FUS in RCC cell lines.9.Protein immunoprecipitation experiment and Cycloheximide(CHX)inhibition experiment were performed to verify that cSLC8A1 plays a functional role through regulate the stability of FUS protein.10.Validate that cSLC8A1 binds to FUS protein to play a functional role,through the presence or absence of cSLC8A1 conditional knockdown or overexpression of FUS by Rescue experiment.Detect of IC50 by CCK-8 experiment to explain the interactions between cSLC8A1 and FUS proteins in sunitinib resistance of RCC Cells.Results1.In vitro experiments proved that the RCC sunitinib resistance cell lines 786-O and ACHN were successfully constructed.We named them Sun-7R(786-O sunitinib resistant cell line)and Sun-AR(ACHN sunitinib resistant cell line),2.RNA-seq results showed that circ RNAs were widely expressed in sunitinib-resistant and sensitive cells.A total of 35 circ RNAs with significant differences which were obtained based on a Fold Change(FC)?2 and FDR <0.05,of which 18 were up-regulated and 17 were down-regulated.3.Select up-regulated circ RNAs of c SPECC1,c MPP6,c SLAIN2,c RANBP9,c PHACTR4,c PAPPA2 and down-regulated cSLC8A1,c LPAR1,c MTCL1,c SCLT1,c DCBLD2,c HIPK2 in the sunitinib-resistant group,and design specific primers for them.qRT-PCR results showed 12 circ RNAs expression had the same trends with sequencing results.The up-regulated c PAPPA2 and the down-regulated cSLC8A1 was significantly different from two groups.4.Validation results in different cell lines of RCC and clinical samples show that cSLC8A1 is related to sunitinib resistance,that is,cSLC8A1 is low-expressed in sunitinib-resistant tissues or cells,and the prognosis of the low-expression group is worse.5.Determine that the circ RNA of interest is cSLC8A1(hsa?circ?0000994 in Circbase database).The circ RNA consists of the back-splicing of the second exon of SLC8A1,which is 1832 bp in length.It has high abundance expression in RCC.cSLC8A1 and has stability characteristics of circ RNA.Cell sub-localization showed that cSLC8A1 can exist widely in the cytoplasm and nucleus.6.After transfection of cSLC8A1 sh RNA lentivirus to 786-O and ACHN cell lines,both cells showed resistance to sunitinib;Sunitinib resistant cell lines restore sensitivity after overexpressing cSLC8A1.In vivo experiments also confirmed that knockdown of cSLC8A1 can promote sunitinib resistance.7.To verify that the downstream mechanism of cSLC8A1,the mass spectrometry and database prediction show that cSLC8A1 has strong binding to the RNA-binding protein FUS.8.Western-blot and immunohistochemical results showed that FUS protein was highly expressed in sunitinib-resistant cells or tissues,but there was no difference in its m RNA expression level;FUS protein expression was inversely correlated with SLC8A1 expression,and patients with high expression of FUS in RCC patients had worse prognosis.Sunitinib-resistant renal cell lines can restore sensitivity after knocking down FUS.9.Further research shows that cSLC8A1 could bind to FUS protein and promotes its ubiquitination,thereby promoting the degradation of FUS protein to reverse sunitinib-resistant.10." Rescue experiment" results showed that knocking down cSLC8A1 can promote resistance of RCC cells to sunitinib,and at the same time,knocking down FUS can restore sensitivity;Overexpression of FUS can promote resistance of RCC cells to sunitinib,at the same time,overexpression of cSLC8A1 can reverse its resistance.ConclusionThere are a large number of circ RNAs,which widely involved in the process of drug resistance in renal cell carcinoma.The difference expression of cSLC8A1 is closely related to drug resistance particularly,that is,cSLC8A1 is low expressed in resistant cells and tissues.Patients are more likely to develop sunitinib-resistant when cSLC8A1 is down-regulated.cSLC8A1 could be a potential biomarker for predicting sunitinib sensitivity in RCC.cSLC8A1 competitively binds to FUS protein,promotes FUS ubiquitination and degrades the protein,thereby reversing sunitinib resistance in RCC.cSLC8A1 may become a new therapy target to reverse sunitinib resistance in RCC.