The Effects Of Exosomal Circular RNA In Cutaneous Squamous Cell Carcinoma And Its Mechanisms

Introduction: Cutaneous squamous cell carcinoma(c SCC)is a vicious skin cancer,with slightly lower malignancy but much higher incidence than cutaneous melanoma.The local recurrence and distant metastasis of c SCC has a close correlation with high risk of death that derived from skin cancers.Early diagnosis and accurate treatment are cable of improving the prognosis of the patients significantly.Exosome is a kind of cell-secreted nano-vesicle that contains genetic material and metabolites,with specific marker proteins on the surface.Most mammalian cells can secrete exosomes which have similarities between each other,but exosomes from different species and different types of cells differ in internal cargoes and superficial marker proteins.Exosomes can bind to recipient cells through their superficial marker and release the internal RNA or DNA,thus play a role in biological processes.It is widely found in a variety of body fluids,such as plasma,urine,saliva and cerebrospinal fluid,as well as tissue and cells.Up to date,the role that exosome plays in tumor invasion and metastasis has drawn attention gradually.Epithelial to mesenchymal transition(EMT)is the process that epithelial cells transform to mesenchymal cells,which is the initial step of tumor invasion and plays a vital role in tumor regulation.Exosome carrying EMT related factors(such as HIF1,MMP13)can accelerate the process of EMT and increase the metastatic tendency of recipient cells.There have been a few researches digging in the relationship between exosome and oral squamous cell cancer or head and neck squamous cell cancer,indicating that exosome takes part in the activation of keratinocyte pathways,cell differentiation,cell signal tranduction and the construction of suitable micro-environment in recipient cells.Nevertheless,what kind of role that exosome plays in cutaneous squamous cell carcinoma remains ambiguous.Circular RNA(circRNA)is a small single-stranded RNA molecule in the shape of closed double-ended.Because of their special structure,they are abundant and exist stably in eukaryotic cells,especially in exosomes.CircRNA was initially referred to as the by-product of aberrant splicing and had no biological function.Until recent couple of years,it has been noticed that circRNAs can serve as sponge of miRNAs and this function can regulate transcription,which made circRNA a popular topic in research fields.CircRNAs can bind miRNAs via their multiple binding sites,just like a sponge absorbing water,circRNAs can "absorb" miRNAs from down-stream target genes.Besides,some circRNAs also have protein binding sites,open reading structure that are capable of protein translation,RNA binding sites that regulates transcriptional splicing and so on.These functions,along with the "sponge effect" of circRNA on miRNA,realizing the process of circRNA regulating gene expression together.In the progress of tumorgenesis and tumor progression,the role of circRNA has been verified gradually by an increasing number of researches.The relationship between circRNA and cutaneous squamous cell carcinoma has been carried out by several researches lately,suggesting that circRNA may participate in cutaneous squamous cell carcinoma by virtue of its "sponge effect" on miRNA.Transduction of Wnt signal is triggered by the combination of Wnt ligands and cellular superficial Fzd family receptor initially,which can regulate the cell proliferation,cell differentiation,cell migration and so on.According to the dependence of ?-catenin,Wnt signaling pathways are divided into Wnt/?-catenin signaling and ?-catenin-independent non-canonical pathways.Abnormal transduction of Wnt/?-catenin is one of common signaling pathways in cutaneous squamous cell carcinoma,canonical Wnt/?-catenin signaling takes part in modulating c SCC cell's proliferation and apoptosis,while non-canonical Wnt5 a pathway plays a role in the tissue invasion of c SCC and in the regulation of EMT,therefore,Wnt5 a can change the process of metastasis in c SCC.But in cutaneous squamous cell carcinoma,whether the differentially expressed circRNAs can alter the cell viability,apoptosis and metastasis via their inducement to abnormal transduction of Wnt/?-catenin signaling has not been studied.In spite of the fact that researches on the association of exosomes and tumor,and researches on the association of circRNA and tumor are both getting more deeper,the researches on correlation of exosomal circRNA and the development of tumor still remains relatively little.The purpose of this study was to find out the differentially expressed exosomal circRNAs and to explore the role of their binding miRNAs in the invasion and metastasis of skin squamous cell carcinoma,as well as the interaction of the differentially expressed circRNA and Wnt/?-catenin pathway,predicting the possible reasons for metastasis and providing the potential targets for determination of prognosis.Methods:1.Experimental materials Clinical sample:Patients that had been diagnosed with cutaneous squamous cell carcinoma in the department of dermatology,NO.1 Hospital of China Medical University were collected.All subjects were volunteered to participate in the experiment with the informed consent.Then their tumor tissues and adjacent non-tumor tissues were collected after pathological examination,and 10 ml fasting venous blood were collected before treatments.As for healthy control group,10 ml fasting venous blood samples were collected with informed consent.All the blood sample was centrifuged to gain plasma sample.This study was approved by ethics committee of the First Hospital of China Medical University(No.AF-SOP-07-1.1-01).Cell lines:Cutaneous squamous cell carcinoma cell lines(human SCL-1 and human HSC-5).2.Extraction and identification of exosomes Exsomes were extracted from plasma by exo Easy Maxi kit,transmission electron microscope was utilized to observe the morphology of exosomes,western blot was applied to verify the marker protein of exosomes and Nano tracking analysis was used to demonstrate the diameter of exosomes.3.High throughput sequencing,bioinformatic analysis and validation Total RNA was extracted from exosomes through Trizol LS Reagent and NEBNext r RNA Depletion kit was used to remove ribosomal RNA in total RNA.In order to construct sequencing library,NEBNext Ultra II Directional RNA Library Prep kit was used,the quality control and quantification of the library were completed by Bio Analyzer2100 system.Library sequencing was performed on an illumina Hiseq instrument with150 bp paired end reads.high quality reads were aligned to the reference genome/transcriptome with the STAR software,and the circRNAs were identified with DCC software.The selected circRNAs were annotated using circ Base,and the differentially expressed circRNAs were identified with the Edge R software,GO and KEGG analyses were then performed for functional annotation.The result of sequencing was validated by q RT-PCR.The interaction between the relevant circRNAs and their target miRNAs were predicted using the Mi Randa,and the network was constructed by Cytoscape software.4.The expression of circRNA(hsa?circ?0060927)and miR-4438 in tumor tissues and non-tumor adjacent tissues was examinated by QRT-PCR5.Cell transfection The over-expressed vector of circRNA(hsa?circ?0060927)and the mimic vector of miR-4438 was applied to transfect SCL-1 and HSC-5 cell lines,transfected cells were divided into four groups:1)control group:without any vector;2)negative control group:transfected with blank vector and NC mimic;3)circ group:transfected with circ vector and NC mimic;4)circ+miRNA group:transfected with circ vector and miRNA mimic.All the transfecting work was complected using Lipofectamine 3000 kit,and the ratio of tranfected cells were confirmed by PCR.6.Cell viability The viability of all groups of transfected cells was evaluated by Cell Counting kit-8.7.Apoptosis ratio The apoptosis ratio of all groups of transfected cells was assessed by flow cytometry8.EMT-related protein expression TGF?1 was added to induce the EMT process and western blot was used to measure the expression level of EMT-related proteins of transfected cells(E-cadherin,Vimentin,Twist1 and Zeb1)..9.Wnt/?-catenin signaling pathway The levels of signaling pathway proteins(Wnt3a and ?-catenin)of each group were demonstrated via western blot;CCK-8 kit and flow cytometry were used to detect cell viability and apoptosis ratio in each group after treatment with the antagonist of Wnt/?-catenin,DKK3;After treating with TGF?1,the levels of EMT-related proteins of each group after treatment with SFRP2 were determined by Western blot..10.Statistical analysis Data collected from experiments were analyzed and diagrammed by Graph Pad Prism 6and Image J.Groups were compared with one-way analysis of variance(ANOVA)and Newman-Keuls.The statistical significance was announced when p<0.05.Results:1.The extracted exosomes from plasma are qualified in morphology,diameter and the expression of superficial marker proteins(CD9,TSG101 and CD63)as previously described in litertures.2.101 significantly differentially expressed circRNAs were selected in total(|log FC|>=2and P<0.05),including 25 up-regulated circRNAs and 76 down-regulated circRNAs,and most of these circRNAs were found to concentrate on the chromosome 1 and chromosome2,80 percent of them were exonic and 2091 circRNAs were newly discovered.Ten circRNAs were randomly selected to verify the accuracy of sequencing results via q RT-PCR,and the PCR results of eight of ten were consistent with the sequencing results,convincing the sequence results.3.The expression of hsa?circ?0060927 in tumor tissues is lower than that in non-tumor adjacent tissues,while the expression of miR-4438 in tumor tissues is higher than that in non-tumor adjacent tissues.4.The circ group(both SCL-1 and HSC-5 cell lines)showed a decreased level in cell viability and an increased level in apoptosis ratio,comparing to those of the control and the negative control groups.The circ+miRNA group remained the decline in cell viability and the advance in apoptosis ratio,yet a slighter range of variation was discovered when comparing with the circ groups.5.The circ group(SCL-1 and HSC-5)expressed more E-cadherin and less Vimentin,Twist,and Zeb1,comparing with the control group and the negative control group,the circ+miRNA group demonstrated the same tendency as the circ group in the expression of EMT-related proteins.But the amplitude of fluctuation of four kinds of EMT proteins in the circ+miRNA group was weakened.6.The circ group exhibited an declined expression of Wnt3 a and ?-catenin comparing to the control group and the negative control group,the circ+miRNA group still showed a reduced expression of Wnt3 a and ?-catenin,yet the degree of reduction was obviously hindered.7.After treated with DKK3,the circ group still showed a lower cell viability and a higher apoptosis ratio than control group and negative control group,but the range of changes between groups was smaller than that of cells without DKK3 treatment.The variation of the circ+miRNA group was similar to that of cells without DKK3 treatment,but the range was smaller than the former groups.8.After treated with SFRP2 and TGF?1,the circ group remained an increased expression in E-cadherin and a decreased expression in Vimentin,Twist1 and Zeb1,yet the changes between groups were smaller than that of cells without SFRP2 treatment.The variation of EMT-related protein in the circ+miRNA group was similar to that of cells without SFRP2 treatment,but the range was smaller than the former groups.Conclusion:1.Differentially expressed circRNAs from exosomes in cutaneous squamous cell carcinoma patients' plasma were identified,a total of 101 circRNAs with significant difference were screened,and the sequencing results were convinced,providing novel targets fors tudies related to cutaneous squamous cell carcinoma.2.The expression level of circRNA(hsa?circ?0060927)in tumor tissues was lower than that in non-tumor adjacent tissues,the expression level of miR-4438 in tumor tissues was higher than that in non-tumor adjacent tissues.3.Over-expressed circRNA(hsa?circ?0060927)can reduce the cell viability,induce cell apotosis and inhibit the process of EMT in c SCC cells.Co-transfected with circRNA(hsa?circ?0060927)and miR-4438 can partly eliminate the deactivating,promoting apoptosis and inhibiting EMT function of circRNA(hsa?circ?0060927)in c SCC cells.4.Overexpressed circRNA(hsa?circ?0060927)can block the transduction of Wnt/?-catenin pathway through impeding miR-4438.Wnt/?-catenin antagonist can attenuate the impacts of circRNA(hsa?circ?0060927)on cell viability,apoptosis and EMT process in c SCC to some extent.