RNA-Seq Profiling Of Circular RNAs And Study On The Carcinogenic Mechanism Of CircPVT1 In Skin Squamous Cell Carcinoma

Objective: Cutaneous squamous cell carcinoma((CSCC))is the second most common cancer in humans,with an estimated incidence of 1 million cases per year in the United States.The incidence continues to rise.In the past three decades,the number of CSCC has increased from 50% to 300%.By 2030,the incidence rate in European countries will double the current level.It is estimated that the risk of developing CSCC in the Caucasian population is 7 to 11 per cent(9 to 14 per cent for men and 4 to 9 per cent for women).Although it usually shows benign clinical features,it can also be locally invasive and metastatic.The 10-year survival rate after CSCC surgery is more than 90%,but when metastasis occurs,the survival rate drops sharply.The rate of lymph node metastasis is about 4%,and the mortality rate is nearly 2%.In view of its high incidence,CSCC has a significant impact on overall mortality.It is the second leading cause of skin cancer death after melanoma and the leading cause of skin cancer death in people over the age of 85.In some parts of the United States,its mortality rate is comparable to that of kidney cancer,oropharyngeal cancer and melanoma.Squamous cell carcinoma is caused by malignant proliferation of epidermal keratinocytes.Advanced age,male,fair skin,immunosuppression and previous history of actinic keratosis(AK)are known important risk factors.Surgery is the cornerstone of the treatment of CSCC,sometimes with radiotherapy.However,some patients with locally advanced and metastatic CSCC may benefit from systematic treatment.In recent decades,the signal pathways involved in the development of CSCC have produced targeted molecules.Circular RNA(circ RNAs)is a kind of single-stranded covalent closed-loop RNA with tissue /development specific expression pattern.circ RNA is stable and plays a carcinogenic or inhibitory role in tumorigenesis,proliferation,apoptosis,metastasis,invasion,chemotherapy resistance and prognosis.circ RNA acts as a mi RNA sponge,protein scaffold,or translation template.There is growing evidence that circ RNA promotes cancer progression by regulating the expression or function of host genes.This study starts with the changes of circ RNA expression profile in skin squamous cell carcinoma and the effect of differential circ RNA on skin squamous cell carcinoma cell line,further explores the effect of circ RNA on skin squamous cell carcinoma and its internal mechanism,and provides more ideas for finding intervention targets,improving curative effect,shortening treatment cycle,reducing recurrence rate and so on.Methods: In this study,human skin squamous cell carcinoma cell lines(A431 cell line,SCL-1 cell line,SCL-12 cell line,),human keratinocytes(Ha Ca T cell line)and skin squamous cell carcinoma tissues were selected as the objects of study.Among them,30 cases of skin squamous cell carcinoma were treated and operated in the Department of Dermatology and sexual Diseases of the first affiliated Hospital of China Medical University from September 2017 to September 2018.All cases were confirmed by pathology.Among the 30 patients,there were 17 males and 13 females,slightly more males than females,23 patients ? 50 years old and 7 patients<50 years old,mainly middle-aged and elderly patients.There were 26 patients whose lesions were located in the exposed site and 4 patients in the non-exposed part.The relevant experimental scheme was approved by the Ethics Committee of the first affiliated Hospital of China Medical University.The tissue samples were collected,the total RNA of the tissue was extracted,and the quality inspection of the total RNA was carried out.Detect the concentration,detect 28S/18 S or 23 Shand 16s,detect RIN,agarose gel electrophoresis to detect the quality of RNA,establish a RNA library,and sequence.The sequencing data were processed to get the circ RNA expression profile and draw the volcano map.Differential expression circ RNA,was screened to draw cluster heat map.Gene ontology and Kyoto encyclopedia of genes and genomes were used to enrich and analyze differentially expressed m RNA and other kinds of RNA-related genes.The interaction between mi RNA-m RNA,mi RNA-circ RNA of the differentially expressed RNA was predicted by using databases such as Target Scan and mi Randa,and the predicted results were plotted into a circ RNA-mi RNA-m RNA network diagram by Cytoscape.According to the predicted network map,the circ RNA was selected for verification by comprehensively considering the genes related to cell proliferation,migration and differentiation.Total RNA,was extracted from cultured A431,SCL-1 and SCL-12 skin squamous cell carcinoma cell lines and human keratinocyte cell lines.The differential circ RNA screened by high-throughput sequencing was verified by real-time quantitative PCR detection.circ RNA expression and differential analysis based on RT-q PCR detection is an effective supplement and verification of sequencing results.According to the closed ring structure of circ RNA,the Divergent primers,designed according to its junction sequence can be effectively distinguished from linear RNA.The primers were strictly verified in many aspects,including RT-q PCR amplification curve,dissolution curve,product agarose gel verification,product sequencing verification,accurate and reliable detection of circ RNA differential expression in tissues and cells.Its stability was verified by RNase R experiment.The expression of circ PVT1 in different cell lines was detected,and the suitable cell line(SCL-1)was selected for further functional experiment.The transient transfection of small interference RNA was constructed,and the transfection efficiency of circ PVT1 was detected by q RT-PCR.The function of circ PVT1 was verified by CCK-8,apoptosis,scratches and Transwell experiments.And combined with the first part of the bioinformatics analysis and previous research,speculate the possible regulation mechanism of circ PVT1.Prediction of circ PVT1--mi R-4663-TRPM(ce RNA mechanism)regulatory axis by bioinformatics.Using circbank database http://www.circbank.cn/search Circ.html)to query the sequence of circ PVT1(circ Base ID),(using Reg RNA2.0 http://regrna2.mbc.nctu.edu.tw/detection?output.php)pre-circ?0001821 to bind mi RNA to has?mi R?4663,3,and using mi Randa software to predict that the mi RNA bound to circ?0001821 is mi R-4663,mi R-3926,mi R-4287,mi R-3660,mi R-181a-2-3p,mi R-595.4.The prediction results of Reg RNA2.0 and the prediction results of mi Randa software are intersected by overlap,so we choose mi R-4663 for follow-up research.5.After ce RNA regulation and control network query,the m RNA targeted by mi R-4663 are MAP3K14,SLC2A7,ATPAF1,ONECUT2 and TRPM3.Then we use the Targetscan database to query the targeted mi RNA,of mi R-4663 and take the intersection.The intersection m RNA is ATPAF1,ONECUT2 and TRPM3.After the fifth step analysis,we find that the intersection m RNA is ATPAF1,ONECUT2 and TRPM3.There are four potential binding sites between mi R-4663 and TRPM3,and the binding score is high.Through literature investigation,we selected TRPM3 as the candidate target gene and selected a suitable cell line(SCL-1)to construct small interference RNA.Each group prepared 6 double holes,and cells were collected 48 hours after transfection.Among them,RNA,q RT-PCR was extracted from 3 wells to detect the expression of circ PVT1,mi R-4663 and TRPM3,and protein was extracted from 3wells,and the protein expression of TRPM3,VEGFA,SNAIL,Vimentin,N-cadherin,E-cadherin,MYC and GAPDH was detected by WB.To explore the effect of circ PVT1 on the expression of mi R-4663 and TRPM3.Results: 1)High throughput sequencing was used to determine the differentially expressed circ RNA in three pairs of CSCC and its adjacent normal tissues.This analysis identified 10669 different circ RNA molecules,and the distribution of this circ RNA on the resulting chromosomes was shown in the Circos map.After conditional screening(p<0.05,multiple change>2.0),we found that 449 circ RNA were differentially expressed between CSCC and normal paracancerous tissues,of which 393 were up-regulated in CSCC and 55 down-regulated in CSCC.The hierarchical clustering heat map and volcano map of these circ RNA expression profiles were drawn,which highlighted the importance of differentially expressed circ RNA.2)We performed GO analysis to identify the functions of differentially expressed circ RNA annotated host genes.The first30 rich GO analyses included DNA replication,endosomal transport and nucleic acid transport,cell composition terms were focus adhesion,cell substrate adhesion junctions and lamellar liposomes,and molecular function GO terms were guanine nucleotide exchange factor activity,magnesium ion binding and RAS guanine nucleotide exchange factor activity.In addition,we also carried out KEGG pathway analysis to further explore the function of host genes on differentially expressed circ RNA.The most abundant pathway identified in the first 15 enrichment pathways is autophagy-animal.Studies have shown that circ RNA can be used as a mi RNA sponge to isolate these molecules and inhibit their ability to regulate the expression of downstream target genes.Therefore,we then construct the circ RNA-mi RNA-m RNA interaction network according to the sequencing results.For this network,we selected the first 10 differentially expressed circ RNA,and then we used Miranda 3.3 to predict the relevant target mi RNAs and m RNAs as well as the specific regulatory network of circ PVT1.These regulatory networks provide ideas for further exploration of mechanisms through which differentially expressed circ RNA may affect skin squamous cell carcinoma.5)in order to explore the functional role of circ PVT1 in skin squamous cell carcinoma,we then constructed A431 and SCL-1 cells transfected with circ PVT1-specific si RNA to interfere with circ PVT1.Then we confirmed that these si RNA constructs targeting circ PVT1 junction sites significantly decreased the level of endogenous circ PVT1 in SCL-1 cells.CCK-8 detection showed that interfering circ PVT1 could significantly inhibit the proliferation of CSCC cells compared with control cells.We studied how circ PVT1 affects the apoptosis of SCL-1 cells by flow cytometry.The results showed that when circ PVT1 was interfered,the apoptosis and death of these cells increased significantly.In addition,in Transwell experiment,circ PVT1 gene knockout inhibited the migration and invasion of SCL-1 cells in vitro.6)according to the specific regulatory network of circ PVT1 and bioinformatics analysis,it was predicted that the regulatory axis of(ce RNA mechanism)was circ PVT1--mi R-4663-TRPM3,.SCL-1 cells were constructed with circ PVT1-specific si RNA,RNA,q RT-PCR was extracted to detect the expression of circ PVT1,mi R-4663 and TRPM3,and protein was extracted and WB was used to detect the protein expression of TRPM3,VEGFA,SNAIL,Vimentin,N-cadherin,E-cadherin,MYC and GAPDH.The construction of si RNA targeting the circ PVT1 junction site significantly decreased the level of endogenous mi R-4663 and increased the level of TRPM3 in SCL-1 cells.Conclusions: 1)The first 10 circ RNA with significant differential expression were regulated by circ RNA-mi RNA-m RNA coexpression network,and it was found that circ PVT1 had more nodes,and it was more likely to have important biological functions.Therefore,circ PVT1 was selected as the object of further study.circ PVT1 was highly expressed in tissue and skin squamous cell carcinoma cell lines,which was consistent with the results of sequencing.The cyclicity and stability of circ PVT1 were confirmed by experiments.2)the expression level of circ PVT1 in skin squamous cell line was higher than that in Hacat cell line,and the difference was statistically significant.We chose SCL-1 cell line for further functional experiment.After Si RNA interference,Q-RT-PCR detection showed that the expression of circ PVT1 in the interference group was significantly lower than that in the control group.The results of CCK-8,apoptosis and Transwell assay showed that the proliferation,migration and invasion ability of cell lines in circ PVT1 interference group were lower than those in control group,and the proportion of apoptosis was increased.3)the abnormally high expression of circ PVT1 may down-regulate the expression of mi R-4663 and relieve the inhibitory effect of mi R-4663 on TRPM3 to some extent,lead to the high expression of TRPM3,change the angiogenesis and epithelial mesenchymal transformation of skin squamous cell carcinoma,and then promote the occurrence and development of skin squamous cell carcinoma.