Circular RNA Expression Profile And Preliminary Study On Function And Mechanism Of Hsa_circ₀000745 In Cervical Cancer

Cervical cancer ranks as the 4th most common malignancy diagnosed and the 4th leading cause of cancer death in women worldwide.In China,there are about 98,900 new cases of cervical cancer each year,accounting for about 20%new cases worldwide,and there are about 30,500 patients died of cervical cancer every year.In the past few decades,researches on the pathogenesis and prevention of cervical cancer have made some progression.Clinical treatment of cervical cancer has been explored such as immunotherapy,chemotherapy and targeted therapy except for surgery and radiotherapy,but cervical cancer is still a global health problem.It has been clarified that persistent infection of high-risk human papillomavirus?HPV?has a carcinogenic effect on cervical cancer.Although HPV vaccination and early screening have a certain effect on reducing the incidence of cervical cancer,but these methods can not work for all cases.Only a small proportion of patients with high-risk HPV infection eventually develop into cervical cancer,and there are also patients with cervical cancer who are not accompanied by HPV infection.Therefore,in addition to the infection of high-risk HPV,there are other factors involved in the development of cervical cancer,but it still needs to be clarified.So it is necessary to explore the pathogenesis of cervical cancer in a more comprehensive and in-depth way to provide ideas for more accurate treatment methods.Circular RNAs?circRNAs?is a type of the noncoding RNAs,named for its unique structure of a covalent closed loop.CircRNAs are indicated to be stable and highly conserved across species and to show stage-specific and tissue-specific features.CircRNAs exists widely and plays an important role in physiological and pathological processes in human.CircRNAs can play a regulatory role in many ways.Firstly,circRNAs can combine with microRNAs?miRNAs?through their miRNA binding sites,which is called miRNA sponges.Then circRNAs can regulate the progression of different diseases by affecting the transcription and translation of target genes.Secondly,circRNAs can regulate RNA alternative splicing or transcription,including competing with linear mRNA in the splicing of precursor mRNA,and regulating the transcription of circRNA's parent genes through interaction with RNA polymerase II complex.Furthermore,circRNAs can interact with RNA binding proteins to regulate the efficiency of mRNA translation.In addition,a few circRNAs have the ability of translation into bioactive peptides.With the widespread use of RNA-sequencing,RNA microarray,and bioinformatics tools in recent years,the role and mechanism of circRNAs in more and more diseases have been gradually elucidated,and its role in malignant tumors has also received much attention.It has been found that circRNAs is differentially expressed in various cancers,and is related to cancer diagnosis and prognosis.CircRNAs is expected to become a new biomarker for diagnosis and prognostic evaluation.CircRNAs regulates gene transcription and translation through different ways mentioned above,thereby promoting or inhibiting the proliferation,apoptosis,infiltration and metastasis of cancer cells.CircRNAs also plays an important role in the progression of cervical cancer.The abnormal expression of circRNAs in cervical cancer has been proved to be related to HPV infection states,the development of cancer and radio-sensitivity of cancer cells.But the study of circRNAs in cervical cancer is still in the primary stage,and its role and mechanism have not been fully defined.The research of circRNAs in cervical cancer will enhance comprehensive understanding of the pathogenesis and exploration of new treatment in cervical cancer.In this study,we detected the expression of circRNAs in cervical cancer tissues by using the human circRNA microarray,aiming to initially clarify the expression profile of circRNAs in cervical cancer and to provide a basis for subsequent research.Through verification and preliminary analysis,hsa_circ₀000745 was selected as the research object from the differentially expressed circRNAs.Studies have found that the level of hsa_circ₀000745 is significantly down-regulated in cancer tissue and peripheral blood of gastric cancer patients,and its expression level is related to some clinicopathological factors of patients.However,the function and mechanisms of hsa_circ₀000745 in gastric cancer are not clear.The role of hsa_circ₀000745 in cervical cancer has not been reported.In this study,we analyzed the expression of hsa_circ₀000745 in cervical cancer tissues,and explored its function and mechanism in cervical cancer,providing new ideas and directions for exploring the pathogenesis of cervical cancer and new methods of clinical treatment.Part ? Identification of differentially expressed circRNAs in cervical cancerObjective:To detect the expression profile of circRNAs in cervical cancer tissues and analyze the differential expression of circRNAs.Methods:Human circRNA microarray was used to detect the expression of circRNAs in 5 pairs of cervical cancer and matched adjacent paracancerous tissues,and to clarify the differentially expressed circRNAs in cervical cancer tissues by comparative analysis to the control group.Ten differentially expressed circRNAs were selected and verified by quantitative reverse transcription-polymerase chain reaction?qRT-PCR?.Results:1.In this study,3008 human circRNAs were detected in cervical cancer tissues and paracanerous tissues by circRNA microarray.Through comparative analysis,178 circRNAs exhibited abnormal expression in cervical cancer tissues with a fold change>2.0,including 77 up-regulated and 101 down-regulated circRNAs.Most of the differentially expressed circRNAs are spliced from exons?149/178?.The report of circRNA microarray has been uploaded to Gene Expression Omnibus?GEO?website?ID:GSE102686?.2.Ten circRNAs were selected from the differentially expressed circRNAs,including hsa circ₀000745,hsa_circ₀000520,hsa_circ₀000515,hsa_circ₀084940,hsa_circ₀006174,hsa_circ₀064735,hsa_circ₀043280,hsa_circ₀062432,hsa_circ₀065898 and hsa_circ₀031027.The results of qRT-PCR showed that compared to paracancerous tissues,hsa circ₀000745,hsa_circ₀000520,hsa circ₀000515,hsa_circ₀084940 and hsa_circ₀006174 were significantly up-regulated in cervical cancer tissues?P<0.05?;while hsa_circ₀064735,hsa_circ₀043280,hsa_circ₀062432,and hsa_circ₀031027 were significantly down-regulated?P<0.01?,which were consistent with the microarray results;but the expression of hsa_circ₀065898 was not statistically significant?P>0.05?,which was not consistent with the microarray results.3.The association between the expression level of the 9 circRNAs and the clinicopathological characteristics of cervical cancer patients was analyzed,which include the clinical stage,tumor differentiation degree and tumor size,depth of interstitial infiltration,lymph node metastasis and vascular/lymphatic invasion states.The results showed that the expression level of hsa_circ₀000745 in patients with poorly differentiated tumors was significantly higher than that in well/moderate differentiated patients?P=0.019?;and the level of hsa_circ₀000745 was much higher in patients with vascular/lymphatic invasion than in patients without vascular/lymphatic invasion?P=0.028?.The expression level of hsa circ₀031027 in patients with deep interstitial infiltration was much lower than that in patients with superficial interstitial infiltration?P<0.001?.There was no association between the expression levels of the other 7 circRNAs and the clinicopathological factors of cervical cancer patients?P>0.05?.Conclusions:1.Compared to the paracancerous tissues,the expression levels of 178 circRNAs in cervical cancer tissues are changed,suggesting that the differential expression of circRNAs is related to cervical cancer.2.The differential expression of hsa_circ₀000745 and hsa_circ₀031027 is related to some clinicopathological factors of patients with cervical cancer,suggesting that hsa_circ₀000745 and hsa_circ₀031027 may be related to the progression of cervical cancer.Part ? The effect of hsa circ₀000745 on the biological behavior of cervical cancer cellsObjective:To detect the expression of hsa_circ₀000745 and SPECC1 mRNA in cervical cancer,analyze the association between hsa_circ₀000745 and clinicopathological factors of patients,and explore the function of hsa_circ₀000745 in cervical cancer.Methods:The expression of hsa_circ₀000745 and SPECC1 mRNA was detected by qRT-PCR in 59 pairs of cervical cancer tissues and paracancerous tissues.And the association between hsa_circ₀000745 and clinicopathological factors of cervical cancer patients was analyzed.The effects of hsa_circ₀000745 on the proliferation,apoptosis,migration and invasion of cervical cancer cells were measured by siRNA and lentivirus transfection,apoptosis and proliferation detection,Transwell assays and animal experiments.Results:1.Compared to the paracancerous tissues?1.025±0.059?,the expression of hsa_circ₀000745 in cervical cancer tissues was significantly up-regulated?3.421±2.307,P<0.001?.The expression of SPECC1 mRNA was also up-regulated in cervical cancer tissues?2.320±1.661?than the control group?1.012±0.018,P<0.001?.Furthermore,hsa_circ₀000745 positively correlated with the expression of SPECC1 mRNA in cervical cancer tissues?r=0.449,P<0.001?.2.Compared to cervical cancer patients with well or moderately differentiated tumors?2.381±1.588?,the expression of hsa_circ₀000745 in patients with poorly differentiated tumors increased significantly?3.960±2.313,P=0.005?.Compared with patients without vascular/lymphatic invasion?2.673±1.482?,the level of hsa_circ₀000745 was higher in patients with vascular/lymphatic invasion?3.900±2.543,P=0.026?.3.After inhibiting the expression of hsa_circ₀000745,the proliferation ability of CaSki,SiHa and HeLa cells was significantly reduced?P<0.05?,and the volume of subcutaneous xenografts in nude mice was significantly smaller than that in the control group?P<0.05?.4.Inhibition of hsa_circ₀000745 significantly reduced the migration and invasion ability of CaSki,SiHa and HeLa cells?P<0.01?.Inhibition of hsa_circ₀000745 had no effect on cell apoptosis?P>0.05?.Conclusions:1.The expression of hsa_circ₀000745 is up-regulated in cervical cancer,and the level of hsa circ 0000745 is related to the degree of tumor differentiation and vascular/lymphatic invasion status of patients,suggesting that hsa_circ₀000745 is involved in the development of cervical cancer.2.After inhibiting the expression of hsa_circ₀000745,the proliferation,migration and invasion of CaSki,SiHa and HeLa cells are significantly reduced,and the tumorigenicity of CaSki cells in nude mice is decreased,suggesting that hsa_circ₀000745 works as a tumor-promotor in the development of cervical cancer.Part ? Study on the mechanism of hsa_circ₀000745 in cervical cancerObjective:To explore the potential target and mechanism of hsa circ 0000745 in cervical cancer.Methods:TargetScan and miRanda databases were used to predict the miRNAs that may bind to hsa_circ₀000745.Through Gene Ontology?GO?and Kyoto Encyclopedia of genes and genes?KEGG?pathway analysis,combined with the function of hsa_circ₀000745 on cervical cancer cells in the second part,the potential target genes and proteins of hsa_circ₀000745 were analyzed and predicted.The predicted miRNAs and target proteins were verified by qRT-PCR,Western blot,cell transfection and Transwell assays.Results:1.Combined with TargetScan and miRanda,the top five miRNAs with the potential to bind with hsa_circ₀000745 were hsa-miR-9-5p,hsa-miR-145-5p,hsa-miR-526b-5p,hsa-miR-578 and hsa-miR-29a-5p.GO and pathway analysis combined with the effect on biological behavior of cervical cancer cells,we predicted that E-cadherin,?-Catenin,Vimentin,p-RAS and p38 MAPK might be the targets of hsa_circ₀000745.2.The results of qRT-PCR showed that inhibiting the expression of hsa_circ₀000745 had no effect on the expressions of hsa-miR-9-5p,hsa-miR-145-5p,hsa-miR-526b-5p,hsa-miR-578 and hsa-miR-29a-5p?P>0.05?.3.Western blot showed that the expression of E-cadherin in CaSki,SiHa and HeLa cells was increased significantly after the inhibition of hsa_circ₀000745?P<0.01?.4.In CaSki,SiHa and HeLa cells with inhibition of hsa_circ₀000745,suppressing the expression of E-cadherin could reverse the inhibition on the migration and invasion of cancer cells?P<0.01?.Conclusions:Hsa_circ₀000745 promotes the migration and invasion of cervical cancer cells by inhibiting the expression of E-cadherin,thereby leading to cancer progression.