CircRNA8924 Regulates Biological Behavior Of Cervical Cancer Cells By Competitively Binding MiR-518d/519-5p Family With CBX8

Objective: Cervical cancer?CC?is one of the most common gynecological cancers worldwide,and it has been proved that persistent infection with high-risk type human papillomavirus?HR-HPV?is an important pathogenic factor in its histogenesis and progression.Nevertheless,higher HPV infection rate is always accompanied by relatively lower incidence of CC,which not only makes CC screening and primary prevention a heavy economic burden of hygienic economics but also hints that there exsit individual genetic differences and other important factors during the progression of CC.Therefore,it is essential to explore the exact pathogenesis of CC and find brand new diagnostic markers as the potential therapeutic targets.Different from mi RNA and lnc RNA with traditional linear structure,circular RNAs?circ RNAs?are a covalently closed continuous loop.Due to its absence of 3',5' end and poly A tail,circ RNAs can avoid the degradative effect of exonuclease and RNase R.Compared with linear RNA,circ RNAs are more conservative and stable and show cell-specific,tissue-specific and stage-specific.Circ RNAs play a significant role in the development and progression of various human cancers.It has been demonstrated that circ RNAs act as competitive endogenous RNAs?ce RNAs?,namely micro RNA sponges,regulating target gene expression.They could also serve as transcriptional regulator or protein-binding RNA,or even be directly translated into proteins under certain circumstances.However,the expression and function of circ RNAs in cervical cancer?CC?have rarely been explored.In this study,human circ RNA Microarray V2.0 was used to screen CC tissues and pair-matched adjacent normal tissues to find the differential expressed circ RNAs and their targets,by the aim to investigate potential markers for the progression of CC and elucidate the possible mechanism involved for new insights of molecular therapy.Methods: Human circ RNA Microarray V2.0 was used to screen CC tissues and three pairmatched adjacent normal tissues to find the differential expressed circ RNAs.By combination of circ RNAs type,fold changes,and expression abundance,we analyzed and screened candidate circ RNA8924.q RT-PCR was applied to velidated the expression of circ RNA8924,and its clinicopathological significance was analyzed.Lentivirus transinfection and RNA interference?si RNA?were used to construct stable cell model with circRNA8924 over-expression or downregulated.CCK 8 was used to observe cell viability,would healing assays were used to test cell migration,and transwell invasion experiments were used to detect cell invasion.Cell cycle was detected by flow cytometry.All methods mentioned above were used to elucidate role of circ RNA8924 on malignant biological behavior of CC cells.q RT-PCR was used to determine m RNA expression of mi R-518d/519-5p and the relationship with circ RNA8924 was analyzed.The dual-luciferase reporter assay was used to determine the targeted binding of circ RNA8924 and mi R-518d/519-5p family.With transient transfection to change the expression of mi RNA,the competitive binding effect of circ RNA8924 on mi R-518d/519-5p was identified by cell functional experiments.Finally,through q RT-PCR,dual-luciferase reporter assay and Western Blot experiment,the targeted effect of mi R-518d/519-5p family on its target gene CBX8 was analyzed,in order to identify the specific mechanism of circ RNA8924 in CC cells.Results: 1.The result of microarray indicated that 591 circ RNAs were differentially expressed between CC tissues and pair-matched adjacent normal tissues?FC?2,P<0.05?,including 447 exonic circ RNAs,72 intronic circ RNAs,46 sense overlapping circ RNAs,18 antisense circ RNAs,and 8 intergenic circ RNAs.Among differentially expressed circ RNAs,237 circ RNAs were upregulated and 354 circ RNAs were downregulated.Identification of 8 candidate circ RNAs by q RT-PCR showed that hsa_circ₀000514,circ RNA8924 were upregulated,hsa_circ₀000301,hsa_circ₀000407,circ RNA11641 were downregulated,consistent with microarray results?P<0.05?;hsa_circ₀078738 was downregulated,which was inconsistent with the results?P<0.05?;while there was no significant difference in expression of hsa_circ₀001831 and hsa_circ₀004225.Among them,circ RNA8924 had the highest fold change.The level of circ RNA8924 expression was significantly correlated with tumor size?P=0.008?,FIGO?International Federation of Gynecology and Obstetrics?stage?P=0.041?and myometrial invasion?P=0.022?,but it had no correlation with patient's age or tumor differentiation or lymph node metastasis.2.The expression of circ RNA8924 could be effectively increased by circ RNA8924 over expression and inhibited by sh-circ RNA8924 knockdown compared with NC?P<0.05?.The CCK-8 assay showed that the proliferation rate of cells could be effectively increased by circ RNA8924 OE and inhibited by sh-circ RNA8924 compared with NC?P<0.001?.Flow cytometry results indicated that compared with the NC group,the proportion of cells in G0/G1 phase of circ RNA8924 OE cells was decreased,and cells in S phase was increased.While,the proportion of cells in G0/G1 phase of sh-circ RNA8924 cells was increased,and cells in S phase was decreased.At the same time,would healing assay and transwell assay indicated that the migration rate and invasion rate of the Si Ha and He La cells of the sh-circ RNA8924 group also decreased significantly?P < 0.01?.However,migration and invasion of the circ RNA8924 OE group were significantly increased.3.The q RT-PCR showed expression of mi R-518d/519-5p was decreased in CC tissues compared with matched adjacent normal tissue samples?P<0.01?,and had a significant negative correlation with circ RNA8924 level?R=-0.542,P=0.0017?.Furthermore,the luciferase activity of circ RNA8924-WT and mi R-519a-5p mimics co-transfection group was significantly decreased compared to NC group?P<0.01?;Nevertheless,the luciferase activity of circ RNA8924-Mut and mi R-519a-5p mimics group was not affected.Additionally,mi R-519a-5p was markedly decreased in Si Ha and He La cells?P<0.05?after transfection of circ RNA8924 OE,and oppositely the mi RNA expression was raised by sh-circ RNA8924 transfection?P < 0.01?.The CCK-8 assay showed that the proliferation rate of Si Ha and He La cells could be effectively inhibited by mi R-519a-5p over expression.The proliferation of circ RNA8924 OE and mi R-519a-5p mimics cotransfection group was not affected.Flow cytometry results indicated that compared with the NC group,the proportion of cells in G0/G1 phase of cells with mi R-519a-5p mimics transfection was increased,and cells in S phase was decreased.circ RNA8924 OE and mi R-519a-5p mimics co-transfection group was not affected.Would healing assay and transwell assay indicated that the migration rate and invasion rate of the CC cells of mi R-519a-5p mimics transtection group was decreased significantly?P<0.01?.However,it was not affected in co-transfection with circ RNA8924 OE and mi R-519a-5p mimics.The level of CBX8 expression was significantly increased in CC tissues than pair-matched adjacent normal tissues?P<0.01?,and was negatively correlated with mi R-519a-5p expression?R=-0.447,P=0.011?,was positively correlated with circ RNA8924 expression?R=0.601, P=0.0007?.Furthermore,the activity of luciferase reporters in CBX8-WT and mi R-519a-5p mimics cotransfection group was significantly decreased?P<0.01?;nevertheless,the luciferase activity of circ RNA8924-Mut group was not affected.m RNA and protein expression of CBX8 were both decreased after transfected with sh-circ RNA8924,the decline trend was partially reversed after the involvement of mir-519a-5p inhibitor,and the difference was statistically significant.Conclusion: 1.591 circ RNAs were differentially expressed between CC tissues and pairmatched adjacent normal tissues.circ RNA8924 was demonstrated to be highly expressed in cervical cancer tissues and was closely associated with tumor size,FIGO stage and myometrial invasion.The expression of circ RNA8924 is closely related to the progression of cervical cancer and can serve as a potential biomarker for cervical cancer.2.circ RNA8924 can significantly promote the proliferation,migration and invasion ability of cervical cancer cells,and interfere with the cell cycle of cervical cancer cells,resulting in a significant increase in the proportion of mitotic cells.circ RNA8924 can participate in the progression of disease by enhancing the malignant biological behavior of cervical cancer cells.3.mi R-518 d /519-5p family is low expressed in cervical cancer tissues and plays an anticancer role.circ RNA8924 can act as mi RNA sponge to exert ce RNA effect and be able to competitively combined with the mi R-518d/519-5p family,upregulated the expression level of CBX8,leading to the enhancement of the malignant biological behavior of CC cells.The circ RNA8924-mi R-518d-5p/519-5p-CBX8 axis provided a brand new insight into molecular treatment of CC,and revealed that the circ RNA8924 might serve as a new biomarker and potential therapeutic target for CC.