CircRPL13 Promotes The Proliferation And Migration Of Cervical Cancer Cell Through Sponging MiR-1285-3p/TLN1 Axis

Objective: Cervical cancer(CC)is one of the most important malignant tumors threatening the life of women all over the world.Despite the huge investment in early screening of cervical cancer worldwide,its morbidity and mortality are still high.Effective clinical intervention for early diagnosis of cervical cancer can significantly improve survival,but there is still a lack of accurate prediction and treatment for advanced and recurrent metastatic cervical cancer.Therefore,it is of great clinical value to reveal the key molecular mechanism of cervical cancer,explore specific molecular biomarkers for early diagnosis,and implement precise treatment and prognosis assessment.Circular RNAs(circ RNAs),as a kind of non-coding RNA with closed loop covalent structure,are more stable than linear RNA and are not easy to degrade.Moreover,because of their strong stability,highly conserved tissue and spatio-temporal specificity,their role in tumor diagnosis,prognosis assessment and targeted therapy is attracting more and more attention.The structure contains mi RNA binding sites,which can bind and remove the inhibiting effect of mi RNA on the expression of target genes,forming the regulatory mechanism of ce RNA and affecting the occurrence and development of tumors.At present,a small number of circ RNAs have been reported on their mechanism of action in cervical cancer.However,compared with a large number of circ RNAs with different functions,there are still insufficient researches on their diagnosis,treatment and mechanism of action in cervical cancer.Therefore,in combination with the results of cervical cancer microarray from our research group and GEO public dataset,this study found abnormally high expression of circ RNAs in cervical cancer tissues and carried out in-depth research on the molecular mechanism of its regulation of cervical cancer progression,providing a theoretical basis for finding molecular bimarkers for early diagnosis of cervical cancer and effective therapeutic targets.Methods: Firstly,based on the previous cervical cancer microarray screening results of our research group and the circ RNA expression profile dataset in GEO public database,circ RPL13 was determined as candidate molecule.According to ce RNA theory,online prediction software was used to construct ce RNA network through bioinformatics so as to screen circ RPL13/mi R-1285-3p/TLN1 as the research target.The expressions of circ RPL13,mi R-1285-3 and TLN1 in paired cervical cancer tissues and adjacent normal tissues were verified by RT-q PCR.ROC curve was used to evaluate the diagnostic value of circ RPL13 in cervical cancer.The expression of TLN1 in cervical cancer and adjacent normal tissues was detected by Western blot.Spearman correlation analysis and multivariable linear regression analysis were used to evaluate the expression correlation of circ RPL13,mi R-1285-3p and TLN1.Nonparametric tests were performed to analyze the correlation between circ RPL13,mi R-1285-3p and TLN1 and clinicopathological characteristics.The relationship between circ RPL13,mi R-1285-3p,TLN1 and lymph node metastasis was evaluated by one-way ANOVA.In vitro experiments,the expression of circ RPL13 in different cervical cancer cell lines was detected by RT-q PCR.The subcellular localization of circ RPL13 in cells was determined by RNA FISH assay.The interference fragment of circ RPL13 was constructed,and the expression of circ RPL13 in cells was silenced by transfection technology,and the transfection efficiency was verified by RT-q PCR.The effect of circ RPL13 on cell proliferation was detected by CCK8 and Ed U assays.The effect of circ RPL13 on migration and invasion of cervical cancer cell lines was detected by scratch and Transwell assays.Transfection efficiency was verified by RT-q PCR using mi R-1285-3p mimic and inhibitor transfection cells.The binding relationship between circ RPL13 and mi R-1285-3p was detected by Dual-luciferase reporter gene assay.RT-q PCR was used to detect the expression level of mi R-1285-3p in cervical cancer cells transfected with si-circ RPL13 to clarify the regulatory relationship.After cotransfection of si-circ RPL13 with mi R-1285-3p inhibitor,CCK-8,Ed U,Transwell and other assays were used to detect the effect of circ RPL13 on proliferation,migration and invasion of cervical cancer cells by targeting mi R-1285-3p.The binding relationship between mi R-1285-3p and TLN1 was detected by Dual-luciferase reporter gene assay.The expression level of TLN1 in cervical cancer cells transfected with si-circ RPL13,mi R-1285-3p and si-circ RPL13+mi R-1285-3p inhibitor was detected by RT-q PCR and Western blot to clarify the regulatory relationship.By constructing TLN1-silenced cervical cancer cell lines,using si-TLN1 and cotransfection of si-TLN1 and mi R-1285-3p inhibitor,CCK-8,Ed U,Transwell and other assays were used to detect the effect of mi R-1285-3p on proliferation,migration and invasion of cervical cancer cells by regulating TLN1.Results: 1.Using the cervical cancer tissue microarray screening results of our research group and the cervical cancer tissue microarray dataset of GSE90737 in GEO public database,12 circ RNAs with abnormally high expression were analyzed.circ RPL13 was selected as the research target through data processing,integration and analysis of the sources and characteristics of the differentially expressed circ RNAs parental genes.In order to explore the mechanism of circ RPL13,the online prediction was used to construct the ce RNA network.It was found that the candidate target gene of circ RPL13 was mi R-1285-3p,and the target gene of mi R-1285-3p was TLN1,and the corresponding binding site was obtained.Tissue experiments showed that the expression level of circ RPL13 in cervical cancer tissues was significantly higher than that in the paired adjacent normal tissues(P<0.01),and the AUC for predicting cervical cancer and non-cancer tissues was 0.750(CI:0.635-0.866),with a sensitivity at 72.2% and specificity at 77.8%.The expression of mi R-1285-3p in cervical cancer tissues was significantly lower than that in adjacent normal tissues(P<0.05),and the expression level of TLN1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues(P<0.01).Univariate analysis showed that the expression of circ RPL13 was significantly negatively correlated with mi R-1285-3p in cervical cancer tissues,and the difference was statistically significant(R=-0.56,P<0.01),while the expression of circ RPL13 was significantly positively correlated with TLN1(R=0.46,P<0.01).However,mi R-1285-3p was significantly negatively correlated with TLN1 expression(R=-0.53,P<0.01).Multivariable linear regression analysis showed that TLN1 m RNA relative expression level was significantly correlated with circ RPL13 and mi R-1285-3p(P=0.009,P=0.046).Correlation analysis showed that the relative expression levels of circ RPL13 and TLN1 were positively correlated with lymph node metastasis in patients with cervical cancer,while the relative expression levels of mi R-1285-3p were positively correlated with lymph node metastasis.2.2.In vitro cell experiment,we found that circ RPL13 was highly expressed in cervical cancer cell lines,especially in Si Ha and Ca Ski cells(P<0.01),which was mainly located in the cytoplasm.Transfection of si-circ RPL13 in Si Ha and Ca Ski cells significantly reduced intracellular circ RPL13 expression.The results of CCK-8 and Ed U assay showed that silenced circ RPL13 expression could significantly inhibit the proliferation of Si Ha and Ca Ski cells(P<0.001,P<0.01).The results of scratching and Transwell assay showed that silencing circ RPL13 expression could significantly inhibit the migration and invasion ability of Si Ha and Ca Ski cells(P<0.05,P<0.01).mi R-1285-3p mimic and inhibitor transfection with Si Ha and Ca Ski cell significantly regulated the expression of mi R-1285-3p(P<0.001,P<0.01).Dual-luciferase reporter gene assay showed that overexpression of mi R-1285-3p inhibited the relative luciferase reporter gene activity in circ RPL13-wt group(P<0.01),while there was no significant change in luciferase reporter gene activity in circr PL13-mut group(P>0.05).After the expression of circ RPL13 was knocked out,the expression level of mi R-1285-3p significantly increased(P<0.01).Cotransfection of mi R-1285-3p inhibitor and si-circ RPL13 significantly reversed the inhibition of silenced circ RPL13 expression on proliferation,migration and invasion of cervical cancer cells(P<0.01).Dual-luciferase assay confirmed that mi R-1285-3p could have a targeted binding relationship with TLN1 3’ UTR region,and overexpression of mi R-1285-3p could inhibit luciferase reporter gene activity in TLN1 3’UTR-wt group(P<0.01).The luciferase reporter gene activity in TLN1 3’ UTR-mut was not inhibited(P>0.05).RT-q PCR and Western blot results showed that knockdown of circ RPL13 and overexpression of mi R-1285-3p could decrease the expression of TLN1 in cervical cancer Si Ha and Ca Ski cells(P<0.01).While silencing mi R-1285-3p expression partially reversed the inhibitory effect of silenced circ RPL13 on TLN1expression(P<0.01).Cervical cancer cells with TLN1 expression silenced were constructed,and the results of RT-q PCR showed that TLN1 expression was significantly decreased(P<0.001).CCK-8 and Ed U results showed that silencing TLN1 expression could significantly inhibit the proliferation of Si Ha and Ca Ski cells(P<0.01).The results of scratch and Transwell assays showed that the expression of silenced TLN1 could significantly inhibit the migration and invasion of Si Ha and Ca Ski cells(P<0.01).After cotransfection with mi R-1285-3p inhibitor,the inhibitory effect of silenced TLN1 expression on Si Ha and Ca Ski was significantly reversed.Conclusions: 1.Through the analysis of cervical cancer tissue microarray of our research group and GEO public dataset,circ RPL13 was selected as the research target.Bioinformatics methods were used to screen out the possible regulatory role of circ RPL13/mi R-1285-3p/TLN1 in cervical cancer.Tissue experiments confirmed that circ RPL13 was high expression in cervical cancer and had good predictive ability in differentiating cervical cancer from non-cancer tissues.TLN1 was also highly expressed in cervical cancer,while mi R-1285-3p was low expressed in cervical cancer.circ RPL13 positively regulated TLN1,while mi R-1285-3p negatively regulated TLN1.The expression levels of circ RPL13 and TLN1 were positively correlated with lymph node metastasis,and mi R-1285-3p was significantly negatively correlated with lymph node metastasis.These results suggest that circ RPL13,mi R-1285-3p and TLN1 may be involved in the progression of cervical cancer due to endogenous competition.2.circ RPL13 was highly expressed in cervical cancer cell lines and mainly located in the cytoplasm.circ RPL13 promoted the proliferation,migration and invasion of cervical cancer cells.Decreasing circ RPL13 expression can significantly inhibit the proliferation,migration and invasion of cervical cancer cells.3.circ RPL13 sponging mi R-1285-3p removed the inhibition of mi R-1285-3p on target gene TLN1 in order to up-regulate the expression of TLN1,thus affecting the proliferation,migration and invasion of cervical cancer cells.