The Effect Of CircTPCN On The Biological Behavior Of Cervical Cancer Hela Cell And Its Mechanism Analysis

Objective:Cervical cancer?CC?is one of the most common gynaecological tumors worldwide,and HR-HPV infection has been proved to be an important pathogenic factor of the development of cervical cancer.In recent years,with the extensive application of HPV vaccine,primary prevention of CC has achieved certain results,and its morbidity and mortality have been effectively controlled in many developed countries.However,higher HPV infection rate is often accompanied by lower CC incidence,which not only makes CC screening and primary prevention a heavy economic burden for health economics,but also indicates that important factors such as individual genetic differences are important factors for the progress of CC.Therefore,it is necessary to explore the exact pathogenesis of CC and find new diagnostic markers as potential therapeutic targets.Less than 2%of the human genome's nucleotide sequence encodes proteins,and most genes are translated into non-coding RNA.In recent years,with the emergence of microRNAs and long non-coding RNAs,circRNAs have become a hot spot of disease research.Unlike conventional linear miRNAs and lncRNAs,circRNAs are continuous rings with covalent bond closure.CircRNAs can avoid the degradation of exonuclease and RNase r due to the lack of 3'5'end and poly A tail.Compared with linear RNA,circRNAs are conserved and stable with cell specificity,tissue specificity and phase specificity.At present,the research on the biological function of circRNAs is still in the exploratory stage.CircRNAs,as competitive endogenous RNAs?ceRNAs?,namely microRNA sponges,have been demonstrated to regulate the expression of target genes.They can also act as transcriptional regulators or protein-binding RNAs and can even be translated directly into proteins under certain conditions.Many potential circRNAs molecular markers and their pathways have been found as miRNA sponges in many tumors,including esophageal squamous cell carcinoma,gastric cancer,pancreatic ductal adenocarcinoma,liver cancer,colorectal cancer,early lung adenocarcinoma,glioma and ovarian cancer.However,the differentially expressed circRNAs and their roles in CC tumorigenesis remain largely unknown.In this current study,we investigated the expression profiles and potential oncogenic mechanisms of circRNAs in CC.In the previous study,circTPCN was found to be highly expressed in cervical cancer tissues.However,the expression and mechanism of circTPCN in cervical cancer is still unclear.Therefore,this study focused on the impact of circTPCN on the biological behavior and mechanism in cervical cancer cell line Hela.Methods:Normal cervical samples and cervical cancer samples were obtained for microarray assay to determine differentially expressed circle RNA between cervical cancer and normal control.Using bioinformatics retrieval method,circTPCN interference sequence si_circTPCN?1?,si_circTPCN?2?,irrelevant sequence si_nc and irrelevant sequence si_nc_FAM with FAM fluorescence were synthesized.The transfection reagents were used to transfect si_circTPCN?1?,si_circTPCN?2?,irrelevant sequence si_nc and irrelevant sequence si_nc_FAM with FAM fluorescence into Hela cells.After transfection,the transfection efficiency of the interference sequence was observed by fluorescence microscopy.After 48 hours of transfection,total RNA was extracted by TRIzol method,and the concentration and purity of RNA were verified,and then transcribed into cDNA.The conditions of RT-qPCR were annealing at 25 for 5 min,elongation at 42 for 60 min,70 for15 min,and storage at 4.The specific primers of circTPCN and SYBR_Green_I fluorescent dye were used for quantitative PCR detection.The specific primers of miR-634?miR-195-5p?miR-455-3p and SYBR_Green_I fluorescent dye were used for quantitative PCR detection.The internal reference was U6.The reaction conditions of fluorescence quantitative PCR were 95?5 min;95?15 s;60?60 s,with 45 cycles.CircRNA was quantitatively determined by 2^-??Ct method.2^-??Ct=2^{-??Ct si_RNA-?Ct si_nc?},?Ct si_RNA=the mean of Ct value of si_RNA group-the mean of Ct value of internal parameter18S or U6,the mean of Ct value of Ct si_nc=si_nc group-the mean of Ct value of internal parameter 18S or U6,and the relative expression of si_nc group was set at 1.24 hours after transfection,cells were collected and counted by cell counting board to detect the proliferation of Hela cells.24 hours after transfection,MTS reagents were added to the transfection system.After incubation,the proliferation of Hela cells was detected by enzyme labeling instrument.48 hours after transfection,cells were collected and Annexin V/FITC was used to detect the proliferation of Hela cells.After incubation,flow cytometry was used to detect the apoptosis of Hela cells after transfection of interfering sequence into Hela cells.After 24 hours of transfection,the scratch test was carried out.After transfection,Hela cells were scratched and supernatant was removed,washed three times with PBS,cultured in serum-free medium at different time points,and observed the scratch changes.24 hours after transfection,cells were collected and inoculated into Transwell chamber,serum-free culture system in the upper chamber and high serum culture system in the lower chamber.After 24 hours of culture,the perforating cells in each group were counted under the microscope.The invasive ability of cells was measured.Results:Transfection efficiency of si_nc_FAM is 27.5pmol:87.5%,55pmol:91.3%;110pmol:93.5%.The amount of the instantaneous transfection interference sequence was determined as 55pmol/well in the 12-well plate.The results of MTS test and cell count showed that the growth ability of Hela cells was significantly reduced after si_circTPCN?1?was inhibited,and the difference was statistically significant.After the Hela cells were transfected with the interfering sequence si_circTPCN?1?,the circTPCN expression was25±1.5%compared with the si_nc group,and the difference was statistically significant.The apoptosis rate of si_circTPCN?1?transfected group was 44.35±2.3%,the apoptosis rate of si_nc control group was 16.35±0.92%,and the apoptosis rate of blank control group was 3.2±0.56%,the difference was statistically significant.Compared with the si_nc transfection group,the scratch spacing of the si_circTPCN?1?group was not significantly reduced.The transfection group Hela cells transfected with si_circTPCN?1?had a transfection number of 48.67±5.51/field,which was significantly lower than that of the si_nc group?90±10/field?and the blank control group?94.33±6.11/field?.Arraystar software was used to predict the potential interaction of circRNA-miRNA and to predict that mir-634/mir-455-3p/mir-195-5p might be the target of circTPCN.The inhibitory effect of si_circTPCN?1?on circTPCN significantly up-regulated mir-634,and the relative expression was up to 45 times that of the control group.Although the relative expression levels of mir-455-3p and mir-195-5p in the si_circTPCN?1?group were significantly up-regulated,the relative expression levels of these two miRNAs were only 2.8 times and 5.5times that of the control group,respectively.The difference was statistically significant.Detection of the effect of circTPCN on mTOR expression in Hela cells showed that circTPCN inhibited mTOR expression significantly.Conclusion:There are a large number of abnormally expressed circRNAs in cervical cancer tissues,and the chemically synthesized circTPCN interference sequence can significantly reduce the circTPCN expression in Hela cells after transfection.The down-regulation of CircTPCN expression can significantly inhibit the proliferation,migration and invasion ability of cervical cancer cell line Hela cells,and promote apoptosis.CircTPCN is a molecular sponge of miR-634,which plays a role in binding and silencing miR-634.The inhibitory effect of si_circTPCN?1?on circTPCN significantly up-regulated the expression of miR-634,and the expression of mTOR was significantly decreased,suggesting that circTPCN significantly reduced the expression of mTOR after inhibiting the expression of miR-634.The results suggest that circTPCN expression is related to the biological behavior of cervical cancer cells and may be an effective therapeutic target for cervical cancer.