Preliminary Study On The Mechanism Of IKCa1 Related MiRNA On Proliferation,Migration And Invasion Of HeLa Cells

Objective:1.To study the effect of the Intermediate-conductance Ca2+-activated K+ channel(IKCa1)on proliferation,migration and invasion of HeLa cells;2.To investigate the effect of IKCa1 on the miRNA expression profile and screen out miRNA related to IKCa1,bioinformatics was used to predict its target gene and analyse the function of its target gene(GO analysis).The differentially expressed miRNA target genes were selected for its signal enrichment analysis,Preliminary exploration of IKCa1 related miRNA and HeLa cell proliferation,invasion and metastasis,It provides clues for further exploration of miRNAs in the prevention and treatment of cervical cancer target molecules.Methods :1.Concentration were set up to 0,10.0,20.0,30.0,40.0 ummol/L TRAM-34(the characteristics of IKCa1 blockers)as the observation group 1-5 groups and Equal volume DMSO group(TRAM-34solvent)to treat HeLa 24 h,48 h and 72 h,we used CCK8 test to detecte the proliferation of HeLa cells and scratch test to measure HeLa cell actual migration distance to detect HeLa cell migration characteristics;2.The invasion characteristics were detected by Transwell invasion in 48 hours after cultured HeLa cells were cultured in the above 6 groups;3.Through preliminary experiments in this study,in combination with screening and scratches CCK8 experiment,we used 0.0,30.0 ummol/L TRAM-34 role HeLa after 48 hours as control group and experimental group respectively,by high throughput sequencing method detection IKCa1 blocking the miRNA expression spectrum before and after the change;4.The results of high throughput sequencing were verified by q RT-PCR method.5.Using miRanda,miRWal K,Targetscan,DIANAm T,m i RDB and other software to predict the differences in screening,the target genes and GO functional analysis of miRNA could be significantly expressed.The miRNA,which is closely related to cervical cancer,was selected as the target miRNA in miRNA,which was closely related to cervical cancer,and the signal pathway was enriched and analyzed.Results:1.CCK8 experiment indicated that TRAM-34 inhibited the proliferation of HeLa cells,and had time and dose dependence.There was no significant difference between the DMSO group,observation group 1 and observation group 1 in cultured cells 24h(p>0.05),The differences between the other groups and the DMSO group and the observation group 1 were statistically significant(p<0.001).After cultured Hela cells 48 and 72 h,the difference between DMSO and observation group 1 was not statistically significant(p>0.05),suggesting that the MDSO concentration in this experiment had no effect on Hela cells.However,the difference between different concentrations of drug groups and observation group 1 and DMSO was statistically significant(p<0.001).The results showed that TRAM-34 inhibited the migration of HeLa cells,and as the concentration increased and the action time was prolonged,the inhibition of migration was more obvious(p< 0.001).2.With the Transwell invasion experiment,the invasion rate of HeLa cells decreased with the concentration of TRAM-34 in a certain concentration range(F=384.050,p< 0.001).3.High-throughput sequencing analysis of the control group and experimental group of micrornas sequencing results,through comparing the different samples of the Clear data,analysis of the common and unique sequences,with a Fold Change≥1.5,qvalue < 0.01 is significantly different expression of screening criteria,extract the 20 difference in 871 differentially expressed significant expression of miRNAs,of which 15 differences of micrornas expression,five differences between the expression of micrornas.4.Combined with related literatures and high-throughput sequencing results,including 15 expression of micrornas in selected four(hsa-miR-451-a,hsa-miR-122-5 P,hsa-miR-143-3 P,hsa-miR-223-3P)and five expression of micrornas in 1(hsa-miR-421)for q RT-PCR test,the results show hsa-miR-451-a,hsa-miR-122-5P,hsa-miR-143-3,the hsa-miR-421 expression quantity change trend and high-throughput sequencing results are consistent,statistically significant difference were observed in the experimental group and control group(P < 0.05),and thus the accuracy of detection of high-throughput sequencing results;5.Through bioinformatics analysis,The target genes of miRNA with significant difference were mainly concentrated in the cell metabolism,transport process,cell components,protein binding,ion binding and catalytic activity.Hsa-miR-143-5p and hsa-miR-421 are closely related to the occurrence and development of various tumors.Through further signal pathway analysis of hsa-miR-143-5p and hsa-miR-421,the target genesof hsa-miR-143-5P were mainly clustered in PI3K-AKT signaling pathway,cancer-related pathway,P53 signaling pathway and MAPK signaling pathway.Conclusion:IKCa1 may further affect the regulation of its target gene in P53 / MAPK / Wnt signaling pathway by affecting the expression of related miRNAs,thus affecting the proliferation,migration and invasion of HeLa cells.Inhibition of IKCa1 channel can inhibit the proliferation,migration and invasion of HeLa cells.