| Background:Breast cancer(BC)is the major cause of cancer death in women worldwide.The pathogenesis of breast cancer is still unclear,and its incidence is rising every year.Breast cancer cell stemness and its biological behavior seriously affect the recurrence rate and survival rate of patients,threatening the quality of survival of breast cancer patients.It is important to investigate the molecular mechanism of breast cancer development and potential therapeutic targets to improve the survival rate of breast cancer patients.Circ RNAs(Circular RNAs)are a type of endogenous non-coding RNAs that exhibit high stability and high specificity in various diseases,are crucial in the formation and progression of human cancer,and play a significant role in tumorigenesis and metastasis.Little is known about the molecular mechanisms by which circ RNAs regulate breast cancer progression.circ RNAs are considered to be ideal candidate biomarkers with potential diagnostic and therapeutic implications.Recent studies have shown that dysregulation of circ＿0006168 is associated with several types of cancers.However,circ＿0006168 has not yet been studied in breast cancer,and the molecular mechanism of its involvement in breast cancer is unclear.Therefore,it is significant to investigate the role of circ＿0006168 in breast cancer and its potential molecular mechanisms of action.mi RNA(micro RNA)is a class of non-coding RNA molecules of 21-22 nt in length,which plays a crucial role in a variety of malignancies by regulating the activity of target genes through post-transcriptional gene silencing.It has been demonstrated that significantly down-regulated in breast cancer tissues and up-regulated in plasma samples,with the potential to detect early breast cancer.mi R-99a-5p can also inhibit breast cancer progression and cell cycle pathways by down-regulating CDC25 A.Therefore,it is necessary to explore the applicability of mi R-99a-5p and its targets as breast cancer therapeutic targets in breast cancer.Frizzled 8(FZD8)is a member of the Frizzled(FZD)receptor family that activates the typical or atypical Wnt/β-catenin pathway.f ZD8 is highly expressed and exerts procancer effects in a variety of cancers,including renal cell carcinoma,thyroid cancer,and gastric cancer,promoting cancer cell proliferation,migration,and invasion.By reviewing the literature and using the KEGG Pathway database,we found that FZD8 can regulate the Wnt pathway to achieve the regulation of cancer progression.According to the study,circ RNA can play a sponge role in competitively binding mi RNAs and deregulating mi RNAs on downstream genes.In this study,we propose that circ＿0006168 participates in breast cancer progression by adsorbing mi R-99a-5p to regulate FZD8/Wnt and thus participate in breast cancer progression,thus regulating breast cancer cell proliferation,migration,invasion,and stemness,and participating in breast cancer progression,to provide novel targets for breast cancer diagnosis and treatment.Objective:1.To explore the expression characteristics and axis relationship of circ＿0006168,mi R-99a-5p,and FZD8 in breast cancer.2.To explore the effect of circ＿0006168 on the stemness and biological behavior(proliferation,migration,invasion)of breast cancer cells.3.To explore the molecular mechanism of circ＿0006168/mi R-99a-5p/FZD8 regulating the stemness and biological behavior of breast cancer cells through the Wnt pathway.Methods:1.The GEO database was used to download the human breast cancer circ RNA microarray data(GSE165884),and GEO2 R was used to identify the circ RNA molecules that were differentially expressed.The q RT-PCR method was then used to identify the expression of these molecules in normal breast cells(MCF-10 A and MCF-7),as well as in the MDA-MB-231 breast cancer cell lines.2.The candidate target mi RNA of circ＿0006168 was predicted using Circinteractome,Inta RNA,with binding sites between mi R-99a-5p,mi R-99a-5p was the candidate target mi RNA of circ＿0006168.using MIRDB,and Targetscan.mir DIP,DIANA,and mi Rmap database to predict the target genes of mi R-99a-5p,and take the intersection,there are 16 target genes,from which we selected FZD8 for the follow-up study.Using the RTq PCR and Western blot techniques,its expression in the normal breast cell lines MCF-10 A and MCF-7 as well as the breast cancer cell line MDA-MB-231 was discovered.3.The circ＿0006168 overexpression vector(circ＿0006168),FZD8 overexpression vector(FZD8)and empty vector(pc DNA),si RNA of circ＿0006168(si-circ＿0006168)and its control(si-NC),mi R-99a-5p mimic(mi R-99a-5p),mi R-99a-5p inhibitor and mi RNA unrelated sequences(mi R-NC)were transfected into breast cancer cells MCF-7,MDA-MB-231,and transfection efficiency was verified using the q RT-PCR technique.The expression changes of mi R-99a-5p were detected using the q RT-PCR technique,and the expression changes of FZD8 at the protein level were detected by the Western blot technique.4.The ability of each group of breast cancer cells to proliferate was assessed using the CCK-8 assay and the cell colony formation assay;the ability of each group to invade was assessed using the Transwell assay;the ability of each group to migrate was assessed using the scratch assay;and the expression of molecules involved in the Wnt signaling pathway,including ALDH1,OCT4,CD133,and related molecules,was assessed using the Western blot method.Western blotting was used to assess the levels of expression of molecules connected to the Wnt signaling pathway,including ALDH1,OCT4,CD133,and CD133.Results:1.The GEO database(GSE165884)was used to download circ RNA microarray data for human breast cancer,and GEO2 R was used to examine the circ RNA molecules that are highly expressed in breast cancer tissues.hsa_circ＿0006168(circ＿0006168)was selected for the study.q RT-PCR results showed that compared with normal breast epithelial cells MCF-10 A,circ＿0006168 expression was significantly higher in breast cancer cell lines MDA-MB-231 and MCF-7 compared to normal breast epithelial cells.2.circ＿0006168 transfection considerably improved MDA-MB-231 and MCF-7 cells’ ability to proliferate,migrate,invade,and retain their stemness as compared to the pc DNA group;the proliferation ability,migration,invasion ability,and stemness of breast cancer cells transfected with si-circ＿0006168 were significantly diminished compared to the si-NC group.3.The experimental results in MDA-MB-231 and MCF-7 cells transfected with circ 0006168 showed that mi R-99a-5p expression level was significantly down-regulated,and the expression of both m RNA level and protein level of FZD8 was increased;After transfection with si-circ 0006168,mi R-99a-5p expression was markedly up-regulated,while FZD8 expression of both m RNA and protein and mi R-99a-5p were both dramatically down-regulated.4.In breast cancer cell lines MDA-MB-231 and MCF-7 cells set up transfection group,the proliferation,migration,invasion ability,and stemness of breast cancer cells were significantly enhanced in si-circ＿0006168+mi R-5p inhibitor group compared to sicirc＿0006168+mi R-NC.5.In MDA-MB-231 and MCF-7 cells,transfection of mi R-99a-5p revealed a decrease in FZD8 expression at the m RNA and protein levels,while transfection of mi R-99a-5p inhibitor resulted in an increase in FZD8 expression at the m RNA and protein levels.6.In breast cancer cell lines MDA-MB-231 and MCF-7 cells set up transfection grouping,the proliferation,migration,invasion ability,and stemness of breast cancer cells were significantly enhanced in the mi R-99a-5p+FZD8 group compared to mi R-99a-5p mimic+pc DNA group.7.In MDA-MB-231 and MCF-7 cells,the protein expression of β-catenin,c-myc,and cyclin D1,the related signaling molecules in the Wnt signaling pathway,were decreased in si-circ＿0006168 group compared to si-NC group;compared to si-circ＿0006168+ mi R-NC group,si-circ＿0006168+ mi R-99a-5p inhibitor group showed elevated protein expression;compared with si-circ＿0006168+ pc DNA group,si-circ＿0006168+FZD8 group showed elevated protein expression.Conclusion:1.circ＿0006168 and FZD8 were highly expressed in breast cancer cells,and mi R-99a-5p was expressed at a lower level in breast cancer cells.2.circ＿0006168 can promotes proliferation,migration,invasive ability,and cell stemness of breast cancer cells.3.circ＿0006168 can promote cell proliferation,migration,invasion,and stemness of breast cancer cells by regulating mi R-9the 9a-5p/FZD8 axis via the Wnt pathway,which can be a potential new target for breast cancer treatment. |
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