The Role And Mechanism Of Hsa＿circ＿0007766 In The Proliferation、Invasion And Migration Of Human Breast Cancer Cells

In 2020,the global incidence of breast cancer in women has surpassed lung cancer as the most common cancer.However,lung cancer is still the main cause of cancer deaths,followed by colorectal cancer,liver cancer,gastric cancer and female breast cancer.Nonetheless,breast cancer worldwide The morbidity and mortality rate are still at a high level every year.Therefore,finding the most effective methods for the diagnosis and treatment of breast cancer patients,studying the molecular mechanisms of breast cancer,understanding the genetic and epigenetic changes of breast cancer,and discovering new diagnostic and treatment strategies is still a top priority.As a newly discovered non-coding RNA(ncRNA),circular RNA(circRNA)has quickly become a hot spot in the field of tumor research through specific biogenesis,high stability and unique functions.This study took the circular RNA hsa＿circ＿0007766 as the research object to explore its role and mechanism in the occurrence and development of breast cancer,with a view to providing new ideas for the diagnosis and treatment of breast cancer.Objective To explore the role and potential molecular mechanism of the circular RNA hsa＿circ＿0007766 in the proliferation,invasion and migration of human breast cancer cells.Method 1.Determine circRNA,downstream miRNA and related target genes through bioinformatics analysis software and GEO database screening,and double luciferase report detection to verify the targeting relationship;2.qRT-PCR detects the three expression in normal human breast epithelial cells MCF10A and breast cancer cell lines(respectively MDA-MB-231,MDA-MB-453,BT-549,HCC38 and MCF7),at the same time,through the plate clone formation test,MTT test,scratch healing test,Transwell test to detect its the effect on cell proliferation,migration and invasion,Western-blot experiment to detect the expression levels of proliferation-related proteins PCNA,E-cadherin and vimentin;3.Methylation-specific polymerase chain reaction(MSP)and qRT-PCR were used to detect the methylation status of miRNA promoters in breast cancer cell lines and the activation in breast cancer cells after treatment with 5 μM and 10 μM decitabine(DAC)The methylation status of the daughter and the expression level of miRNA;4.Cell function salvage experiments to further verify the regulatory relationship between circRNA,downstream miRNA and related target genes.Results 1.Hsa＿circ＿0007766 was screened out by bioinformatics analysis of relevant literature and GEO database.It is highly expressed in breast cancer cell lines by qRT-PCR.After silence,it can inhibit the proliferation,migration and invasion of breast cancer cells MDA-MB-231 and MCF7.Western blot results showed that silencing hsa＿circ＿0007766 inhibited the protein expression of PCNA and Vimentin and promoted the protein expression of E-cadherin(P<0.05);2.Using bioinformatics software analysis to determine that miR-1301-3p has a binding site with hsa＿circ＿0007766 as a follow-up Research subjects,qRT-PCR detection found that the expression of miR-1301-3p is down-regulated in breast cancer cells,and its overexpression can inhibit the proliferation,migration and invasion of breast cancer cells,as well as the expression of PCNA and Vimentin,while promoting the expression of E-cadherin(P<0.05);3.MSP results showed that compared with normal breast epithelial cells MCF10A,the promoter region of miR-1301-3p gene in breast cancer cells MDA-MB-231 and MCF7 showed a high methylation level;48h after DAC treatment,The methylation level of the promoter region of miR-1301-3p gene was significantly reduced,while its expression level was significantly higher than that of the control group(P<0.001).It is speculated that the low expression of miR-1301-3p may be related to the methylation level.DNA methyltransferase 3B(DNMT3B)was selected for follow-up studies through bioinformatics software analysis and screening and methylation detection results.The dual luciferase reporter method confirmed the difference between miR-1301-3P and DNMT3 B.There is a targeted binding site between.The results of qRT-PCR showed that DNMT3 B is highly expressed in breast cancer cell lines.Silencing DNMT3 B can promote the expression of miR-1301-3p,while inhibiting the proliferation and migration of breast cancer cells(P<0.05);5.Cell function salvage test results It is shown that co-transfection of si-hsa＿circ＿0007766+miR-1301-3p inhibitor and sh-DNMT3B+miR-1301-3p inhibitor can reverse the inhibitory effect of si-hsa＿circ＿0007766 on the proliferation,migration and invasion of breast cancer cells.Western blot results show Co-transfection of si-hsa＿circ＿0007766+miR-1301-3p inhibitor can reverse the inhibition of si-hsa＿circ＿0007766 cell proliferation marker PCNA and mesenchymal cell marker Vimentin expression,and at the same time,reverse si-hsa＿circ＿0007766 markers of epithelial cells The promotion of E-cadherin expression(P<0.05).Conclusion In summary,hsa＿circ＿0007766 regulates the expression of DNMT3B through sponge of miR-1301-3p,promoting the proliferation,migration and invasion of breast cancer cells.In addition,the expression of miR-1301-3p is regulated by DNA methylation,and there is feedback regulation with DNMT3B.Therefore,hsa＿circ＿0007766 can be silenced and DAC combined therapy can be used to inhibit breast cancer cell proliferation,migration and invasion.