Research On The Role Of RNA CircWWC3 Mediated By Transcription Factor ZEB1 In The Progression Of Breast Cancer And Its Immune-related Mechanism

Part 1 Screening and validation of circWWC3 and its regulation by transcription factor ZEB1Objective:To screen circ RNAs regulated by the transcription factor ZEB1 in breast cancer cells and study their biological functions in breast cancer.Methods:1. Transfected ZEB1 in MDA-MB-231 and used Arraystar circ RNA chip technology to screen differentially expressed circ RNA.2.RT-PCR was used to detect the expression of two circular RNAs in breast cancer tissues and cells.RNase R and Actinomycin D experiments verified their characteristics in breast cancer cells.3.qRT-PCR was used to detect the expression of hsa_circ₀001910 and hsa_circ₀089866 in breast cancer cells and tissues.4.Sanger sequencing identified the circularized splice junction sequence of circWWC3,and FISH experiments verified the main presence of circ-WWC3 in the cells.5.Ch IP-PCR and dual luciferase reporter gene experiments were used to verify the direct binding ability of ZEB1 to WWC3 promoter.6.The effects of knockdown of circWWC3 on the proliferation,migra-tion and invasion of breast cancer cells were tested using CCK-8 experi-ments,plate clone formation experiments,scratch healing experiments,and Transwell experiments.Results:1.Two circular RNAs?hsa_circ₀089866 and hsa_circ₀001910?which are derived from a same gene WWC3 were up-regulated after ZEB1 trans-fection.2. RT-PCR results showed that using breast cancer tissues and cells cDNA and g DNA as templates,circular transcripts could only be amplified in c DNA by divergent primers,while linear transcripts could be amplified in both c DNA and g DNA by polymerizing primers.The results of RNase R and actinomycin D experiments showed that hsa_circ₀001910 and hsa_circ₀009866 were more resistant to the digestion of RNase R than their corresponding linear transcripts.After treated with Actinomycin D,circular RNA were more stable and the half-life time were longer.3. qRT-PCR showed that hsa_circ₀001910 content was significantly higher in breast cancer cells and tissues than hsa_circ₀009866.Therefore,hsa_circ₀001910 was selected for subsequent research,which was called circWWC3.4.Sanger sequencing results of the PCR products verified the circularized splice junction sequence of circWWC3.FISH experiments confirmed that circWWC3 mainly existed in the cytoplasm.5.Ch IP-PCR and dual luciferase reporter experiments confirmed that ZEB1 and WWC3 promoter has the directly binding site.6.CCK-8 experiments and plate clone formation experiments showed that knockdown of circWWC3 could inhibit the proliferation of breast cancer cells?P<0.05?.The results of scratch healing experiment and Transwell experiment showed that knockdown of circWWC3 could inhibit the migration and invasion ability of breast cancer cells?P<0.05?.Summary:After MDA-MB-231 cells were transfected with ZEB1,breast cancer cells were screened and detected by circ RNA chip,and circWWC3 was selected as the research target.Circ WWC3 could be combined with ZEB1.The high expression of Circ WWC3 were positively correlated with the clinical stage.Survival analysis showed that the increased expression of circWWC3indicated a poor overall survival of breast cancer patients.Circ WWC3 could promote breast cancer cells proliferation,migration and invasion.Part 2 Study on the related mechanism of circWWC3 regulating on PD-L1 in breast cancerObjective:To investigate the possible mechanism by which circWWC3regulates its downstream immune-related pathways by acting as a molecular sponge of miRNA.Method:1.Miranda and Targetscan online databases were used to predict miRNA molecules and their binding sites that could interact with circWWC3.2.qRT-PCR and FISH experiments verified the presence of circWWC3,miR-26b-3p and miR-660-3p in breast cancer cells.3. RIP experiments and dual luciferase reporter experiments were used to verify whether circWWC3 could interact with miR-26b-3p and miR-660-3p.4. The effects of overexpression or knockdown of miR-26b-3p and miR-660-3p on the proliferation,migration,and invasion of breast cancer cells were tested using CCK-8 experiments,plate clone formation experiments,scratch healing experiments,and Transwell experiments.5. The effect of knockdown of circWWC3 and miR-26b-3p and miR-660-3p on the proliferation,migration and invasion of breast cancer cells was tested by rescue experiments.6. qRT-PCR was used to find downstream immune related genes.7.The Targetscan database was used to predict STAT3 as a downstream target gene,and the experiment was verified with a dual luciferase reporter gene.8.qRT-PCR and Western blot were used to detect the expression of STAT3,p-STAT3,PD-L1 in breast cancer cells after knocking down circWWC3 and overexpressing miR-26b-3p and miR-660-3p.Results:1.By analyzing the Miranda and Targetscan databases,the top two miRNAs were miR-26b-3p and miR-660-3p based on the positions of the estimated binding sites in the circWWC3 sequence.2.qRT-PCR and FISH results showed that circWWC3 and miR-26b-3p or miR-660-3p were mainly located in the cytoplasm.3.The results of the RIP experiment and the dual luciferase reporter gene experiment showed that circWWC3 could interact with miR-26b-3p and miR-660-3p in breast cancer cells?P<0.05?.4.The results of CCK-8 experiments and plate clone formation experiments showed that overexpression of miR-26b-3p and miR-660-3p could inhibit the proliferation of MDA-MB-453 cells?P<0.05?;While knocking down miR-26b-3p and miR-660-3p could enhance the proliferation of MDA-MB-231 and BT-549 cells?P<0.05?.The results of scratch healing experiments and Transwell experiments showed that overexpression of miR-26b-3p and miR-660-3p could inhibit the migration and invasion ability of MDA-MB-453 cells?P<0.05?;While knocking down miR-26b-3p and miR-660-3p could enhance the migration and invasion ability of MDA-MB-231 and BT-549 cells?P<0.05?.5.The rescue experiment results showed that circWWC3 knockdown could inhibit proliferation,migration,and invasion,while miR-26b-3p and miR-660-3p knockdown could rescue circWWC3 knockdown-mediated inhibition of cell proliferation,migration,and invasion.6. qRT-PCR results showed that PD-L1 decreased after knocking down circWWC3 and overexpressing miR-26b-3p and miR-660-3p?P<0.05?.7. Targetscan database showed that miR-26b-3p and miR-660-3p had direct binding sites with STAT3,and was verified by dual luciferase reporter experiments.8. qRT-PCR didn't show changes of STAT3 m RNA after miR-26b-3p and miR-660-3p were overexpressed.Western blot experiments showed that the expressions of STAT3,p-STAT3,and PD-L1 were reduced after circ-WWC3 was knocked down and miR-26b-3p and miR-660-3p were overexpressed.Results:Circ WWC3 has directly binding sites with miR-26b-3p and miR-660-3p,which can inhibit the proliferation,migration and invasion of breast cancer.STAT3 is the downstream target of miR-26b-3p and miR-660-3p.Circ WWC3can sponge the miR-26b-3p and miR-660-3p and activate STAT3 to act on PD-L1 to play an immune escape role.Part 3 ZEB1 promotes breast cancer growth and metastasis by regulating circWWC3 in vivo Objective:To verify the effect of ZEB1 on breast cancer by regulating circWWC3in vivoMethods:Female nude mice were randomly divided into four groups with 5mice/group and 1x10⁷ MDA-MB-231-luc cells infected with?1?Empty vector+sh-scramble,?2?ZEB1+sh-scramble,?3?Empty vector+sh-circWWC3,or?4?ZEB1+sh-circWWC3 were injected into mice subcutaneously or via tail vein.Tumors were evaluated weekly using in vivo imaging system?IVIS?.Result:1.In nude mouse xenograft models,ZEB1 overexpression could accelerate breast cancer proliferation,and sh RNA-mediated knockdown of circWWC3 could partially reverse ZEB1-induced breast cancer growth.2.For the metastasis models,lung and liver metastases increased significantly after ZEB1 overexpression,while the number of metastases in the ZEB1 and sh-circWWC3 co-transfection group decreased,indicating that ZEB1 overexpression increased breast cancer metastasis,and sh RNA-mediated knockdown of circWWC3 leads to ZEB1-induced breast cancer metastasis.3.The survival analysis revealed that high expression of ZEB1 plus circWWC3 indicated a poor prognosis survival of breast cancer patients.Summary:ZEB1 overexpression promoted the growth and metastasis of breast cancer tumors,while sh RNA mediated knockdown of circWWC3 partially reversed the growth and metastasis of breast cancer tumors induced by ZEB1.Conclusion:The main conclusions of our study are as follows:?1?we identified a novel circular RNA,circWWC3 that is induced by ZEB1 and promotes the growth and metastasis of breast cancer.?2?we found that circWWC3 can act as a sponge for miR-26b-3p and miR-660-3p and inhibit their functions in breast cancer.?3?miR-26b-3p and miR-660-3p play an inhibitory role in breast cancer.?4?we demonstrated that circWWC3 activates STAT3,the direct target of miR-26b-3p and miR-660-3p,through spongy miR-26b-3p and miR-660-3p,and the activation of STAT3 can affect the immune escape function of PD-L1.