Mechanism Of Channel Protein ANO6 Affecting Breast Cancer Prognosis Through Ferroptosis Pathway

Breast cancer(BC)is a highly heterogeneous malignancy of the epithelial tissue of the breast ranked first in new cases of oncology in the world according to cancer statistic 2022.The development of breast cancer is influenced by both genetic and environmental factors,and the clinical management of breast cancer remains challenging due to its diverse pathology and plummeting survival rate after metastasis.Therefore,it is important to discover new mechanisms that influence the development of BC and its prognosis,and subsequently to find new tumor markers for the clinical management of BC.Transmembrane Protein 16F(TMEM16F),also known as Anoctamin(ANO)6,is a calcium-activated chloride channel that is widely expressed in many cells and has cellular regulatory functions.ANO6 has a dual function as a channel protein and a phospholipid flipping enzyme,is involved in ferroptosis death pathway,is detected in a variety of cancer cells and is closely associated with the proliferation,migration and invasion of cancer cells.Firstly the TCGA database was used to analyse the differences in hANO6 expression in BC tumour tissues and paracancerous tissues and to construct the corresponding prognostic models,which were finally validated using the GEO database.That hANO6 was found to be lowly expressed in BC and associated with poor prognosis in BC.Subsequently,clinical tissue samples were collected to verify the differences in hANO6 expression in BC tumor tissues by qRT-PCR and IHC,and the expression of hANO6 in different breast cancer cell lines was examined by qRT-PCR.This will provide new ideas for clinical treatment.METHODS:1.The expression of hANO6 in BC was analyzed from the TCGA database by bioinformatics method,the prognostic model was constructed,and the analysis results and model reliability were verified by using the GEO dataset.2.Fresh tumors and paracancerous tissues of BC patients were collected,RNA was extracted,the expression difference of hANO6 was detected by qRT-PCR,paraffin tissue sections were made,and the difference in hANO6 expression was verified by IHC.At the same time,different breast cancer cell lines MDA-MB-231,MDA-MB-453,MDA-MB-468,MCF10A,MCF-7 RNA were extracted,and the mRNA expression difference of hANO6 was detected by qRT-PCR.3.Stable construction of breast cancer cell lines overexpressing hANO6.The lentiviral plasmid pCDH-FH-hANO6 was co-transfected with the corresponding packaged plasmids(VSVG,MDLG,REV)to generate lentivirus.MDA-MB-231 cell lines were infected with lentivirus,and was screened by puro to construct stable cell lines that overexpressed hANO6.The expression of hANO6 at the gene level and protein level in the above cell lines were analyzed by qRT-PCR and IFA assays.The successfully constructed overexpression cell lines were named 231-FH and 231-hANO6 respectively.4.The effect of hANO6 overexpression on the proliferation ability of human breast cancer cell lines was analyzed by plate cloning and MTT proliferation assays;the effect of hANO6 overexpression on the migration ability of human breast cancer cell lines was analyzed by scratch assays;the effect of hANO6 overexpression on the invasion ability of human breast cancer cell lines was evaluated by transwell assays.5.The expression levels of EMT-related proteins E-cadherin,N-cadherin,snail and Vimentin,as well as iron death detection protein GPX4 and ERK in hANO6 overexpression cell line 231-hANO6 were detected by western blot analysis,to investigate the mechanism by which ANO6 affects breast cancer progression and prognosis through the ferroptosis pathway The study was conducted to investigate the mechanism by which ANO6 affects breast cancer progression and prognosis through the iron death pathway.RESULTS1.By analysing the gene sequencing information of breast cancer patients in the TCGA database,hANO6 was found to be lowly expressed in BC tissues,and a prognostic model was constructed with four other ferroptosis-related genes,ALOX15B,CHAC1,IFNG and TP63,as comprehensive markers.Patients in the high-risk group had a significantly poorer prognosis compared to those in the low-risk group,and hANO6 was relatively highly expressed in the high-risk group.Subsequent analysis of the GEO dataset GSE20685 validated the expression of hANO6 and the accuracy of the prognostic model,and the results were consistent and reliable.2.qRT-PCR and IHC results showed that hANO6 was low expressed in BC tumor tissues.Compared with the control breast cell line MCF-10A,there was no significant difference in the expression of hANO6 mRNA in the breast cancer cell line MCF-7,slightly higher expression in MDA-MB-231 and MDA-MB-468,and significantly increased expression in MDA-MB-453.3.qRT-PCR and IFA experimental results showed that the stable hyperexpression of hANO6 breast cancer cell line 231-hANO6 was successfully constructed.The results of plate cloning and MTT experiments showed that hANO6 had little effect on the proliferation of cell line 231-hANO6 after overexpression,and the results of cell scratch experiment and transwell experiment showed that the migration and invasion ability of 231-hANO6 were significantly enhanced after overexpression of hANO6.It is suggested that hANO6 has the effect of promoting breast cancer progression in breast cancer cell lines.4.Western blot results showed that in the MDA-MB-231 breast cancer cell line of hANO6,the expression of EMT-related proteins N-cadherin and snail was upregulated,the expression of Vimentin and E-cadherin was not significantly changed,the expression of the ferroptosis detection protein GPX4 was up-regulated,and the expression of ERK was up-regulated.CONCLUSIONhANO6 was lowly expressed in breast cancer tumor tissues,where patients in the high-risk score group with relatively high hANO6 expression had a worse prognosis;overexpression of hANO6 significantly increased the invasive and migration ability of BC cell lines with upregulated expression levels of the EMT-related proteins snail and N-cadherin.It is hypothesized that hANO6 may inhibit the ferroptosis pathway in BC cells by upregulating the expression levels of ERK and GPX4,thereby promoting BC progression and poor prognosis.