Expression Of CLDN6 In Breast Cancer And Its Role In Inducing Autophagy In Breast Cancer Cells

Claudin-6(CLDN6)has high organ-tissue specificity.Overexpression of CLDN6 inhibits the proliferation and induces apoptosis of breast cancer cells,suggesting that CLDN6 may be a suppressor gene and may be a marker for the prediction and diagnosis of breast cancer.CLDN6 is a membrane protein that may participate in cell autophagy,and then affects the occurrence of breast cancer through autophagy.Predicting and analyzing the structure,subcellular localization and related signals of CLDN6 will help to study the cellular biological behavior of CLDN6-related.In the early works about high throughput screening of CLDN6 regulatory target genes,it was found that the expression of autophagy-related genes was up-regulated.At the same time,when CLDN6 is overexpressed,there are the mitochondrial swelling and endoplasmic reticulum dilatation in breast cancer MCF-7 cells,suggesting that CLDN6 has an effect on autophagy,which may be related to its localization and expression in cells.In this study,we first analyzed the correlation between CLDN6 expression and clinicopathological characteristics of breast cancer patients with different molecular subtypes,and its prognostic significance.We also discussed the clinical significance of CLDN6 expression in ductal breast cancer and evaluated its clinical prognostic value.Then,we analyzed the regulatory region,protein structure and regulatory network of CLDN6 gene,and predicted that CLDN6 might be related to autophagy in the occurrence of breast cancer.Finally,the effect of autophagy on overexpression of CLDN6 cells and its possible mechanism were discussed.This will enrich the basic information of CLDN6 and provide potential targets for early diagnosis,prognosis and treatment of breast cancer.Methods:1.Expression of CLDN6 in breast cancer with different molecular subtypes and its relationship with clinicopathological data(1)TCGA and UALCAN online databases were used to collate breast cancer data and analyze the correlation between CLDN6 expression level and clinicopathological parameters of different types of breast cancer patients,and the impact on survival of patients.(2)The expression of CLDN6 in HBL-100 cells and breast cancer cells MCF-7 was detected by RT-PCR and Western blot,and the expression and localization of CLDN6 protein in 160 breast cancer tissue microarrays were detected by immunohistochemistry.2.Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions(1)The sequence of 5’regulatory region of human CLDN6 gene was obtained by BLAST comparison,and the promoter,EMBOSS and CPG Island Searcher were analyzed by online analysis platform software,neural network promoter prediction software and ROMOTER 2.0.The potential transcription factor binding sites were analyzed by TFSEARCH.(2)Inter Pro was used to analyze protein function;WOLFPsort and PSORT II were used to predict protein subcellular location in eukaryotic cells;KEGG database platform was used to analyze CLDN6-related proteins and their signaling pathways and functions.3.The role of CLDN6-induced autophagy in breast cancer MCF-7 cells3.1 Establishment of autophagy in breast cancer cell clones with stable CLDN6 expression(1)Establishment and screening of stably expressing CLDN6 breast cancer MCF-7 cells clones: To construct of p IRES2-EGFP-CLDN6 eukaryotic expression vector,and to transfect p IRES2-EGFP-CLDN6 into breast cancer MCF-7 cells by liposome transfection method,to select single cell clones by G418;the expression level of CLDN6 was identified by RT-PCR and western blot.The expression of CLDN6 was observed by immunofluore scence staining.(2)Determination of autophagy level of stably expressing CLDN6 cells clones: vectors and clone cells were divided into two groups,autophagy structure was observed by transmission electron microscope,autophagy about section parameters were analyzed by stereological measurement,localized expression of LC3 II was observed by immunofluorescence staining,and intracellular tropism was observed by acridine orange staining.The expression of autophagy related proteins LC3 II,beclin1 and P62 was detected by Western blot.3.2 Effects of autophagy on apoptosis of CLDN6-induced breast cancer MCF-7 cellsThe effects of 3-MA on the apoptosis of MCF-7 cells of stably expressing CLDN6: The cells were divided into vector,clone and 3-MA(5 mmol/L,4 h)groups.Cell apoptosis and autophagy were observed by immunofluorescence,hoechst 33342 and flow cytometry respectively.3.3 Mechanism of CLDN6-induced autophagy in breast cancer MCF-7 cellsCells were divided into vector,clone,and 3-MA groups.The expression of p38,CHOP,Bcl-2 signaling pathway molecules and autophagy-related proteins LC3 II and beclin1 were detected by Western blot.After clone cells were treated with p38 inhibitor SB203580,the expression of pathway proteins and the expression of autophagy-related proteins LC3 II and beclin1 were further detected by Western blot.Results:1.Expression of CLDN6 in breast cancer with different molecular subtypes and its relationship with clinicopathological data(1)The results of TCGA analysis showed that the expression of CLDN6 in invasive ductal breast cancer patients was higher than that of Luminal,HER2 and TNBC-UNS,and the expression level of CLDN6 in TNBC-LAR breast cancer was significantly lower than that of Luminal,HER2 and TNBC-UNS(P < 0.05).It is related to the gender,menstrual status and the type of breast cancer,and affects the prognosis of patients.(2)In breast cell lines HBL and MCF-7,the high expression level of CLDN6 m RNA and protein was consistent with that of them.In 160 breast cancer tissue microarrays,the expression of CLDN6 protein was mainly located in cytoplasm or nucleus,71.6% was low,28.4% was high,which was lower than that in adjacent tissues.Survival analysis showed that the expression of CLDN6 was significantly different from that of age,pathological grade,TNM stage and TNBC type breast cancer(P < 0.05).The survival rate of breast cancer patients with low expression of CLDN6 was related to the type of breast cancer.The expression level of CLDN6 was related to the expression of ER and Ki67 in cell nuclear(P < 0.05)and CK5/6 in cytoplasm(P < 0.001).2.Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions(1)CLDN6 is regulated by a diverse set of transcription factors(SP1,SPR,AML-1a,Cdx A,CRE-BP and CREB)and associated with the levels of methylation of Cp G islands in promoters.(2)CLDN6 is composed of 220 amino acids,the molecular formula is C1054H1682N268O291S16,the molecular weight is 23.2775 k Da,PI 8.32.CLDN6 is hydrophobic,and its secondary structure includes 26-42 bp,102-112 bp,131-139 bp,147-150 bp,222-238 bp,176-194 bp,196-220 bp irregular curl overlap region.(3)Signal peptide cleavage sites are located between 21 and 22 loci.CLDN6 is a four-time transmembrane protein,mainly transported through the endoplasmic reticulum(66.7%)and mitochondria(33.3%)and localized through secretory pathways.(4)KEGG signaling pathway showed that hsa04514,hsa04530,hsa04670,hsa05160 were related to CLDN6.3.The role of autophagy by CLDN6-induced in breast cancer MCF-7 cells3.1 Establishment of autophagy in human cervical carcinoma cell clones with stable CLDN6 expressionThe clones were detected by RT-PCR and Western blot.Compared with vectors,the expression of CLDN6 was up-regulated in clone group(P < 0.05),and the expression level of CLDN6 in clone cells group was higher than that in vector cells group.The cells were divided into vector and clone cells groups.The cells were observed by transmission electron microscopy.The typical autophagy and apoptotic bodies,mitochondria swelling and the formation of TJ structure between adjacent cells were observed in clone cells group.Immunofluorescence staining showed that LC3 B aggregated in the cytoplasm of clone cells group,the volume and quantity of LC3 B increased,and the orange-red eosinophilic vesicles in clone cells increased.Compared with vectors,the results showed that LC3II/LC3 I ratio was significantly up-regulated,expression level of beclin1 was up-regulated.The expression level of P62 was down regulated,and the difference was statistically significant(P < 0.05).3.2 Effects of autophagy on apoptosis of CLDN6-induced breast cancer MCF-7 cellsThe effect of 3-MA on the cell apoptosis of MCF-7 cells of stably expressing CLDN6: the cell activity in clone cells group was decreased,the OD value was increased in cells-treated with 3-MA group significantly and the expression levels of LC3 B were down-regulated,as well as,the apoptosical rate was decreased significantly.3.3 Mechanism of CLDN6-induced autophagy in breast cancer MCF-7 cellsThe expression level of the key signal molecules such as p38,CHOP and Bcl-2 in clone cells group were up-regulation,and autophagy-related proteins LC3 II and beclin1 were activation,however,in cells-treated with 3-MA group,the changes were opposite,the level of signal molecules and LC3 II and beclin1 in cell-treated with SB203580 group were decreased.Conclusions:1.The expression of CLDN6 in the overexpression of HER2,TNBC-UNS and Luminal breast cancer occurred significant changes,which were correlated with menstrual status and breast cancer type,and affected the prognosis and survival of patients,suggesting that CLDN6 can be used as a non-independent prognostic marker for breast cancer.2.CLDN6 protein is transported to cell membranes by secretory pathway in endoplasmic reticulum.The structure and function of CLDN6 are related to endoplasmic reticulum and p38/CHOP pathway.3.Autophagy of CLDN6-overexpression cells may related to p38/CHOP pathway.