KLF9 inhibits breast cancer metastasis by up-regulating E-cadherin

KLF9 inhibits breast cancer metastasis by up-regulating E-cadherin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article KLF9 inhibits breast cancer metastasis by up-regulating E-cadherin Mengyao Pang, Jie Zhang, Mengjie Zhang, Rui Ni, Mei Zhang, Ranru Wei, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4005329/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Krüppel-like factor 9 (KLF9) plays an inhibitory role in the process of breast cancer metastasis. The metastasis of tumor cells is often related to epithelial-mesenchymal transition (EMT). Among them, the gradual decrease of E-cadherin expression on cell surface is an important feature of EMT process. However, the concrete mechanism involved in this process remains largely unknown. In order to explore the mechanism, we have done relevant research. Through the analysis of transcriptome data in TCGA database and immunohistochemistry, we found that KLF9 was at a low expression level in breast cancer patients. The expression of KLF9 was positively correlated with the expression of E-cadherin in breast cancer cells. Functionally, KLF9 transcriptionally up-regulated E-cadherin expression and inhibited breast cancer metastasis, depending on its DBD domain. Mechanistically, KLF9 promoted E-cadherin promoter activity by binding to the CACCC motif (-12 to + 8), increasing the mRNA and protein level of E-cadherin. We also found that KLF9 can compete with SNAI1 to bind to the promoter region(-206 to + 47)of E-cadherin, and inhibit the transcriptional activity of SNAI1, leading to the activation of E-cadherin in breast cancer cells. Taken together, these results confirmed a new mechanism by which KLF9 could up-regulate E-cadherin expression to inhibit breast cancer metastasis, providing a research support for the prevention and treatment of breast cancer. Krüppel-like factor 9 Human breast cancer E-cadherin SNAI1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among females( 1 ). The majority of deaths from breast cancer are not due to the primary tumor, but the result of metastasis at distant sites in the body( 2 ). Recent studies have shown that aberrant activation of epithelial–mesenchymal transition (EMT) has been implicated in this process( 3 – 5 ). During this process, epithelial tumor cells may lose cell-to-cell adhesion and polarity characteristics, accompanied by cytoskeleton rearrangements( 6 ). They may acquire a motile and invasive fibroblast-like mesenchymal phenotype( 7 – 9 ). Silencing of E-cadherin gene or protein expression in epithelial cells is sufficient to induce a full EMT, because direct suppression of E-cadherin causes a decrease in cell-cell adhesion and increased invasion and motility( 10 – 12 ). Hence, E-cadherin is seemed as a hallmark of EMT( 13 – 15 ). E-cadherin is the prototypical cadherin, mediating cell–cell adhesion complexes anchored to the actin cytoskeleton via its cytoplasmic domain and β-catenin and α-catenin, thereby forming the core of the epithelial adherens junction( 16 , 17 ). Several transcription factors have been implicated in the repression of E-cadherin expression, including SNAI1( 18 – 20 ), SNAI2( 21 ), ZEB1( 22 – 24 ) and ZEB2( 25 ), all of which promote EMT through direct binding to the E-box of the E-cadherin promoter. SNAI1 has been one of the important classical EMT transcription factors in cancer research( 26 , 27 ). It mediates the repression of E-cadherin by direct recruitment of a co-repressor complex containing HDAC1 and HDAC2 (HDAC1/2)( 28 , 29 ) and Sin3A( 29 , 30 ). EMT is modulated by many factors, finding and understanding the regulation of novel factors would provide important insight into the molecular mechanisms implicated in EMT. Krüppel-like factor 9 (KLF9) is a member of the KLF family of transcription factors. All members of the KLF family have three highly conserved zinc finger motifs DNA-binding domain (DNA-binding domain, DBD) at the C-terminal of the proteins, recognizing GC-rich sequences with a preference for the 5′-CACCC-3′ core motif in the promoters and enhancers( 31 ). Expression profiling of migrated and invaded breast cancer cells reveals KLF9 as a potential suppressor of invasion in breast cancer( 32 – 34 ). Recently, our studies have found KLF9 could inhibit breast cancer metastasis by transcriptionally inhibiting MMP9( 35 ). However, detailed molecular mechanism by which KLF9 regulates EMT remains largely unknown. Here, we showed that KLF9 was at a low expression level in breast cancer patients through the analysis of transcriptome data in TCGA database and immunohistochemistry and we found that KLF9 inhibited the metastasis of breast cancer cells, at least in part, by up-regulating E-cadherin expression. Mechanistically, KLF9 promoted the transcription level of E-cadherin by binding to the CACCC motif of E-cadherin promoter. We also concluded that KLF9 could compete with SNAI1 to bind to the promoter region of E-cadherin and inhibit the transcriptional activity of SNAI1, leading to the activation E-cadherin in breast cancer cells. These studies indicate that KLF9 play an important role in restraining metastatic activity of breast cancers. Methods Data acquisition TCGA(The Cancer Genome Atlas) is a cancer genome project launched by the National Cancer Institute and the National Human Genome Research Institute in 2005, which is dedicated to collecting all tumor genome data, and it is a landmark successful attempt( 36 , 37 ). The Cancer Genome Atlas (TCGA) data is public repository. The database contains genome (gene expression, variation, methylation, single nucleotide polymorphism, etc.), transcriptome (mRNA, miRNA expression, etc.), epigenetics, proteome and other data of 33 kinds of human tumors( 38 , 39 ).The publicly available cancer genome data set provided by TCGA provides convenience for researchers to further explore tumor-related mechanisms and new ideas of anti-tumor diagnosis and treatment. Its data can be obtained by visiting https://portal.gdc.cancer.gov/ , and TCGA data is also included on UCSC Xena website. RNA sequence transcriptome data and clinical information for 1096 Breast Cancer and 112 non-tumor counterparts were acquired via TCGA. Statistical analysis Download the gene expression data set (gene expression rnaseq-illuminahiseq) and patient phenotype data set of TCGA Breast Cancer from the website, and import them into R software for processing. The relevant data of KLF9 (Ensemble ID: ENSG00000119138) were selected to screen and sort out the data, remove the missing values and standardize the gene expression data. According to the sample type, pathological grade of breast cancer and PAM50 subtype of breast cancer, the data of patients were classified and the expression difference was analyzed. Kaplan-Meier survival curve was drawn by statistical software, and the results were statistically tested by Log-rank test. Correlation analysis was carried out on the expression data sets of different genes. All data processing was done in the R language (version 4.2.1). All statistical P values were two-tailed, with p < 0.05 as statistical significance. Data were expressed as means ± SDs from at least three independent experiments. Un-paired t-test was used when the results from two groups were compared. Statistical analyses were carried out by one-way analysis of variance with Bonferroni's multiple comparison correction for comparison among three or more groups. Statistical significance was considered at the p < 0.05 level. Immunohistochemistry (IHC) All experimental protocols for this study were performed in accordance with established guidelines and regulations from the Biological and Medical Ethics Committee of Dalian University of Technology (NO. 2020–027). A total of 20 samples comprising 4 normal tissues and 16 breast cancer tissues were examined. Paraffin-embedded tissues were retrieved from department of pathology of Liaoning Cancer Hospital, and tissue microarray slides were prepared according to a published method. Anti-KLF9 antibodies were used for immunohistochemistry. The samples were fixed in 4% para- formaldehyde (Solarbio, Beijing, CHN) for 24–48 h and then embedded in paraffin. The samples were sectioned into 5-µm sections and stained with antibody as previously described. Images of the samples were obtained at 100× magnification (Nikon, Tokyo, JPN) and analyzed by ImageJ (NIH, Bethesda, MD, USA). Cell culture and transfection Human breast cancer cell line MCF-7, T47D, ZR-75-30 have been used in our previous study and were cultured as previously described( 40 , 41 ). Cells were grown in a humidified incubator with 5% CO 2 at 37℃, and transfected by Lipofectamine 2000 (Invitrogen, Auckland, New Zealand) according to the manufacturer’s specifications. MCF-7 human breast cancer cell (product number: SNL-060) was purchased from WuHan SHANER Biotechnology Company( http://www.sunnbio.com/ ). T47D human breast ductal cancer cell (product number: BNCC339608) and ZR-75-30 human breast cancer cell (product number: BNCC100125)were purchased from Bena Biological Company༈ https://www.bncc.com/༉ . Plasmids and antibodies Human KLF9 was cloned from a human cDNA library using the forward primer KLF9-F:5’-cggaattcctccgcggccgcctac-3’ and the reverse primer KLF9-R:5’-ccgctcgagcggtcacaaagcgttggc-3’ and the amplified KLF9 DNA fragment was inserted into the expression vector pcDNA3.1-3×Flag at the EcoRI and XhoI sites. Flag-tagged truncated KLF9 (KLF9-∆DBD) was constructed according to standard PCR-based cloning procedure using Flag-KLF9 plasmid as a template. PCR fragments were inserted into pcDNA3.1-3×Flag at the EcoRI and XhoI sites. pEGFP-SNAI1, pGL3 vector and pGL3-E-cadherin-Luc were acquired as previously described( 42 , 43 ). Small interfering RNAs (siRNAs) corresponding to the following sequences for knockdown of Ctrl and KLF9 were synthesized in GenePharma (Shanghai, China): siCtrl: 5’-UUCUCCGAACGUGUCACGUTT-3’ (forward) and 5′-ACGUGACACGUUCGGAGAATT-3′(reverse); siKLF9: 5’-GGCCCUUUCCCUGCACGTT-3’ (forward) and 5′-CGUGCAGGGAAAGGGCCTT − 3′ (reverse). Rabbit anti-Flag, anti-GFP, anti-KLF9 antibodies were purchased from Sigma (Sigma, Saint Louis, MO, USA). Mouse anti-E-cadherin was obtained from Abcam (Abcam, Cambridge, MA, USA). Rabbit anti-SNAI1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, CA, USA). Luciferase reporter assay Promoter activity was determined by a luciferase assay system. ZR-75-30 cells were seeded in a 24-well plate at a density of 1×10 5 and cultured for 24 h. Then the cells were transfected with corresponding plasmids using Lipofectamine 2000 according to the company’s specification. Twenty-four hours after transfection, luciferase activity was determined using Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA). RNA extraction and RT‑PCR Total RNA was extracted from ZR-75-30 cells using Takara RNAiso Reagent (Takara, Dalian, China). Total RNA (3µg) was reverse transcribed by oligo (dT) primer using a Reverse Transcription System (Takara). The single-stranded cDNA was amplified by PCR using specific primers: E-cadherin:5′-CTTCGGAGGAGAGCGGTG-3′ (forward) and 5′-CTAGTCGTCCTCGCCGCC-3′ (reverse); SNAI1: 5′-CGCGCTCTTTCCTCGTCAG-3′ (forward) and 5′-TCCCAGATGAGCATTGGCAG-3′ (reverse); KLF9: 5′-AACTGCTTTTCCCCAGTGTG-3′(forward) and 5′-TCCCATCTCAAAGCCCATTA-3′ (reverse); GAPDH:5′-TGAAGGTCGGAGTCAACGG − 3′ (forward) and 5′- CCTGGAAGATGGTGATGGG − 3′ (reverse). The PCR products were analyzed by electrophoresis using a 1.5% agarose gel. Real-time PCR Appropriate plasmids were transfected into ZR-75-30 cells and 24 h after transfection, the total cDNA was synthesized as described above. Relative mRNA levels were determined using the ABI Prism 7500 sequence detection system with SYBR premix Ex Taq (Takara) as previously described( 44 ). Expression of target genes was determined according to the 2-∆∆ CT method using GAPDH as a reference gene. The following primer sequences were used: E-cadherin: 5′-CTTCGGAGGAGAGCGGTG-3′ (forward) and 5′-CTAGTCGTCCTCGCCGCC-3′ (reverse); SNAI1: 5′- CGCGCTCTTTCCTCGTCAG − 3′ (forward) and 5′-TCCCAGATGAGCATTGGCAG-3′ (reverse); KLF9: 5′-AACTGCTTTTCCCCAGTGTG-3′ (forward) and 5′-TCCCATCTCAAAGCCCATTA-3′ (reverse); and GAPDH: 5′-TGAAGGTCGGAGTCAACGG-3′ (forward) and 5′- CCTGGAAGATGGTGATGGG − 3′ (reverse). Western blot, ChIP Western blot and chromatin immunoprecipitation (ChIP) assays were conducted as previously described( 45 , 46 ). The primers of E-cadherin promoter used in the ChIP PCR analysis were as follows: 5′-AGAGGGTCACCGCGTCTATG-3′ (forward) and 5′-TCCGCAAGCTCACAGG-3′ (reverse) for E-cad-c and 5′- TGAAGGTCGGAGTCAACGG − 3′ (forward) and 5′- CCTGGAAGATGGTGATGGG − 3′ (reverse) for GAPDH. Transwell assays ZR-75-30 were transfected with appropriate plasmids. After 24h transfection, the cells were suspended in serum-free medium and counted. The suspension containing 3×10 5 cells was inoculated in the upper chamber with (invasion) or without (migration) Matrigel (BD Biosciences, San Jose, CA, USA). After 24 or 48 h, cells remained on the top surface of the filter were removed with a cotton swab, while cells migrated or invaded into the bottom surface were washed with PBS, fixed with 4% paraformaldehyde for 15 min. Cells were stained with Crystal Violet solution for 20 min and then photographed. Wound‑healing assay Scratch wound-healing were performed as previously described( 4 ).Transfected cells were seeded into the six-well plates cells with a density of 3.0×10 5 cells. And after 24 h, multiple wounds were created by using a 200-µl pipette tip. Photographs were recorded at 0 h and 24 h and the wound healing was measured from these images. Statistical analysis Data were expressed as means ± SDs from at least three independent experiments. Un-paired t-test was used when the results from two groups were compared. Statistical analyses were carried out by one-way analysis of variance with Bonferroni's multiple comparison correction for comparison among three or more groups. Statistical significance was considered at the p < 0.05 level. Results Down-regulation of KLF9 RNA and protein levels in breast cancer patients. To explore the potential role of KLF9 in the occurrence and development of breast cancer, we downloaded RNA sequence transcriptome data and clinical information data of 1096 breast cancer cases and 112 corresponding normal tissues from TCGA database. Next, the differential gene expression data and clinical information data of breast cancer tissues and normal tissues are analyzed. The results showed that the expression level of KLF9 in breast cancer tissues was lower than that in normal tissues (Fig. 1 A). To explore whether the expression of KLF9 is related to the clinical stage of patients, we analyzed the relevant data. The results showed that the expression of KLF9 in breast cancer tissue samples was related to the clinical stages of breast cancer patients, and the expression of KLF9 in late clinical patients was lower than that in early clinical patients (Fig. 1 B). We also analyzed the expression of KLF9 in patients of different age groups, and found that the expression of KLF9 in patients aged > 65 was lower than that in patients aged ≤ 65 (Fig. 1 C). TNM staging system is the most common tumor staging system in the world at present. TNM staging system was put forward by Frenchman Pierre Denoix from 1943 to 1952, and then gradually improved by American Joint Committee on Cancer (AJCC) and union for international cancer control (UICC). In 1968, the first edition of TNM Classification of Malignant Tumors was officially published. The TNM staging system of each tumor is different, so the meaning of letters and numbers in TNM staging is different in different tumors. At present, TNM staging system has become the standard method for clinicians and medical scientists to stage malignant tumors( 47 ). In TNM staging system, "T" (tumor) refers to the situation of the primary tumor, which is represented by T1 ~ T4 in turn with the increase of tumor volume and adjacent tissue involvement. "N" (node) refers to the involvement of Regional Lymph Node. When lymph nodes are not involved, they are denoted by N0, and with the increase of the degree and scope of lymph node involvement, they are denoted by N1 ~ N3 in turn. "M" (Metastasis) refers to distant metastasis (usually hematogenous metastasis). Those without distant metastasis are denoted by M0, and those with distant metastasis are denoted by M1. Therefore, a specific tumor stage can be expressed by the combination of three indicators of TNM. The TNM stage of breast cancer is shown in Table 1 ( 48 , 49 ). The results showed that the expression of KLF9 in breast cancer tissue samples was significantly correlated with some different T stages, different N stages and different M stages of breast cancer patients, and the low expression of KLF9 in breast cancer tissue was associated with tumor volume increase and distant metastasis (Fig. 1 D-F). Table 1 TNM staging of breast cancer T(Tumor) N(Lymph Node) M(Metastasis) T0: No primary cancer was detected. N0: There are no enlarged lymph nodes in the ipsilateral armpit. M0: No distant metastasis. Tis: Preinvasive carcinoma. N1: There are enlarged lymph nodes in the ipsilateral armpit, which can be pushed. M1: Distant metastasis. T1: The tumor size is 0−2cm. N2: The enlarged lymph nodes in the ipsilateral armpit fuse or adhere to the surrounding tissues. T2: The tumor size is 2−5cm. N3: There were ipsilateral parasternal lymph node metastasis and ipsilateral supraclavicular lymph node metastasis. T3: Tumors larger than 5cm. T4: The tumor has penetrated the skin or attached to the chest wall. At present, the diagnosis of breast cancer mainly depends on the combination of clinical physical examination, imaging auxiliary examination and pathological examination, and pathological examination is still the main "gold standard" of diagnosis. The expression of Estrogen Receptor (ER), Progesterone Receptor (PR), human epidermal growth factor-2 (HER-2) and Ki67 in cells were detected by whole gene expression profile, and breast cancer was divided into five subtypes. They are Basal-like, HER2 overexpression (HER2+), Luminal-A, Luminal-B and Normal like( 50 , 51 ). Luminal-A breast cancer is hormone receptor positive (estrogen receptor or progesterone receptor positive), HER2 negative, and the protein Ki-67 level is low, which helps to control the growth rate of cancer cells. Luminal-A cancer is a low-grade cancer, which tends to grow slowly and has the best prognosis. Luminal-B breast cancer is hormone receptor positive (estrogen receptor or progesterone receptor positive), HER2 positive or HER2 negative, and the protein Ki-67 level. Luminal-B cancer usually grows slightly faster than luminal-A cancer, and its prognosis is slightly worse. Basal breast cancer is hormone receptor negative (estrogen receptor and progesterone receptor negative) and HER2 negative, so it is also called Triple-negative breast cancer. This type of cancer is more common in women with BRCA1 gene mutation. HER2 overexpression breast cancer is hormone receptor negative (estrogen receptor and progesterone receptor negative) and HER2 positive. HER2-enriched cancers tend to grow faster and have a worse prognosis than intracavitary cancers. For this kind of breast cancer, targeted therapy for HER2 protein is usually used in combination with corresponding drugs, such as Herceptin (chemical name: trastuzumab), Perjeta (chemical name: pertuzumab), Tykerb (chemical name: lapatinib), Nerlynx (chemical name: neratinib) and Kadcyla (chemical name: T-DM1 or ADO). Normal breast cancer, similar to Luminal-A, is hormone receptor positive (estrogen receptor or progesterone receptor positive), HER2 negative, and the level of protein Ki-67 is low, which helps to control the growth of cancer cells. However, although the prognosis of normal breast cancer is good, its prognosis is slightly lower than that of lumen cancer( 52 – 54 ). See Table 2 for the classification of breast cancer subtypes. Table 2 Subtype of breast cancer Subtype ER PR HER2 Ki−67 Prognosis Luminal-A + + - < 14% Better Luminal-B + + - ≥ 14% Good + + + Any Good HER2+ - - + Any Poor Triple-negative/Basal-like - - - Any Poor Normal like + + - < 14% Good Next, we explore whether the expression of KLF9 is related to the subtypes of breast cancer patients. The results showed that the expression of KLF9 was significantly different among different subtypes, and there was a certain relationship between the low expression of KLF9 in breast cancer samples and the poor prognosis of breast cancer patients (Fig. 1 G). Kaplan-Meier survival curve of breast cancer patients was generated based on TCGA data set. The results showed that there was no significant correlation between KLF9 expression in breast cancer samples and the overall survival rate of patients (Fig. 1 H). The correlation analysis of gene expression between KLF9 and CDH1 (encoding E-cadherin) showed that there was a significant negative correlation between the expression levels of KLF9 and CDH1 (Fig. 1 I). Twenty tissue samples of 4 breast cancer adjacent tissues and 16 breast cancer tissues were specifically labeled with anti-KLF9 antibody, and the images of the samples were obtained at 100× magnification. It is obvious from the image that the expression of KLF9 in breast cancer tissue is lower than that in breast cancer adjacent tissue (Fig. 1 J). We then randomly select the images of cell areas from each IHC slice, and use Image-J software to quantify the average optical density (AOD) of the images. The AOD data is plotted and visualized in the GraphPad Prism 9.4.0 software. The results showed that the AOD value of breast cancer tissue was significantly lower than that of breast cancer adjacent tissue, which indicated that the expression of KLF9 protein in breast cancer tissue was significantly lower than that in breast cancer adjacent tissue (Fig. 1 K). Through the analysis of transcriptome data and immunohistochemistry in TCGA database, we found that KLF9 was at a low expression level in breast cancer patients. Therefore, we have reason to believe that KLF9 has a certain correlation with the occurrence of breast cancer, and we will continue to explore it through follow-up experiments. KLF9 inhibits the migration and invasion of human breast cancer cells Recent studies have shown that KLF9 acts as a cancer-inhibitory effector in breast cancer cells( 32 , 35 ). The diffusion of tumor cells is often related to Epithelial interstitial transformation (EMT) process. EMT is a multifunctional cell process, which is accompanied by the characteristics of cell polarity loss, adhesion decline, cytoskeleton change, cell migration and cell mobility enhancement. Among them, the gradual decrease of E-cadherin expression on the cell surface is an important feature of EMT process. Besides, KLF9 could transcriptionally down-regulate MMP9 expression and inhibited the metastasis of breast cancer cells( 35 ). However, the relationship between KLF9 and E-cadherin, an important marker of EMT, remains largely unknown. In order to explore the association between KLF9 and E-cadherin in the occurrence and development of breast cancer, we determined the protein level in breast cancer cell lines containing MCF-7, T47D, ZR-75-30 cells. Western blot analysis revealed almost no endogenous KLF9 was detected in ZR-75-30 cells, which displayed mesenchymal-like morphology and lesser cell–cell adhesion. Relatively higher-level expression of KLF9 was identified in MCF-7 cells than in T47D cells, consistent with the expression of E-cadherin in breast cancer cells (Fig. 2 A and 2 B). The protein expression of KLF9 in ZR-75-30 breast cancer cell line of Luminal-B subtype is lower than that of T47D and MCF7 breast cancer cell lines of Luminal-A subtype, which is consistent with the transcription level analysis of KLF9 in different breast cancer subtypes in TCGA database. These studies suggested that KLF9 expression is higher in non-invasive cancer cells and may inhibit breast cancer by increasing cell-cell adhesion. To verify the role of KLF9 in cell motility in breast cancer cells, scratch wound-healing and transwell assays were carried out to demonstrate the migration and invasion capacity. Compared with control cells, overexpressing of KLF9 markedly inhibited cell motility (Fig. 2 C and 2 D) and reduced the cell numbers that migrated through the Matrigel-coated membrane (Fig. 2 G and 2 H) in ZR-75-30 cells. In contrast, knockdown of endogenous KLF9 in ZR-75-30 cells significantly enhanced cell migration ability (Fig. 2 E and 2 F) and increased the capacity of cells to traverse the Matrigel-coated membrane (Fig. 2 I and 2 J), compared with control cells. These results indicated that KLF9 can inhibit the invasion and metastasis in breast cancer cells. KLF9 increases E-cadherin expression via transcriptional activation in breast cancer cells As E-cadherin is an important inhibitor in the process of EMT( 55 , 56 ), which has the same function as KLF9, we speculate whether KLF9 inhibits breast cancer metastasis by regulating the expression of E-cadherin. As we predicted, Reverse transcription-PCR(RT-PCR) showed that overexpression of KLF9 increased the mRNA level of E-cadherin compared with the control group in ZR-75-30 cells (Fig. 3 A and 3 B). In contrast, the mRNA level of E-cadherin decreased after siKLF9 (pRNA T-U6.1 vector) was transfected in ZR-75-30 cells, suggesting that KLF9 could promote the transcription of E-cadherin (Fig. 3 A and 3 B). Western blot results showed that increased level of E-cadherin protein by overexpression of KLF9, and decreased level of E-cadherin protein following siKLF9 (pRNAT-U6.1 vector), consistent with the change of mRNA level (Fig. 3 C and 3 D). These data clearly indicated that increased E-cadherin protein expression was mediated by KLF9, occurred mainly at the mRNA level regulation. Luciferase reporter gene assay also revealed that increasing doses of KLF9 induced a dose-dependent growth of E-cadherin-promoter-driven luciferase activity (Fig. 3 E), while knockdown of KLF9 resulted in a decrease in E-cadherin-promoter-driven luciferase activity in ZR-75-30 cells (Fig. 3 F). These data showed that KLF9 could activate the E-cadherin promoter. Taken together, these results indicated that KLF9 could up-regulate the level of E-cadherin protein by transcriptionally activating the activity of the E-cadherin promoter. KLF9 binds to the CACCC motif of the E-cadherin promoter through its DNA binding domain To explore the specific sites and regions of KLF9 acting on E-cadherin promoter, we constructed four truncated E-cadherin promoter and fused each promoter with luciferase reporter gene to yield a reporter construct (Fig. 4 A). The reporter construct comprising E-cad-a promoter (-999 ~ + 47) increased more than two-fold of the reporter activity when ZR-75-30 cells overexpressed KLF9 compared with control. Interestingly, the reporter activity disappeared when deleted nucleotide from − 206 to + 47 (E-cad-b), suggesting that KLF9 binding site might be existed in nucleotide − 206 to + 47. The result showed that after the deletion of nucleotide − 999 to -206 (E-cad-c), the activation multiple of KLF9 overexpression was almost the same as that of the E-cad-a, which proved that nucleotide − 999 to -206 (E-cad-b) probably contained no regulatory element. These data indicate that nucleotide − 206 to + 47 (E-cad-c) on the E-cadherin promoter was contain response elements required for KLF9 to activate its transcriptional activity. Furthermore, due to KLF9 recognize sequences with a preference for the 5′-CACCC-3′ core motif in the promoters and enhancers( 31 ), nucleotide − 12 to + 8 "CACCC" located within nucleotide − 206 to + 47 was mutated into "CATTT" (E-cad-d), the activation of KLF9 was almost completely lost, indicating that KLF9 was exactly bound to the "CACCC" sequence of E-cadherin promoter (Fig. 4 B). Since KLF9 DNA-binding domain (zinc finger domain, ZNF) could recognize the CACCC element, we speculated that DNA-binding domain of KLF9 might bind to the E-cadherin promoter to regulate the transcription activity of E-cadherin. Therefore, to verify this hypothesis, we constructed a deletion of KLF9 DNA binding domain (pcDNA3.1-3×Flag-KLF9△DBD) (Fig. 4 C). CHIP experiment further showed that KLF9 can specifically bind to the E-cad-c promoter, which included "CACCC" sequence, consistent with the results of previous reporter gene assay (Fig. 4 D, top). KLF9-∆DBD cannot achieve specific binding to E-cad-c promoter. We also amplified the GAPDH fragment as a control (Fig. 4 D, bottom). Taken together, these results suggest that KLF9 may promote E-cadherin transcription through its DNA-binding domain (DBD) interaction with CACCC motif located within nucleotides − 206 to + 47 of E-cadherin promoters. Consistently, compared to full length KLF9, the activation of E-cad -c promoter by KLF9△DBD was disappeared (Fig. 4 E and 4 F ), suggesting that the DNA-binding domain (DBD) of KLF9 might be responsible for the E-cadherin promotion. This was subsequently confirmed by ChIP assay. ChIP assay also showed that the KLF9△DBD experimental group, compared with the wild-type KLF9, did not detect specific DNA bands on E-cad -c promoter, indicating that KLF9 DNA-binding domain specifically bind to E-cad-c promoter (Fig. 4 D). Collectively, these results suggested that KLF9 binds to the CACCC motif of E-cadherin promoter through its DNA binding domain and the DNA-binding domain of KLF9 is essential for its tumor-suppressive role in breast cancer cells. Furthermore, MCF-7 cells were tested for their motility, migration ability and invasion ability by wound healing and transwell experiment respectively. When KLF9 is overexpressed, the migration ability of cells is obviously reduced (Fig. 4 G and 4 H), and the ability of cells to pass through the matrix gel coating film is also reduced (Fig. 4 I and 4 J). However, when Flag-KLF9-△DBD was overexpressed, compared with the control group, the migration ability of cells did not change significantly (Fig. 4 G and 4 H), and the ability of cells to pass through the matrix gel coating film remained unchanged (Fig. 4 I and 4 J). To sum up, after deleting the DNA binding region of KLF9, the inhibitory effect of overexpression of KLF9 on the motility, migration and invasion of MCF7 cells disappeared, which proved that the DNA binding region of KLF9 was the necessary region to inhibit the motility, migration and invasion of breast cancer cells. KLF9 affects the transcriptional regulation of E-cadherin by competing with SNAI1 The transcription factor SNAI1 has been recognized as a direct repressor of E-cadherin expression, which recruits HDAC and the corepressor mSin3A to form a multimolecular complex to repress E-cadherin( 6 , 26 , 29 ). Because of the target sites located in E-cad-c, we speculated that KLF9 may be involved in coordinating the interaction between SNAI1 and E-cadherin. Firstly, we utilized luciferase reporter detection to further evaluate our hypothesis. Overexpression of SNAI1 alone inhibited E-cadherin-Luc activity, whereas overexpression of SNAI1 and KLF9 relieved this inhibition (Fig. 5 A). Moreover, western blot results also showed that overexpression of SNAI1 alone repressed the expression of E-cadherin protein, whereas overexpression of SNAI1 and KLF9 relieved this repression (Fig. 5 B and 5 C). Quantitative RT-PCR showed that E-cadherin mRNA levels were significantly decreased after transfection of SNAI1 alone, and KLF9 partially rescued the decrease of E-cadherin mRNA, consistent with the change of protein level (Fig. 5 D and 5 E). To determine whether KLF9 affects the regulation of E-cadherin promoter by SNAI1, ChIP assays were carried out using ZR-75-30 cells. Overexpression of KLF9 effectively prevented the recruitment of SNAI1 to the E-cad - c promoter (Fig. 5 F). Taken together, these findings revealed that KLF9 may enhance the activity of E-cadherin by alleviating its transcriptional suppression exerted by SNAI1. To further confirm, using scratch wound-healing and transwell assays to demonstrate the cell motility, migration and invasion capacity respectively in ZR-75-30 cells. When the ZR-75-30 cells co-overexpressed KLF9 and SNAI1, the migration ability of cells are significantly reduced (Fig. 5 G and 5 H), and the ability of cells to pass through the matrix gel coating film are reduced (Fig. 5 I and 5 J), compared to the cells expressed SNAI1 alone. Collectively, these results indicate that SNAI1 is involved in KLF9-mediated suppression of breast cancer invasion and metastasis. Discussion Through the analysis of TCGA database and immunohistochemistry, we found that the RNA and protein expression levels of KLF9 in breast cancer patients were lower than those in non-breast cancer patients. The expression of KLF9 in breast cancer tissue samples is related to the clinical stages of breast cancer patients, and the expression of KLF9 in patients with advanced clinical stage is lower than that in patients with early clinical stage. The expression of KLF9 was significantly different among different subtypes, and there was a certain relationship between the low expression of KLF9 in breast cancer samples and the poor prognosis of breast cancer patients. In this study, we identified that E-cadherin is a novel KLF9 transcriptional target gene, which may provide a new mechanism for KLF9 in the regulation of human breast cancer metastases. First, KLF9 inhibits migration and invasion in breast cancer cells. Second, KLF9 could up-regulate E-cadherin expression, depending on DBD domain, by promoting the transcription level of E-cadherin and such promotion is via CACCC motif in the promoter. Third, we found the underlying mechanism is that KLF9 could compete with SNAI1 to bind the E-cadherin promoter, further activating the expression of E-cadherin. Finally, we demonstrated that KLF9 could suppress the invasiveness of breast cancer cells, at least in part, through up-regulating the expression and activity of E-cadherin. This study revealed the important role for KLF9 in regulating the invasion and metastasis of ER-positive breast cancer cells, providing further theoretical support for the treatment and prevention of ER-positive breast cancer, and we plan to select ER-negative breast cancer cell lines for related experimental exploration in the future. Breast cancer has surpassed lung cancer to become the most common cancer in the world, and the invasion and metastasis of breast cancer are the main reasons for the poor prognosis and low survival rate of patients( 57 – 60 ). Studies have shown that EMT is associated with increased cell migration, invasion, and metastasis( 7 , 61 , 62 ). E-cadherin, the cellular adhesion molecule that forms the cell–cell adhesion junctions of epithelial cells, is essential for the cells to maintain their epithelial phenotype( 7 , 61 , 62 ). During the EMT, E-cadherin is cleaved at the plasma membrane and subsequently degraded, resulting in loss of epithelial adherent junctions( 55 ). Thus, downregulation or loss of E-cadherin expression is considered to be the hallmark event of EMT( 14 , 55 , 56 ). The first discovered and most important transcriptional repressor of E-cadherin is SNAI1( 26 , 63 , 64 ).SNAI1 repressed E-cadherin expression by interacting with a co-repressor complex SIN3A/HDAC1/HDAC2 and modification of local chromatin structure(29, 65). In the present study, we found that the KLF9 alleviated the transcriptional inhibition and protein downregulation of E-cadherin by SNAI1. Whether KLF9 regulates the expression of E-cadherin by binding to the E-box of the promoter requires further study. Therefore, we suspected that KLF9 may compete with SNAI1 to regulate E-cadherin. Further study is needed to clarify this issue. The Krüppel-like transcription factor family may emerge as a new type of transcription factors that control the transcription of E-cadherin. KLF4 binds to and activates the E-cadherin promoter, which is necessary to maintain the epithelial phenotype in mammary epithelial cells( 46 , 63 , 66 ). Indeed, KLF6, another KLF member and tumor suppressor, has been shown to directly bind and activate E-cadherin gene promoter in ovarian cancer( 67 ). KLF8 is a novel repressor of E-cadherin in epithelial cells and plays a large part in the loss of E-cadherin expression in human breast carcinoma cells and their invasiveness( 42 , 67 ). KLF6 activates E-cadherin transcription similar to KLF4 while KLF8 binding results in repression of the E-cadherin promoter. These data demonstrate the distinct abilities of individual Krüppel-like factors to modulate expression of the same target genes. As suggested in our study, E-cadherin is a novel transcriptional target of KLF9 and KLF9 could directly bind to the CACCC motif of E-cadherin promoter to increase E-cadherin expression. This regulation is necessary to inhibit the migration and invasion capabilities of breast cancer cells. Consistent with our research, KLF9 has been regarded as a transcription repressor in several types of tumor. For example, in colorectal cancer, KLF9 may as a transcriptional repressor of a module of IFN-stimulated genes and specifically ISG15 to prevent tumor cell survival and growth by promoting apoptosis( 68 ). In Esophageal squamous cell carcinoma, KLF9 inhibited the cancer by regulating Cyr61 and negatively modulating the beta-catenin/TCF signaling( 69 ). KLF9 suppresses gastric cancer metastasis through directly inhibiting transcriptional expression of MMP28( 70 ). Nevertheless, KLF9 in ovarian cancer may be different from other cancer. Expression of KLF9 is up-regulated in ovarian cancer and knockdown KLF9 by lentivirus inhibits the growth of ovarian cancer cell( 71 ). It indicates KLF9 plays different roles in different cancer tissues. Furthermore, KLF9 has been found to play a potential suppressor role in breast cancer( 32 , 35 ). The researchers found that the expression of KLF9 was significantly lower in breast cancer tissue than that in normal tissue, suggesting that KLF9 may be a potential breast cancer metastasis inhibitory factor. Recent studies have shown that KLF9 suppressed the metastasis of breast cancer by down-regulating the expression of MMP9( 35 ). Our study further confirmed KLF9 could inhibit breast cancer cells by promoting E-cadherin expression and competing with snail to bind to the promoter region(-206 ~ + 47)of E-cadherin. Although our results indicate that E-cadherin transcription is promoted by KLF9 during breast cancer metastasis, we speculate that might other target genes are involved in this process due to the complexity of TF regulatory networks. Therefore, further research is required to clarify the transcriptional regulation network mediated by KLF9 during breast cancer metastasis. In summary, our study reported KLF9 could inhibit breast cancer metastasis by increasing the expression of E-cadherin at mRNA and protein levels. Further mechanistic details that came to KLF9 binds to the CACCC motif of E-cadherin promoter. Furthermore, forced expression of KLF9 in highly expressed SNAI1 was sufficient to rescue E-cadherin expression and cell motility and invasion. These findings define E-cadherin regulation by KLF9 as a potentially critical pathway to prevent epithelial-to-mesenchymal transition and support a metastasis suppressive role for KLF9 in breast cancer. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material The datasets used, analyzed, or both during the current study are available from the corresponding author on reasonable request. Competing Interests The authors have declared that no competing interests exist. Funding This work was supported by grants 81301504 to Miao Wang from National Natural Science Foundation of China, grants DUT21LK26 to Miao Wang from the Fundamental Research Funds for the Central Universities, and grants XMMC-FCTM202105 to Mei Zhang from the Key laboratory of functional and clinical translational medicine for Fujian province university. The funders had no roles in study design, data collection, and analysis, the decision to publish, or preparation of the manuscript. Authors' Contributions Miao Wang, Mengyao Pang and Jie Zhang conceived and designed the experiments; Miao Wang, Mengyao Pang, Jie Zhang, Mengjie Zhang, Rui Ni, Mei Zhang, Ranru Wei, Guohui Li, Ying Tang, Liming Ma and Xiaoyan Li performed the experiments; Miao Wang, Mengyao Pang, Jie Zhang, Mengjie Zhang, Rui Ni, Mei Zhang, Ranru Wei, Guohui Li, Ying Tang, Liming Ma and Xiaoyan Li analyzed the data; Miao Wan, Mengyao Pang and Mei Zhang wrote the manuscript. All authors have read and approved the manuscript. Data Availability Statement In this study, data in Fig.1 were downloaded from TCGA database (https://portal.gdc.cancer.gov/). We confirmed that all data are original and have not been published elsewhere. Acknowledgement Not Applicable. References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4005329","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276962550,"identity":"b5d6b160-b96b-44d6-8b30-1109f5648cf0","order_by":0,"name":"Mengyao Pang","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mengyao","middleName":"","lastName":"Pang","suffix":""},{"id":276962551,"identity":"133ea8a4-d60a-4429-9e5b-11fe046ecb41","order_by":1,"name":"Jie Zhang","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhang","suffix":""},{"id":276962552,"identity":"4c2ddb7a-4b8c-482f-bf2d-ae8873038d19","order_by":2,"name":"Mengjie Zhang","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mengjie","middleName":"","lastName":"Zhang","suffix":""},{"id":276962553,"identity":"cadbc385-46d4-4151-af7f-d162eeed0439","order_by":3,"name":"Rui Ni","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Ni","suffix":""},{"id":276962554,"identity":"ec2c3bad-19a7-49f3-9f0e-61bffbcb1722","order_by":4,"name":"Mei Zhang","email":"","orcid":"","institution":"Fujian province university, Xiamen Medical College XIaMen Fujian Province","correspondingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Zhang","suffix":""},{"id":276962555,"identity":"643c04a4-bb74-4f3c-b268-1a1dd2bbdbeb","order_by":5,"name":"Ranru Wei","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ranru","middleName":"","lastName":"Wei","suffix":""},{"id":276962556,"identity":"bd559baa-7f7f-447f-a18c-a9646a701db2","order_by":6,"name":"Guohui Li","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Guohui","middleName":"","lastName":"Li","suffix":""},{"id":276962557,"identity":"364af052-de0f-4703-bf73-73a221ae1b82","order_by":7,"name":"Ying Tang","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Tang","suffix":""},{"id":276962559,"identity":"c908c122-73f5-4cac-a583-c69aeb737203","order_by":8,"name":"Liming Ma","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Liming","middleName":"","lastName":"Ma","suffix":""},{"id":276962564,"identity":"662d510b-8257-474d-9397-4aabc6281b74","order_by":9,"name":"Xiaoyan Li","email":"","orcid":"","institution":"Cancer Hospital of China Medical University, Liaoning Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Li","suffix":""},{"id":276962565,"identity":"90d18c6f-43c1-443f-b87a-a63bd5d80352","order_by":10,"name":"Miao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYNCCCmYZMM1DvJYzzDwMbCRpYWwjRYvB8bOHX92cZ81jcL+B8cHbNgZ5c4JazuSlWeduS+cxOMbAbDi3jcFwZwMBLWYHcsyMc7cdBmlhk+ZtY0gwOEBIy/k3QC1zwFrYfxOn5UaO8ePcBogtzERpsb/xxow551g6j+SxxGbJOeckDDcQ0iLZn2P8OafGWo7v8OGDH96U2cgTtAUI2CQgNGMDkJAgrB4ImD8QpWwUjIJRMApGLgAAgpQ84iVpCv8AAAAASUVORK5CYII=","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Miao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-03-02 03:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4005329/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4005329/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52452380,"identity":"75d02c84-10d9-45ef-9435-fd33cbdbe6bf","added_by":"auto","created_at":"2024-03-11 19:16:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1939656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTCGA database analysis and immunohistochemical analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Gene expression difference of KLF9 in normal tissues and breast cancer tissues. The samples of normal tissues and breast cancer tissues were 112 and 1096. The expression data were standardized and logarithmic, and the unit was log10(count+1). The mean difference between groups was analyzed by two-sided T-test. (B) Expression data of KLF9 in different stages of breast cancer patients. (C) The expression difference of KLF9 in patients of different age groups. The sample numbers of patients ≤65 years old and patients \u0026gt; 65 years old were 764 and 297 respectively. (D) The expression data of KLF9 in the tumor of patients with T1-T4 breast cancer, the number of samples is 280, 625, 138 and 39 respectively. (E) The expression data of KLF9 in breast cancer patients with stage N0-N3 were 512, 358, 119 and 76 respectively. (F) The expression data of KLF9 in M0 and M1 breast cancer patients, the number of samples is 902 and 21, respectively. (G) The expression difference of KLF9 in different breast cancer subtypes. The samples are Basal-like, HER2+, Luminal-A, Luminal-B and Normal-like, and the number of samples is 140, 185, 421, 194 and 24 respectively. (H) Kaplan-Meier survival curve of breast cancer patients based on TCGA data set, through Log-rank test, the difference of survival time distribution is not statistically significant (p= 0.625). (I) Scatter plot of KLF9 and CDH1 gene expression in breast cancer patients, using Spearman correlation test, the Spearman correlation coefficient is-0.1535 (****: P≤ 0.0001). (J) Immunohistochemical detection of KLF9 expression in breast cancer adjacent tissues and breast cancer tissues. Some photos are shown in the picture, and the magnification is 100 X. (K) Image-J software is used to quantify the average optical density (AOD) of the image, and the AOD data is plotted and visualized in GraphPad Prism 9.4.0 software.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4005329/v1/c251b539a990e2c348f6fab1.png"},{"id":52452392,"identity":"cf20fd27-13cb-4e3e-9db9-36a61ea17913","added_by":"auto","created_at":"2024-03-11 19:16:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":969037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of KLF9 on the cellular motility of breast cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Western blot analysis of KLF9 and E-cadherin expression in MCF-7, T47D and ZR-75-30 cell lines. Experiments were repeated at least three times. (C-F) Scratch wound-healing assay evaluating the effects of KLF9 overexpression or knockdown on the cellular motility and invasion ability of ZR-75-30 cells. (G-J)Transwell migration and invasion assays assessing the effects of KLF9 overexpression or knockdown on the motility of ZR-75-30 cells. Scale bars, 100 mm. Cell migration and invasion assays were performed in 24-well chambers without or with Matrigel. Cells (1 × 10\u003csup\u003e5\u003c/sup\u003e per well) were transfected with control vector or Flag-KLF9; and siCtrl or siKLF9 (pRNA T-U6.1 vector) plasmid respectively then plated in the upper chamber. After 24h of incubation, the migrating and invading cells on the lower surface of the filter were stained and counted. The bar graphs show the number of migrating and invading cells for each category of cells repeated three times. Data are presented as mean ± SD (p \u0026lt; 0.05, significant; ns, not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4005329/v1/98803e0c49c091c460937997.png"},{"id":52452298,"identity":"ccb0f3ab-cbe5-4490-a816-2306b288c8b5","added_by":"auto","created_at":"2024-03-11 19:16:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":271211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLF9 up-regulates E-cadherin mRNA and protein levels through increasing its promoter activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) RT-PCR showing the effects of KLF9 overexpression and knockdown on the mRNA levels of E-cadherin in ZR-75-30 cells. ZR-75-30 cells were transfected with control vector or Flag-KLF9; and siCtrl or siKLF9 (pRNAT-U6.1 vector) plasmid. (C-D) ZR-75-30 cells were transfected with Flag-KLF9 or control vector, and siCtrl or siKLF9 plasmid. Cell lysates were subjected to western blot analysis with the indicated antibodies. (E-F) Reporter-gene assay showing E-cadherin promoter-driven luciferase reporter activity was regulated by KLF9 in ZR-75-30 cells. Cells were co-transfected with E-cadherin-Luc and Flag-KLF9, siKLF9 (pRNAT-U6.1 vector) or control vector, and then cultured for 24h before harvest. The bar graph shows the fold changes in relative luciferase activity normalized against β-galactosidase activity. Data are presented as means ± SD (p \u0026lt; 0.05, significant; ns, not significant).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4005329/v1/b10685104fda65f39f1fe04f.png"},{"id":52452314,"identity":"a701560c-2816-45a9-98ae-e2f29d5b2b9a","added_by":"auto","created_at":"2024-03-11 19:16:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5301637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLF9 binds to the CACCC motif of the E-cadherin promoter through its DNA binding domain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the E-cadherin promoter and the deletion or mutant constructs (E-cad a~d), indicating the position of potential regulatory control elements. (B) ZR-75-30 cells were transiently co-transfected with E-cad-a~d promoter reporter plasmids, and either control vector or Flag-KLF9 for 24 hours, and luciferase activities were measured. Activity of the different constructs was analyzed in ZR-75-30 cells transiently transfected with Flag-KLF9 or with the empty expression vector. (C) Schematic diagram of the deletion mutant of KLF9 DNA binding domain (pcDNA3.1-3×Flag-KLF9△DBD) (D) left: ChIP assay showing the interaction between KLF9 and the E-cad-c promoter (top) or GAPDH promoter (bottom) in ZR-75-30 cells. Cross-linked chromatin was extracted from the cells and subjected to immunoprecipitation with anti-Flag or IgG antibody, and the DNA was used for amplification of E-cad-c fragments shown in (A). IgG was used as a negative control; right: Regions of KLF9 physically associated with E-cad-c promoter were analyzed using ChIP assay. ChIP assays were done using anti-Flag antibody to screen KLF9-bound or KLF9-∆DBD E-cad-c promoter for PCR amplification in ZR-75-30 cells. IgG was used as a negative control. (E-F) ZR-75-30 cells were co-transfected with E-cadherin-Luc and wild-type KLF9 or DNA-binding domain deleted KLF9 (KLF9-∆DBD) constructs or control vector. Luciferase activity was measured after 24 hours incubation. For comparison, the E-cadherin-Luc activity level of control cells was set to 1. Data are presented as means ± SD (p \u0026lt; 0.05, significant; ns, not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01). (G-H) The motility of MCF-7 cells that overexpressed KLF9 and KLF9-∆DBD were evaluated by scratch wound healing assay. (I-J) Transwell assay was performed to assess the invasion and of migration in MCF-7 cells transfected with KLF9 and KLF9-∆DBD. Scale bars, 100 mm.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4005329/v1/ab85d59a998e55560894b6f1.png"},{"id":52452464,"identity":"0b4d59c9-bd83-49cc-bdb7-264b60747b3d","added_by":"auto","created_at":"2024-03-11 19:17:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16886597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLF9 affects the transcriptional regulation of E-cadherin by competing with SNAI1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Reporter-gene assay showing the regulation of E-cadherin-luciferase activity by KLF9 and SNAI1 overexpression in ZR-75-30 cells. Cells were co-transfected with E-cadherin-Luc, KLF9 and SNAI1. Luciferase activity was measured after 24 h incubation. All reporter gene bar graphs show the fold change of relative luciferase activity normalized to β-galactosidase activity. (B-C) Western blot analysis showing the expression of E-cadherin in ZR-75-30 cells were transfected without or with KLF9 and SNAI1 overexpression, E-cadherin and β-Tubulin in the cells were analyzed using the corresponding antibodies. (D-E) RT-PCR analysis showing the level E-cadherin transcript in the different groups of cells in b. The mRNA levels of E-cadherin are expressed relative to GAPDH transcripts. (F) ChIP experiments with KLF9 and SNAI1 overexpression on the\u003cem\u003e \u003c/em\u003eE-cad\u003cem\u003e-\u003c/em\u003ec (region shown in Figure 4A) promoter in ZR-75-30 cells. Without KLF9 was also evaluated for comparison purpose in which SNAI1 had been overexpressed. (G-H) The motility of ZR-75-30 cells that overexpressed SNAI1 and KLF9 were evaluated by scratch wound healing assay. (I-J) Transwell assay was performed to assess the invasion and of migration in ZR-75-30 cells transfected with KLF9 and SNAI1. Scale bars, 100 mm.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4005329/v1/8d3aad6bbccd2644926c5596.png"},{"id":53056533,"identity":"bdf53c15-a69e-4abc-ac95-ba462e1cad9c","added_by":"auto","created_at":"2024-03-20 06:23:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2555069,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4005329/v1/cdb031f5-8db6-4a97-9586-ff0fe8d51231.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"KLF9 inhibits breast cancer metastasis by up-regulating E-cadherin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among females(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The majority of deaths from breast cancer are not due to the primary tumor, but the result of metastasis at distant sites in the body(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Recent studies have shown that aberrant activation of epithelial\u0026ndash;mesenchymal transition (EMT) has been implicated in this process(\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). During this process, epithelial tumor cells may lose cell-to-cell adhesion and polarity characteristics, accompanied by cytoskeleton rearrangements(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). They may acquire a motile and invasive fibroblast-like mesenchymal phenotype(\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Silencing of E-cadherin gene or protein expression in epithelial cells is sufficient to induce a full EMT, because direct suppression of E-cadherin causes a decrease in cell-cell adhesion and increased invasion and motility(\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Hence, E-cadherin is seemed as a hallmark of EMT(\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eE-cadherin is the prototypical cadherin, mediating cell\u0026ndash;cell adhesion complexes anchored to the actin cytoskeleton via its cytoplasmic domain and β-catenin and α-catenin, thereby forming the core of the epithelial adherens junction(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Several transcription factors have been implicated in the repression of E-cadherin expression, including SNAI1(\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), SNAI2(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), ZEB1(\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and ZEB2(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), all of which promote EMT through direct binding to the E-box of the E-cadherin promoter. SNAI1 has been one of the important classical EMT transcription factors in cancer research(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). It mediates the repression of E-cadherin by direct recruitment of a co-repressor complex containing HDAC1 and HDAC2 (HDAC1/2)(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and Sin3A(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). EMT is modulated by many factors, finding and understanding the regulation of novel factors would provide important insight into the molecular mechanisms implicated in EMT.\u003c/p\u003e \u003cp\u003eKr\u0026uuml;ppel-like factor 9 (KLF9) is a member of the KLF family of transcription factors. All members of the KLF family have three highly conserved zinc finger motifs DNA-binding domain (DNA-binding domain, DBD) at the C-terminal of the proteins, recognizing GC-rich sequences with a preference for the 5\u0026prime;-CACCC-3\u0026prime; core motif in the promoters and enhancers(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Expression profiling of migrated and invaded breast cancer cells reveals KLF9 as a potential suppressor of invasion in breast cancer(\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Recently, our studies have found KLF9 could inhibit breast cancer metastasis by transcriptionally inhibiting MMP9(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). However, detailed molecular mechanism by which KLF9 regulates EMT remains largely unknown.\u003c/p\u003e \u003cp\u003eHere, we showed that KLF9 was at a low expression level in breast cancer patients through the analysis of transcriptome data in TCGA database and immunohistochemistry and we found that KLF9 inhibited the metastasis of breast cancer cells, at least in part, by up-regulating E-cadherin expression. Mechanistically, KLF9 promoted the transcription level of E-cadherin by binding to the CACCC motif of E-cadherin promoter. We also concluded that KLF9 could compete with SNAI1 to bind to the promoter region of E-cadherin and inhibit the transcriptional activity of SNAI1, leading to the activation E-cadherin in breast cancer cells. These studies indicate that KLF9 play an important role in restraining metastatic activity of breast cancers.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eData acquisition\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTCGA(The Cancer Genome Atlas) is a cancer genome project launched by the National Cancer Institute and the National Human Genome Research Institute in 2005, which is dedicated to collecting all tumor genome data, and it is a landmark successful attempt(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The Cancer Genome Atlas (TCGA) data is public repository. The database contains genome (gene expression, variation, methylation, single nucleotide polymorphism, etc.), transcriptome (mRNA, miRNA expression, etc.), epigenetics, proteome and other data of 33 kinds of human tumors(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).The publicly available cancer genome data set provided by TCGA provides convenience for researchers to further explore tumor-related mechanisms and new ideas of anti-tumor diagnosis and treatment. Its data can be obtained by visiting \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, and TCGA data is also included on UCSC Xena website.\u003c/p\u003e \u003cp\u003eRNA sequence transcriptome data and clinical information for 1096 Breast Cancer and 112 non-tumor counterparts were acquired via TCGA.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eDownload the gene expression data set (gene expression rnaseq-illuminahiseq) and patient phenotype data set of TCGA Breast Cancer from the website, and import them into R software for processing. The relevant data of KLF9 (Ensemble ID: ENSG00000119138) were selected to screen and sort out the data, remove the missing values and standardize the gene expression data. According to the sample type, pathological grade of breast cancer and PAM50 subtype of breast cancer, the data of patients were classified and the expression difference was analyzed. Kaplan-Meier survival curve was drawn by statistical software, and the results were statistically tested by Log-rank test. Correlation analysis was carried out on the expression data sets of different genes. All data processing was done in the R language (version 4.2.1). All statistical P values were two-tailed, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as statistical significance.\u003c/p\u003e \u003cp\u003eData were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs from at least three independent experiments. Un-paired t-test was used when the results from two groups were compared. Statistical analyses were carried out by one-way analysis of variance with Bonferroni's multiple comparison correction for comparison among three or more groups. Statistical significance was considered at the p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemistry (IHC)\u003c/b\u003e \u003c/p\u003e \u003cp\u003e All experimental protocols for this study were performed in accordance with established guidelines and regulations from the Biological and Medical Ethics Committee of Dalian University of Technology (NO. 2020\u0026ndash;027). A total of 20 samples comprising 4 normal tissues and 16 breast cancer tissues were examined. Paraffin-embedded tissues were retrieved from department of pathology of Liaoning Cancer Hospital, and tissue microarray slides were prepared according to a published method. Anti-KLF9 antibodies were used for immunohistochemistry. The samples were fixed in 4% para- formaldehyde (Solarbio, Beijing, CHN) for 24\u0026ndash;48 h and then embedded in paraffin. The samples were sectioned into 5-\u0026micro;m sections and stained with antibody as previously described. Images of the samples were obtained at 100\u0026times; magnification (Nikon, Tokyo, JPN) and analyzed by ImageJ (NIH, Bethesda, MD, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and transfection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHuman breast cancer cell line MCF-7, T47D, ZR-75-30 have been used in our previous study and were cultured as previously described(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Cells were grown in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃, and transfected by Lipofectamine 2000 (Invitrogen, Auckland, New Zealand) according to the manufacturer\u0026rsquo;s specifications.\u003c/p\u003e \u003cp\u003eMCF-7 human breast cancer cell (product number: SNL-060) was purchased from WuHan SHANER Biotechnology Company(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.sunnbio.com/\u003c/span\u003e\u003cspan address=\"http://www.sunnbio.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). T47D human breast ductal cancer cell (product number: BNCC339608) and ZR-75-30 human breast cancer cell (product number: BNCC100125)were purchased from Bena Biological Company༈\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bncc.com/༉\u003c/span\u003e\u003cspan address=\"https://www.bncc.com/༉\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmids and antibodies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHuman KLF9 was cloned from a human cDNA library using the forward primer KLF9-F:5\u0026rsquo;-cggaattcctccgcggccgcctac-3\u0026rsquo; and the reverse primer KLF9-R:5\u0026rsquo;-ccgctcgagcggtcacaaagcgttggc-3\u0026rsquo; and the amplified KLF9 DNA fragment was inserted into the expression vector pcDNA3.1-3\u0026times;Flag at the EcoRI and XhoI sites. Flag-tagged truncated KLF9 (KLF9-∆DBD) was constructed according to standard PCR-based cloning procedure using Flag-KLF9 plasmid as a template. PCR fragments were inserted into pcDNA3.1-3\u0026times;Flag at the EcoRI and XhoI sites. pEGFP-SNAI1, pGL3 vector and pGL3-E-cadherin-Luc were acquired as previously described(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Small interfering RNAs (siRNAs) corresponding to the following sequences for knockdown of Ctrl and KLF9 were synthesized in GenePharma (Shanghai, China): siCtrl: 5\u0026rsquo;-UUCUCCGAACGUGUCACGUTT-3\u0026rsquo; (forward) and 5\u0026prime;-ACGUGACACGUUCGGAGAATT-3\u0026prime;(reverse); siKLF9: 5\u0026rsquo;-GGCCCUUUCCCUGCACGTT-3\u0026rsquo; (forward) and 5\u0026prime;-CGUGCAGGGAAAGGGCCTT \u0026minus;\u0026thinsp;3\u0026prime; (reverse).\u003c/p\u003e \u003cp\u003eRabbit anti-Flag, anti-GFP, anti-KLF9 antibodies were purchased from Sigma (Sigma, Saint Louis, MO, USA). Mouse anti-E-cadherin was obtained from Abcam (Abcam, Cambridge, MA, USA). Rabbit anti-SNAI1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, CA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLuciferase reporter assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePromoter activity was determined by a luciferase assay system. ZR-75-30 cells were seeded in a 24-well plate at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e and cultured for 24 h. Then the cells were transfected with corresponding plasmids using Lipofectamine 2000 according to the company\u0026rsquo;s specification. Twenty-four hours after transfection, luciferase activity was determined using Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction and RT‑PCR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal RNA was extracted from ZR-75-30 cells using Takara RNAiso Reagent (Takara, Dalian, China). Total RNA (3\u0026micro;g) was reverse transcribed by oligo (dT) primer using a Reverse Transcription System (Takara). The single-stranded cDNA was amplified by PCR using specific primers: E-cadherin:5\u0026prime;-CTTCGGAGGAGAGCGGTG-3\u0026prime; (forward) and 5\u0026prime;-CTAGTCGTCCTCGCCGCC-3\u0026prime; (reverse); SNAI1: 5\u0026prime;-CGCGCTCTTTCCTCGTCAG-3\u0026prime; (forward) and 5\u0026prime;-TCCCAGATGAGCATTGGCAG-3\u0026prime; (reverse); KLF9: 5\u0026prime;-AACTGCTTTTCCCCAGTGTG-3\u0026prime;(forward) and 5\u0026prime;-TCCCATCTCAAAGCCCATTA-3\u0026prime; (reverse); GAPDH:5\u0026prime;-TGAAGGTCGGAGTCAACGG \u0026minus;\u0026thinsp;3\u0026prime; (forward) and 5\u0026prime;- CCTGGAAGATGGTGATGGG \u0026minus;\u0026thinsp;3\u0026prime; (reverse). The PCR products were analyzed by electrophoresis using a 1.5% agarose gel.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReal-time PCR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAppropriate plasmids were transfected into ZR-75-30 cells and 24 h after transfection, the total cDNA was synthesized as described above. Relative mRNA levels were determined using the ABI Prism 7500 sequence detection system with SYBR premix Ex Taq (Takara) as previously described(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Expression of target genes was determined according to the 2-∆∆ CT method using GAPDH as a reference gene. The following primer sequences were used: E-cadherin: 5\u0026prime;-CTTCGGAGGAGAGCGGTG-3\u0026prime; (forward) and 5\u0026prime;-CTAGTCGTCCTCGCCGCC-3\u0026prime; (reverse); SNAI1: 5\u0026prime;- CGCGCTCTTTCCTCGTCAG \u0026minus;\u0026thinsp;3\u0026prime; (forward) and 5\u0026prime;-TCCCAGATGAGCATTGGCAG-3\u0026prime; (reverse); KLF9: 5\u0026prime;-AACTGCTTTTCCCCAGTGTG-3\u0026prime; (forward) and 5\u0026prime;-TCCCATCTCAAAGCCCATTA-3\u0026prime; (reverse); and GAPDH: 5\u0026prime;-TGAAGGTCGGAGTCAACGG-3\u0026prime; (forward) and 5\u0026prime;- CCTGGAAGATGGTGATGGG \u0026minus;\u0026thinsp;3\u0026prime; (reverse).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot, ChIP\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWestern blot and chromatin immunoprecipitation (ChIP) assays were conducted as previously described(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The primers of E-cadherin promoter used in the ChIP PCR analysis were as follows: 5\u0026prime;-AGAGGGTCACCGCGTCTATG-3\u0026prime; (forward) and 5\u0026prime;-TCCGCAAGCTCACAGG-3\u0026prime; (reverse) for E-cad-c and 5\u0026prime;- TGAAGGTCGGAGTCAACGG \u0026minus;\u0026thinsp;3\u0026prime; (forward) and 5\u0026prime;- CCTGGAAGATGGTGATGGG \u0026minus;\u0026thinsp;3\u0026prime; (reverse) for GAPDH.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranswell assays\u003c/b\u003e \u003c/p\u003e \u003cp\u003eZR-75-30 were transfected with appropriate plasmids. After 24h transfection, the cells were suspended in serum-free medium and counted. The suspension containing 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells was inoculated in the upper chamber with (invasion) or without (migration) Matrigel (BD Biosciences, San Jose, CA, USA). After 24 or 48 h, cells remained on the top surface of the filter were removed with a cotton swab, while cells migrated or invaded into the bottom surface were washed with PBS, fixed with 4% paraformaldehyde for 15 min. Cells were stained with Crystal Violet solution for 20 min and then photographed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWound‑healing assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eScratch wound-healing were performed as previously described(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).Transfected cells were seeded into the six-well plates cells with a density of 3.0\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells. And after 24 h, multiple wounds were created by using a 200-\u0026micro;l pipette tip. Photographs were recorded at 0 h and 24 h and the wound healing was measured from these images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs from at least three independent experiments. Un-paired t-test was used when the results from two groups were compared. Statistical analyses were carried out by one-way analysis of variance with Bonferroni's multiple comparison correction for comparison among three or more groups. Statistical significance was considered at the p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDown-regulation of KLF9 RNA and protein levels in breast cancer patients.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the potential role of KLF9 in the occurrence and development of breast cancer, we downloaded RNA sequence transcriptome data and clinical information data of 1096 breast cancer cases and 112 corresponding normal tissues from TCGA database. Next, the differential gene expression data and clinical information data of breast cancer tissues and normal tissues are analyzed. The results showed that the expression level of KLF9 in breast cancer tissues was lower than that in normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To explore whether the expression of KLF9 is related to the clinical stage of patients, we analyzed the relevant data. The results showed that the expression of KLF9 in breast cancer tissue samples was related to the clinical stages of breast cancer patients, and the expression of KLF9 in late clinical patients was lower than that in early clinical patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We also analyzed the expression of KLF9 in patients of different age groups, and found that the expression of KLF9 in patients aged\u0026thinsp;\u0026gt;\u0026thinsp;65 was lower than that in patients aged\u0026thinsp;\u0026le;\u0026thinsp;65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTNM staging system is the most common tumor staging system in the world at present. TNM staging system was put forward by Frenchman Pierre Denoix from 1943 to 1952, and then gradually improved by American Joint Committee on Cancer (AJCC) and union for international cancer control (UICC). In 1968, the first edition of TNM Classification of Malignant Tumors was officially published. The TNM staging system of each tumor is different, so the meaning of letters and numbers in TNM staging is different in different tumors. At present, TNM staging system has become the standard method for clinicians and medical scientists to stage malignant tumors(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In TNM staging system, \"T\" (tumor) refers to the situation of the primary tumor, which is represented by T1\u0026thinsp;~\u0026thinsp;T4 in turn with the increase of tumor volume and adjacent tissue involvement. \"N\" (node) refers to the involvement of Regional Lymph Node. When lymph nodes are not involved, they are denoted by N0, and with the increase of the degree and scope of lymph node involvement, they are denoted by N1\u0026thinsp;~\u0026thinsp;N3 in turn. \"M\" (Metastasis) refers to distant metastasis (usually hematogenous metastasis). Those without distant metastasis are denoted by M0, and those with distant metastasis are denoted by M1. Therefore, a specific tumor stage can be expressed by the combination of three indicators of TNM. The TNM stage of breast cancer is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). The results showed that the expression of KLF9 in breast cancer tissue samples was significantly correlated with some different T stages, different N stages and different M stages of breast cancer patients, and the low expression of KLF9 in breast cancer tissue was associated with tumor volume increase and distant metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTNM staging of breast cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT(Tumor)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN(Lymph Node)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM(Metastasis)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT0: No primary cancer was detected.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN0: There are no enlarged lymph nodes in the ipsilateral armpit.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM0: No distant metastasis.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTis: Preinvasive carcinoma.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN1: There are enlarged lymph nodes in the ipsilateral armpit, which can be pushed.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM1: Distant metastasis.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1: The tumor size is 0\u0026minus;2cm.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN2: The enlarged lymph nodes in the ipsilateral armpit fuse or adhere to the surrounding tissues.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2: The tumor size is 2\u0026minus;5cm.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN3: There were ipsilateral parasternal lymph node metastasis and ipsilateral supraclavicular lymph node metastasis.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3: Tumors larger than 5cm.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT4: The tumor has penetrated the skin or attached to the chest wall.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAt present, the diagnosis of breast cancer mainly depends on the combination of clinical physical examination, imaging auxiliary examination and pathological examination, and pathological examination is still the main \"gold standard\" of diagnosis. The expression of Estrogen Receptor (ER), Progesterone Receptor (PR), human epidermal growth factor-2 (HER-2) and Ki67 in cells were detected by whole gene expression profile, and breast cancer was divided into five subtypes. They are Basal-like, HER2 overexpression (HER2+), Luminal-A, Luminal-B and Normal like(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Luminal-A breast cancer is hormone receptor positive (estrogen receptor or progesterone receptor positive), HER2 negative, and the protein Ki-67 level is low, which helps to control the growth rate of cancer cells. Luminal-A cancer is a low-grade cancer, which tends to grow slowly and has the best prognosis. Luminal-B breast cancer is hormone receptor positive (estrogen receptor or progesterone receptor positive), HER2 positive or HER2 negative, and the protein Ki-67 level. Luminal-B cancer usually grows slightly faster than luminal-A cancer, and its prognosis is slightly worse. Basal breast cancer is hormone receptor negative (estrogen receptor and progesterone receptor negative) and HER2 negative, so it is also called Triple-negative breast cancer. This type of cancer is more common in women with BRCA1 gene mutation. HER2 overexpression breast cancer is hormone receptor negative (estrogen receptor and progesterone receptor negative) and HER2 positive. HER2-enriched cancers tend to grow faster and have a worse prognosis than intracavitary cancers. For this kind of breast cancer, targeted therapy for HER2 protein is usually used in combination with corresponding drugs, such as Herceptin (chemical name: trastuzumab), Perjeta (chemical name: pertuzumab), Tykerb (chemical name: lapatinib), Nerlynx (chemical name: neratinib) and Kadcyla (chemical name: T-DM1 or ADO). Normal breast cancer, similar to Luminal-A, is hormone receptor positive (estrogen receptor or progesterone receptor positive), HER2 negative, and the level of protein Ki-67 is low, which helps to control the growth of cancer cells. However, although the prognosis of normal breast cancer is good, its prognosis is slightly lower than that of lumen cancer(\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). See Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for the classification of breast cancer subtypes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSubtype of breast cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubtype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHER2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKi\u0026minus;67\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePrognosis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuminal-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBetter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuminal-B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAny\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAny\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePoor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriple-negative/Basal-like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAny\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePoor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNext, we explore whether the expression of KLF9 is related to the subtypes of breast cancer patients. The results showed that the expression of KLF9 was significantly different among different subtypes, and there was a certain relationship between the low expression of KLF9 in breast cancer samples and the poor prognosis of breast cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Kaplan-Meier survival curve of breast cancer patients was generated based on TCGA data set. The results showed that there was no significant correlation between KLF9 expression in breast cancer samples and the overall survival rate of patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). The correlation analysis of gene expression between KLF9 and CDH1 (encoding E-cadherin) showed that there was a significant negative correlation between the expression levels of KLF9 and CDH1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eTwenty tissue samples of 4 breast cancer adjacent tissues and 16 breast cancer tissues were specifically labeled with anti-KLF9 antibody, and the images of the samples were obtained at 100\u0026times; magnification. It is obvious from the image that the expression of KLF9 in breast cancer tissue is lower than that in breast cancer adjacent tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). We then randomly select the images of cell areas from each IHC slice, and use Image-J software to quantify the average optical density (AOD) of the images. The AOD data is plotted and visualized in the GraphPad Prism 9.4.0 software. The results showed that the AOD value of breast cancer tissue was significantly lower than that of breast cancer adjacent tissue, which indicated that the expression of KLF9 protein in breast cancer tissue was significantly lower than that in breast cancer adjacent tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eThrough the analysis of transcriptome data and immunohistochemistry in TCGA database, we found that KLF9 was at a low expression level in breast cancer patients. Therefore, we have reason to believe that KLF9 has a certain correlation with the occurrence of breast cancer, and we will continue to explore it through follow-up experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKLF9 inhibits the migration and invasion of human breast cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRecent studies have shown that KLF9 acts as a cancer-inhibitory effector in breast cancer cells(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The diffusion of tumor cells is often related to Epithelial interstitial transformation (EMT) process. EMT is a multifunctional cell process, which is accompanied by the characteristics of cell polarity loss, adhesion decline, cytoskeleton change, cell migration and cell mobility enhancement. Among them, the gradual decrease of E-cadherin expression on the cell surface is an important feature of EMT process. Besides, KLF9 could transcriptionally down-regulate MMP9 expression and inhibited the metastasis of breast cancer cells(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). However, the relationship between KLF9 and E-cadherin, an important marker of EMT, remains largely unknown.\u003c/p\u003e \u003cp\u003eIn order to explore the association between KLF9 and E-cadherin in the occurrence and development of breast cancer, we determined the protein level in breast cancer cell lines containing MCF-7, T47D, ZR-75-30 cells. Western blot analysis revealed almost no endogenous KLF9 was detected in ZR-75-30 cells, which displayed mesenchymal-like morphology and lesser cell\u0026ndash;cell adhesion. Relatively higher-level expression of KLF9 was identified in MCF-7 cells than in T47D cells, consistent with the expression of E-cadherin in breast cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The protein expression of KLF9 in ZR-75-30 breast cancer cell line of Luminal-B subtype is lower than that of T47D and MCF7 breast cancer cell lines of Luminal-A subtype, which is consistent with the transcription level analysis of KLF9 in different breast cancer subtypes in TCGA database. These studies suggested that KLF9 expression is higher in non-invasive cancer cells and may inhibit breast cancer by increasing cell-cell adhesion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the role of KLF9 in cell motility in breast cancer cells, scratch wound-healing and transwell assays were carried out to demonstrate the migration and invasion capacity. Compared with control cells, overexpressing of KLF9 markedly inhibited cell motility (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and reduced the cell numbers that migrated through the Matrigel-coated membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) in ZR-75-30 cells. In contrast, knockdown of endogenous KLF9 in ZR-75-30 cells significantly enhanced cell migration ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) and increased the capacity of cells to traverse the Matrigel-coated membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ), compared with control cells. These results indicated that KLF9 can inhibit the invasion and metastasis in breast cancer cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKLF9 increases E-cadherin expression via transcriptional activation in breast cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs E-cadherin is an important inhibitor in the process of EMT(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e), which has the same function as KLF9, we speculate whether KLF9 inhibits breast cancer metastasis by regulating the expression of E-cadherin. As we predicted, Reverse transcription-PCR(RT-PCR) showed that overexpression of KLF9 increased the mRNA level of E-cadherin compared with the control group in ZR-75-30 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, the mRNA level of E-cadherin decreased after siKLF9 (pRNA T-U6.1 vector) was transfected in ZR-75-30 cells, suggesting that KLF9 could promote the transcription of E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Western blot results showed that increased level of E-cadherin protein by overexpression of KLF9, and decreased level of E-cadherin protein following siKLF9 (pRNAT-U6.1 vector), consistent with the change of mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These data clearly indicated that increased E-cadherin protein expression was mediated by KLF9, occurred mainly at the mRNA level regulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLuciferase reporter gene assay also revealed that increasing doses of KLF9 induced a dose-dependent growth of E-cadherin-promoter-driven luciferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), while knockdown of KLF9 resulted in a decrease in E-cadherin-promoter-driven luciferase activity in ZR-75-30 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These data showed that KLF9 could activate the E-cadherin promoter.\u003c/p\u003e \u003cp\u003eTaken together, these results indicated that KLF9 could up-regulate the level of E-cadherin protein by transcriptionally activating the activity of the E-cadherin promoter.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKLF9 binds to the CACCC motif of the E-cadherin promoter through its DNA binding domain\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the specific sites and regions of KLF9 acting on E-cadherin promoter, we constructed four truncated E-cadherin promoter and fused each promoter with luciferase reporter gene to yield a reporter construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe reporter construct comprising E-cad-a promoter (-999\u0026thinsp;~\u0026thinsp;+\u0026thinsp;47) increased more than two-fold of the reporter activity when ZR-75-30 cells overexpressed KLF9 compared with control. Interestingly, the reporter activity disappeared when deleted nucleotide from \u0026minus;\u0026thinsp;206 to +\u0026thinsp;47 (E-cad-b), suggesting that KLF9 binding site might be existed in nucleotide \u0026minus;\u0026thinsp;206 to +\u0026thinsp;47. The result showed that after the deletion of nucleotide \u0026minus;\u0026thinsp;999 to -206 (E-cad-c), the activation multiple of KLF9 overexpression was almost the same as that of the E-cad-a, which proved that nucleotide \u0026minus;\u0026thinsp;999 to -206 (E-cad-b) probably contained no regulatory element. These data indicate that nucleotide \u0026minus;\u0026thinsp;206 to +\u0026thinsp;47 (E-cad-c) on the E-cadherin promoter was contain response elements required for KLF9 to activate its transcriptional activity. Furthermore, due to KLF9 recognize sequences with a preference for the 5\u0026prime;-CACCC-3\u0026prime; core motif in the promoters and enhancers(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), nucleotide \u0026minus;\u0026thinsp;12 to +\u0026thinsp;8 \"CACCC\" located within nucleotide \u0026minus;\u0026thinsp;206 to +\u0026thinsp;47 was mutated into \"CATTT\" (E-cad-d), the activation of KLF9 was almost completely lost, indicating that KLF9 was exactly bound to the \"CACCC\" sequence of E-cadherin promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSince KLF9 DNA-binding domain (zinc finger domain, ZNF) could recognize the CACCC element, we speculated that DNA-binding domain of KLF9 might bind to the E-cadherin promoter to regulate the transcription activity of E-cadherin. Therefore, to verify this hypothesis, we constructed a deletion of KLF9 DNA binding domain (pcDNA3.1-3\u0026times;Flag-KLF9△DBD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). CHIP experiment further showed that KLF9 can specifically bind to the E-cad-c promoter, which included \"CACCC\" sequence, consistent with the results of previous reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, top). KLF9-∆DBD cannot achieve specific binding to E-cad-c promoter. We also amplified the GAPDH fragment as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, bottom). Taken together, these results suggest that KLF9 may promote E-cadherin transcription through its DNA-binding domain (DBD) interaction with CACCC motif located within nucleotides \u0026minus;\u0026thinsp;206 to +\u0026thinsp;47 of E-cadherin promoters. Consistently, compared to full length KLF9, the activation of E-cad\u003cem\u003e-c\u003c/em\u003e promoter by KLF9△DBD was disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF ), suggesting that the DNA-binding domain (DBD) of KLF9 might be responsible for the E-cadherin promotion. This was subsequently confirmed by ChIP assay. ChIP assay also showed that the KLF9△DBD experimental group, compared with the wild-type KLF9, did not detect specific DNA bands on E-cad\u003cem\u003e-c\u003c/em\u003e promoter, indicating that KLF9 DNA-binding domain specifically bind to E-cad-c promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eCollectively, these results suggested that KLF9 binds to the CACCC motif of E-cadherin promoter through its DNA binding domain and the DNA-binding domain of KLF9 is essential for its tumor-suppressive role in breast cancer cells.\u003c/p\u003e \u003cp\u003eFurthermore, MCF-7 cells were tested for their motility, migration ability and invasion ability by wound healing and transwell experiment respectively. When KLF9 is overexpressed, the migration ability of cells is obviously reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), and the ability of cells to pass through the matrix gel coating film is also reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). However, when Flag-KLF9-△DBD was overexpressed, compared with the control group, the migration ability of cells did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), and the ability of cells to pass through the matrix gel coating film remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). To sum up, after deleting the DNA binding region of KLF9, the inhibitory effect of overexpression of KLF9 on the motility, migration and invasion of MCF7 cells disappeared, which proved that the DNA binding region of KLF9 was the necessary region to inhibit the motility, migration and invasion of breast cancer cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKLF9 affects the transcriptional regulation of E-cadherin by competing with SNAI1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe transcription factor SNAI1 has been recognized as a direct repressor of E-cadherin expression, which recruits HDAC and the corepressor mSin3A to form a multimolecular complex to repress E-cadherin(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Because of the target sites located in E-cad-c, we speculated that KLF9 may be involved in coordinating the interaction between SNAI1 and E-cadherin.\u003c/p\u003e \u003cp\u003eFirstly, we utilized luciferase reporter detection to further evaluate our hypothesis. Overexpression of SNAI1 alone inhibited E-cadherin-Luc activity, whereas overexpression of SNAI1 and KLF9 relieved this inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Moreover, western blot results also showed that overexpression of SNAI1 alone repressed the expression of E-cadherin protein, whereas overexpression of SNAI1 and KLF9 relieved this repression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Quantitative RT-PCR showed that E-cadherin mRNA levels were significantly decreased after transfection of SNAI1 alone, and KLF9 partially rescued the decrease of E-cadherin mRNA, consistent with the change of protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether KLF9 affects the regulation of E-cadherin promoter by SNAI1, ChIP assays were carried out using ZR-75-30 cells. Overexpression of KLF9 effectively prevented the recruitment of SNAI1 to the E-cad\u003cem\u003e-\u003c/em\u003ec promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Taken together, these findings revealed that KLF9 may enhance the activity of E-cadherin by alleviating its transcriptional suppression exerted by SNAI1.\u003c/p\u003e \u003cp\u003eTo further confirm, using scratch wound-healing and transwell assays to demonstrate the cell motility, migration and invasion capacity respectively in ZR-75-30 cells. When the ZR-75-30 cells co-overexpressed KLF9 and SNAI1, the migration ability of cells are significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), and the ability of cells to pass through the matrix gel coating film are reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), compared to the cells expressed SNAI1 alone. Collectively, these results indicate that SNAI1 is involved in KLF9-mediated suppression of breast cancer invasion and metastasis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThrough the analysis of TCGA database and immunohistochemistry, we found that the RNA and protein expression levels of KLF9 in breast cancer patients were lower than those in non-breast cancer patients. The expression of KLF9 in breast cancer tissue samples is related to the clinical stages of breast cancer patients, and the expression of KLF9 in patients with advanced clinical stage is lower than that in patients with early clinical stage. The expression of KLF9 was significantly different among different subtypes, and there was a certain relationship between the low expression of KLF9 in breast cancer samples and the poor prognosis of breast cancer patients. In this study, we identified that E-cadherin is a novel KLF9 transcriptional target gene, which may provide a new mechanism for KLF9 in the regulation of human breast cancer metastases. First, KLF9 inhibits migration and invasion in breast cancer cells. Second, KLF9 could up-regulate E-cadherin expression, depending on DBD domain, by promoting the transcription level of E-cadherin and such promotion is via CACCC motif in the promoter. Third, we found the underlying mechanism is that KLF9 could compete with SNAI1 to bind the E-cadherin promoter, further activating the expression of E-cadherin. Finally, we demonstrated that KLF9 could suppress the invasiveness of breast cancer cells, at least in part, through up-regulating the expression and activity of E-cadherin. This study revealed the important role for KLF9 in regulating the invasion and metastasis of ER-positive breast cancer cells, providing further theoretical support for the treatment and prevention of ER-positive breast cancer, and we plan to select ER-negative breast cancer cell lines for related experimental exploration in the future.\u003c/p\u003e \u003cp\u003eBreast cancer has surpassed lung cancer to become the most common cancer in the world, and the invasion and metastasis of breast cancer are the main reasons for the poor prognosis and low survival rate of patients(\u003cspan additionalcitationids=\"CR58 CR59\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Studies have shown that EMT is associated with increased cell migration, invasion, and metastasis(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). E-cadherin, the cellular adhesion molecule that forms the cell\u0026ndash;cell adhesion junctions of epithelial cells, is essential for the cells to maintain their epithelial phenotype(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). During the EMT, E-cadherin is cleaved at the plasma membrane and subsequently degraded, resulting in loss of epithelial adherent junctions(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Thus, downregulation or loss of E-cadherin expression is considered to be the hallmark event of EMT(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). The first discovered and most important transcriptional repressor of E-cadherin is SNAI1(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).SNAI1 repressed E-cadherin expression by interacting with a co-repressor complex SIN3A/HDAC1/HDAC2 and modification of local chromatin structure(29, 65). In the present study, we found that the KLF9 alleviated the transcriptional inhibition and protein downregulation of E-cadherin by SNAI1. Whether KLF9 regulates the expression of E-cadherin by binding to the E-box of the promoter requires further study. Therefore, we suspected that KLF9 may compete with SNAI1 to regulate E-cadherin. Further study is needed to clarify this issue.\u003c/p\u003e \u003cp\u003eThe Kr\u0026uuml;ppel-like transcription factor family may emerge as a new type of transcription factors that control the transcription of E-cadherin. KLF4 binds to and activates the E-cadherin promoter, which is necessary to maintain the epithelial phenotype in mammary epithelial cells(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Indeed, KLF6, another KLF member and tumor suppressor, has been shown to directly bind and activate E-cadherin gene promoter in ovarian cancer(\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). KLF8 is a novel repressor of E-cadherin in epithelial cells and plays a large part in the loss of E-cadherin expression in human breast carcinoma cells and their invasiveness(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). KLF6 activates E-cadherin transcription similar to KLF4 while KLF8 binding results in repression of the E-cadherin promoter. These data demonstrate the distinct abilities of individual Kr\u0026uuml;ppel-like factors to modulate expression of the same target genes. As suggested in our study, E-cadherin is a novel transcriptional target of KLF9 and KLF9 could directly bind to the CACCC motif of E-cadherin promoter to increase E-cadherin expression. This regulation is necessary to inhibit the migration and invasion capabilities of breast cancer cells.\u003c/p\u003e \u003cp\u003eConsistent with our research, KLF9 has been regarded as a transcription repressor in several types of tumor. For example, in colorectal cancer, KLF9 may as a transcriptional repressor of a module of IFN-stimulated genes and specifically ISG15 to prevent tumor cell survival and growth by promoting apoptosis(\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). In Esophageal squamous cell carcinoma, KLF9 inhibited the cancer by regulating Cyr61 and negatively modulating the beta-catenin/TCF signaling(\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). KLF9 suppresses gastric cancer metastasis through directly inhibiting transcriptional expression of MMP28(\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). Nevertheless, KLF9 in ovarian cancer may be different from other cancer. Expression of KLF9 is up-regulated in ovarian cancer and knockdown KLF9 by lentivirus inhibits the growth of ovarian cancer cell(\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). It indicates KLF9 plays different roles in different cancer tissues.\u003c/p\u003e \u003cp\u003eFurthermore, KLF9 has been found to play a potential suppressor role in breast cancer(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The researchers found that the expression of KLF9 was significantly lower in breast cancer tissue than that in normal tissue, suggesting that KLF9 may be a potential breast cancer metastasis inhibitory factor. Recent studies have shown that KLF9 suppressed the metastasis of breast cancer by down-regulating the expression of MMP9(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Our study further confirmed KLF9 could inhibit breast cancer cells by promoting E-cadherin expression and competing with snail to bind to the promoter region(-206\u0026thinsp;~\u0026thinsp;+\u0026thinsp;47)of E-cadherin. Although our results indicate that E-cadherin transcription is promoted by KLF9 during breast cancer metastasis, we speculate that might other target genes are involved in this process due to the complexity of TF regulatory networks. Therefore, further research is required to clarify the transcriptional regulation network mediated by KLF9 during breast cancer metastasis.\u003c/p\u003e \u003cp\u003eIn summary, our study reported KLF9 could inhibit breast cancer metastasis by increasing the expression of E-cadherin at mRNA and protein levels. Further mechanistic details that came to KLF9 binds to the CACCC motif of E-cadherin promoter. Furthermore, forced expression of KLF9 in highly expressed SNAI1 was sufficient to rescue E-cadherin expression and cell motility and invasion. These findings define E-cadherin regulation by KLF9 as a potentially critical pathway to prevent epithelial-to-mesenchymal transition and support a metastasis suppressive role for KLF9 in breast cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used, analyzed, or both during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants 81301504 to\u0026nbsp;Miao Wang\u0026nbsp;from National Natural Science Foundation of China, grants DUT21LK26 to\u0026nbsp;Miao Wang\u0026nbsp;from the Fundamental Research Funds for the Central Universities, and grants\u0026nbsp;XMMC-FCTM202105 to\u0026nbsp;Mei Zhang\u0026nbsp;from the\u0026nbsp;Key laboratory of functional and clinical translational medicine\u0026nbsp;for\u0026nbsp;Fujian province university.\u0026nbsp;The funders had no roles in study design, data collection, and analysis, the decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiao Wang, Mengyao Pang and Jie Zhang conceived and designed the experiments; Miao Wang, Mengyao Pang, Jie Zhang, Mengjie Zhang, Rui Ni, Mei Zhang, Ranru Wei, Guohui Li, Ying Tang, Liming Ma and Xiaoyan Li performed the experiments; Miao Wang, Mengyao Pang, Jie Zhang, Mengjie Zhang, Rui Ni, Mei Zhang, Ranru Wei, Guohui Li, Ying Tang, Liming Ma and Xiaoyan Li analyzed the data; Miao Wan, Mengyao Pang and Mei Zhang wrote the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, data in Fig.1 were downloaded from TCGA database (https://portal.gdc.cancer.gov/). We confirmed that all data are original and have not been published elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin [Journal Article]. 2018;68(6):394\u0026ndash;424. 2018-11-01.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeigelt B, van Peterse JL. T VL. Breast cancer metastasis: markers and models. NAT REV CANCER. 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[Journal Article]. 2015 2015-02-01;291(2):377\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Krüppel-like factor 9, Human breast cancer, E-cadherin, SNAI1","lastPublishedDoi":"10.21203/rs.3.rs-4005329/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4005329/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKr\u0026uuml;ppel-like factor 9 (KLF9) plays an inhibitory role in the process of breast cancer metastasis. The metastasis of tumor cells is often related to epithelial-mesenchymal transition (EMT). Among them, the gradual decrease of E-cadherin expression on cell surface is an important feature of EMT process. However, the concrete mechanism involved in this process remains largely unknown. In order to explore the mechanism, we have done relevant research. Through the analysis of transcriptome data in TCGA database and immunohistochemistry, we found that KLF9 was at a low expression level in breast cancer patients. The expression of KLF9 was positively correlated with the expression of E-cadherin in breast cancer cells. Functionally, KLF9 transcriptionally up-regulated E-cadherin expression and inhibited breast cancer metastasis, depending on its DBD domain. Mechanistically, KLF9 promoted E-cadherin promoter activity by binding to the CACCC motif (-12 to +\u0026thinsp;8), increasing the mRNA and protein level of E-cadherin. We also found that KLF9 can compete with SNAI1 to bind to the promoter region(-206 to +\u0026thinsp;47)of E-cadherin, and inhibit the transcriptional activity of SNAI1, leading to the activation of E-cadherin in breast cancer cells. Taken together, these results confirmed a new mechanism by which KLF9 could up-regulate E-cadherin expression to inhibit breast cancer metastasis, providing a research support for the prevention and treatment of breast cancer.\u003c/p\u003e","manuscriptTitle":"KLF9 inhibits breast cancer metastasis by up-regulating E-cadherin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 19:15:37","doi":"10.21203/rs.3.rs-4005329/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6e393eae-8837-4c9c-9d20-8a0ed63b59c9","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-20T06:15:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-11 19:15:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4005329","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4005329","identity":"rs-4005329","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}