PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression

Lung cancer is the major cause of death worldwide. Activation of oncogenes or inhibition of tumor suppressors causes cancer formation. Previous studies have indicated that PTEN, as a tumor suppressor, inhibits cancer formation. In this study, we studied the role of PTEN in EGFRL858R-induced lung cancer in vivo. Interestingly, loss of PTEN increased bronchial cell hyperplasia but decreased alveolar cell hyperplasia in EGFRL858R*PTEN-/--induced lung cancer. Systematic analysis of gene expression by RNA-seq showed that several genes related to ciliogenesis were upregulated in EGFRL858R*PTEN-/--induced lung cancer and subsequently showed that bronchial ciliated cells were hyperplastic. Several critical ciliogenesis-related genes, such as Mucin5A, DNAI2, and DNAI3, were found to be regulated by NR2F1. Next, NR2F1 was found to be inhibited by overexpression of PTEN, indicating that PTEN negatively regulates NR2F1, thereby inhibiting the expression of ciliogenesis-related genes and leading to the inhibition of bronchial cell hyperplasia during EGFRL858R-induced lung cancer progression. In addition, we also found that PTEN decreased AKT phosphorylation in A549, KRAS mutant, and H1299 cells but increased AKT phosphorylation in PC9, EGFRL858R, and H1299L858R cells, suggesting that PTEN may function as a tumor suppressor and an oncogene in lung cancers with KRAS mutation and EGFR mutation, respectively. PTEN acts as a double-edged sword that differentially regulates EGFRL858R-induced lung cancer progression in different genomic backgrounds. Understanding the PTEN in lung cancer with different genetic backgrounds will be beneficial for therapy in the future.


Introduction
Lung cancer, which includes non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), is the leading cause of cancer-related death worldwide.Lung cancer is classi ed into two main, non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC), based on their histological appearance and cellular origin.Epidermal Growth Factor Receptor (EGFR) and Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) mutations were associated with the development of NSCLC.
Pten, a well-known tumour suppressor gene, has been reported to be involved in inactivation of the PI3K/AKT signalling pathway, leading to inhibition of cell adhesion, migration, and invasion.It was observed that the absence of Pten protein expression resulted in increased phosphorylation of Akt and increased PI3K signalling in the breast cancer cell lines MDA-MB-468 and BT-549 (1).In lung cancer cells, overexpression of Pten suppressed cell proliferation and induced cell cycle arrest by reducing the Skp2 protein level (2).Recent studies have indicated that Pten may be a biomarker for the response to immunotherapy, suggesting that Pten regulates not only cancer cells but also other cell types in the tumour microenvironment (3).Understanding the role and mechanisms of Pten in cancer progression will be bene cial for the success of cancer therapies using precision medicine.
Ciliated airway epithelial cells have an elongated columnar structure and isolated contact with the basement membrane.Generally, cilia consist of a microtubule, a ciliary membrane, a basal body, and an axoneme (4,5).In lung organs, ciliated cells are located in bronchioles with club-cells, goblet cells and basal cells.Previous studies have shown that PTEN interacts with Dishevelled (DVL) to inhibit WNT pathway-mediated ciliogenesis (6).Recently, studies have indicated that ciliogenesis in the mouse trachea and ependyma was requires Pten (7,8).In Xenopus embryos with conditional knockout of Pten driven by the Foxj1 promoter, ciliogenesis was signi cantly decreased compared with that in control embryos.However, the role of Pten in ciliogenesis during cancer progression needs to be clari ed.In addition, NR2F1 has been demonstrated to promote cancer cell dormancy in several malignant tumours, thereby leading to recurrence and metastasis during cancer progression (9).Numerous studies have investigated the role of NR2F1-AS1 as a sponge to inhibit the expression of many miRNAs in turn promote the formation of several cancer (10)(11)(12).In this study, we found that Pten can negatively regulate a transcription factor, NR2F1, to decrease the expression of genes related to ciliogenesis, thereby causing bronchial epithelial cell hyperplasia.

Results
Knockout of Pten enhances bronchial cell growth but inhibits alveolar cell growth in EGFR L858R -induced lung cancer Pten function as a tumour suppressor by inhibiting the PI3K pathway (13).What is the role of Pten in bronchial and alveolar cells in EGFR L858R -induced lung cancer?In this study, we established an animal model of induced lung cancer, the EGFR L858R *Pten -/-model, in which doxycycline treatment is used to induce the formation of a complex between TetO and scgb1a1-driven rtTA to express EGFR L858R and tamoxifen treatment is used to activate scgb1a1-driven Cre to cut the Lox P sites in the 5th exon of the Pten gene, namely, its DNA binding motif (Fig. 1A).Genotyping was used to con rm the presence of EGFR L858R /rtTA, Cre and Lox P (Fig. 1B).After 2 months of induction by doxycycline treatment, the levels of Pten in the lungs of EGFR L858R *Pten -/-mice (n = 8) were decreased compared to those in the lungs of EGFR L858R mice (n = 4) (Fig. 1C and 1D).The lungs of EGFR L858R *Pten -/-and EGFR L858R mice were larger in size than those of normal (RO) mice (Fig. 1E).In studying lung tissue pathology in EGFR L858R *Pten -/- and EGFR L858R mice, we found that a larger lung area was occupied by cancer cells in EGFR L858R mice than in EGFR L858R *Pten -/-mice (Fig. 1F, a).However, when we evaluated the lung tissues in detail (Fig. 1F, b), we found that knockout of Pten increased the hyperplasia of bronchial cells but inhibited the growth of alveolar cells in mice with EGFR L858R -induced lung cancer, implying a differential effect of Pten on the tumour burden in bronchial and alveolar cells in mice with EGFR L858R -induced lung cancer.Finally, the level of the hyperplasia marker CCSP was measured in EGFR L858R *Pten -/-and EGFR L858R mice (Fig. 1G).
The data indicated that the CCSP level was increased, primarily in bronchial cells, in EGFR L858R *Pten -/- mice compared to EGFR L858R mice but was decreased in the alveolar region in the lungs.In summary, Pten might play different roles in bronchial and alveolar cells in EGFR L848R -induced lung cancer.
Functional knockout of Pten promotes ciliogenesis in EGFR L858R *Pten -/--induced lung cancer The molecular mechanism by which Pten regulates bronchial and alveolar cells in EGFR L858R -induced lung cancer was next studied (Fig. 2).First, total RNA was isolated from lung tissues of EGFR L858R *Pten -/- and EGFR L858R *Pten +/+ mice to systematically study the gene expression, and bioinformatics analysis was then performed (Fig. 2).Quality control indicated that the distribution of gene expression was similar between EGFR L858R *Pten -/-and EGFR L858R *Pten +/+ mice, suggesting that the RNA-Seq data were of good quality (Suppl.Figure 1).In Pten knockout mice, 288 genes were upregulated and 97 genes were downregulated in EGFR L858R -induced lung cancer tissues (Fig. 2A).In the Circus plot, most of the Ptenregulated genes can be seen to be involved in three pathways, namely, ciliary plasm, axoneme and cilium movement, implying that Pten might regulate ciliated bronchial cells in EGFR L858R -induced lung cancer (Fig. 2B and Supplementary Table 1).The list of the top 30 terms identi ed ALL GO enrichment analysis shows that Pten was involved in most of the pathways, such as cilium movement, microtubule-based movement, and cilium organization, indicating that Pten inhibits hyperplasia of ciliated cells in EGFR L858R -induced lung cancer (Fig. 2C).In addition, based on statistical signi cance, the dot plot of the ALL GO analysis results revealed that knockout of Pten resulted in enrichment of pathways related to ciliary organization and movement (Fig. 2D).Next, we also used Upset analysis to study the related genes in the different pathways (Fig. 2E).The data indicated that several genes were involved in most of the pathways related to cilium organization and movement (Fig. 2E).Next, the bar plot of the DEG All KEGG Enrichment Pathways analysis results suggested that loss of PTEN activated several pathways involved in circadian rhythms, drug metabolism, metabolism of xenobiotics, lysosomes and so on (Fig. 2F).Finally, the bar plot of the DEG All DisGeNET Enrichment analysis results indicated that loss of PTEN positively regulated myocardial ischaemia, lung diseases, preeclampsia, thrombosis, endothelial dysfunction and so on (Fig. 2G).In summary, loss of PTEN positively regulates a number of genes related to ciliogenesis, which may increase the hyperplasia of bronchial ciliated cells in EGFR L858R -induced lung cancer.
Next, we prepared more mice to study the effect of Pten on these cilia-related genes (Fig. 3).Indeed, the results indicated that Pten knockout signi cantly increased the mRNA levels of most of the ciliated genes, namely, Cep126, Dcdc2a, Dnaic2, Dnah5, FOXJ1, Hydin, Tctex1d4, Spag16, and Wdr63, implying that loss of Pten may be involved in ciliogenesis during EGFR L858R -induced lung cancer progression (Fig. 3A).Next, we used Alcian blue staining to study the ciliated cells among bronchial epithelial cells from mice with EGFR L858R *Pten +/+ -and EGFR L858R *Pten -/--induced lung cancer (Fig. 3B).The data indicated that Pten knockout strongly increased the Alcian blue signal in bronchial epithelial cells, suggesting that loss of Pten stimulates hyperplasia of bronchial epithelial cells in the EGFR L858R *Pten -/-mouse model of lung cancer (Fig. 3B).Finally, we also used a marker of cilia, mucin5a, to study the effect of Pten on ciliogenesis in EGFR L858R -induced lung cancer (Fig. 3C).The data indicated that mucin5a was more abundant in the bronchial of EGFR L858R *Pten -/-mice compared to EGFR L858R mice (Fig. 3C).In summary, Pten inhibits most of cilia-related genes to regulate ciliogenesis, thereby suppressing bronchial epithelial cell hyperplasia in EGFR L858R -induced cancer.

Pten inhibits NR2F1 expression to decrease ciliogenesisrelated gene expression in EGFR L858R -induced lung cancer
Next, we studied how Pten regulates the expression of cilia-related genes (Fig. 4).We investigated the transcription factors possibly recruited to the promoters of all the Pten-regulated cilia-related genes and then checked the read numbers of these transcription factors in the RNA-seq data.The results showed that NR2F1 can be recruited to the promoters of most cilia-related genes and increased their read numbers in mice with EGFR L858R *Pten -/--induced lung cancer, implying that Pten may inhibit NR2F1 to in turn inhibit the expression of cilia-related genes.First, we examined the effect of Pten on the expression of cilia-related genes in BEAS-2B (Fig. 4A).The data indicated that Pten knockout increased the mRNA levels of DNAI2, DNAI3 and MUC5AC in BEAS-2B cells, a bronchial cell line.Knockdown of NR2F1 abolished the effect of Pten on DNAI2 and DNAI3 expression but not MUC5AC expression (Fig. 4A).Next, the protein and mRNA levels of NR2F1 were found to be increased in Pten knockdown BEAS-2B cells (Fig. 4B).The NR2F1 level was also increased in EGFR L858R *Pten -/-mice compared to that of EGFR L858Rmice (Fig. 4C), indicating that Pten negatively regulates NR2F1 in vitro and in vivo.Pten negatively regulates the Akt signalling pathway, which is involved in cancer progression (14).Thus, knockout of Pten increased the levels of phosphor-Akt in EGFR L858R *Pten -/-mice compared to EGFR L858R mice (Fig. 4D).Additionally, inhibition of Akt phosphorylation by MK2206 treatment abolished the effect of Pten on the expression of DNAI2, DNAI3 (Fig. 4E) and NR2F1 (Fig. 4F).Finally, loss of Pten increased the recruitment of NR2F1 to the promoter of DNAI2 (Fig. 4G, a and b) and increased the luciferase activity driven by the promoter of NR2F1 (2K bp), indicating that NR2F1 can regulate DNAI2 directly.In summary, Pten suppresses Akt activation, thereby inhibiting NR2F1 expression and subsequently decreasing cilia-related gene expression, leading to inhibition of bronchial epithelial hyperplasia in EGFR L858R -induced lung cancer.

Pten promotes alveolar cell growth in EGFR L858R -induced lung cancer
Thus far, we have elucidated the mechanism by which Pten negatively regulates hyperplasia of bronchial cells in EGFR L858R -induced lung cancer.However, what is the molecular mechanism by which Pten positively regulates alveolar cellular growth in EGFR L858R -induced lung cancer (Fig. 5)?In addition, is the positive effect of Pten in EGFR L858R mice is EGFR L858R dependent?We used two alveolar lung cancer cell lines, A549 and PC9, which have Kras and EGFR mutations, respectively, to study the role of Pten in cell proliferation (Fig. 5A).Overexpression of GFP-Pten inhibited A549 cell growth but promoted PC9 cell growth (Fig. 5A, a and b).Knockdown of Pten increased A549 cell growth and decreased PC9 cell growth (Fig. 5A, b and c), indicating that Pten negatively regulates Kras-mutant cell growth but positively regulates EGFR L858R -mutant cell growth.In addition, overexpression of GFP-Pten increased the level of phosphorylated Akt (p-Akt) in PC9 and H1299 L858R cells but not in A549 and H1299 cells, suggesting that the EGFR L858R mutation is critical for the effect of Pten on alveolar cell growth (Fig. 5B and Fig. 5C).
Finally, the cell cycles in PC9 and A549 cells with or without GFP-PTEN overexpression was studied by ow cytometry (Fig. 5D).The data indicated that overexpression of GFP-Pten increased the percentage of G1/S-phase PC9 cells (71.1%/78.6%)but decreased the percentage of G1/S-phase A549 cells (60.8%/54.9%),indicating that Pten may have differential effects on alveolar cells with different mutation backgrounds, a nding that is expected to be bene cial for precision therapy in the future (Fig. 5D).In summary, differential effects of PTEN were found on EGFR-mut and Kras-mut lung cancer cell lines, implying that the PTEN status might be important for clinical lung cancer patients with EGFR mutations or Kras mutations.
The clinical relationships between the survival rate and the expression of Pten and ciliogenesis-related proteins Next, we used online data, animal samples and clinical specimens to study the relationship among survival, Pten, NR2F1 and cilia-related proteins (Fig. 6 and Fig. 7).First, the levels of NR2F1 and DNAI2 in EGFR L858R *Pten -/-and EGFR L858R *Pten +/+ mice were measured (Fig. 6A).The data indicated that the levels of both NR2F1 and DNAI2 were dramatically higher in EGFR L858R *Pten -/-mice, implying that loss of PTEN increases NR2F1 expression, thereby promoting DNAI2 expression and leading to cilium formation (Fig. 6A).Next, the related information of non-small cell lung cancer (NSCLC) cohorts from TCGA was used to study the relationships between the survival rate and the expression of cilium-related genes (Fig. 6B-6E).First, the survival rate in the lung cancer cohorts with EGFR mutations was found to be lower than that in the cohort with wild-type EGFR (Fig. 6B).MUC5B expression was decreased in the EGFR-mut and PTEN-mut cohorts (Fig. 6C).In addition, SCGB1A1 expressionwas also inhibited in the PTEN-mut cohort (Fig. 6D, a).NR2F1 was highly expressed in the PTEN-mut cohorts (Fig. 6D, b).There was a positive correlation between NR2F1 and DNAI2 expression in all the NSCLC patients (Fig. 6D, c).Finally, the Kras level was different in the PTEN-WT and PTEN-mut cohorts, but no difference was found in the EGFR level, implying that the effect of PTEN on Kras-mutant and EGFR-mutatnt cancers is different (Fig. 6E).
Finally, we collected lung cancer clinical specimens to compare the level of DNAI2 in tissues of different stages (Fig. 7A).The results indicated that DNAI2 was increased in the early stage of NSCLC but was decreased in the late stage, indicating that DNAI2 might be involved in lung tumorigenesis (Fig. 7A).

Discussion
In this study, we found that loss of Pten increases NR2F1 expression and subsequently increases DNAI2 expression, leading to bronchial cell hyperplasia but inhibiting alveolar cell hyperplasia during EGFR L858Rinduced lung cancer progression, a nding expected to be bene cial for the precision medicine in the treatment of lung cancer in the future (Fig. 7B).
Interestingly, Pten is well known as a tumour suppressor in the various cancer types.However, we found that Pten may act as a double-edged sword in lung cancer progression.In this study, we found that Pten knockout induced the hyperplasia of bronchial epithelial cells but inhibited the growth of alveolar cells in EGFR L858R -induced lung cancer.Previous studies revealed that Pten knockout also induced the hyperplasia of bronchial epithelial cells but did not change the growth of alveolar epithelial cells in Kras G12D -induced lung cancer, in which Pten inactivation cooperated with oncogenic Kras G12D to promote lung cancer progression (15).First, loss of Pten was found to increase the hyperplasia of bronchial epithelial cells in EGFR L858R -and Kras G12D -induced lung cancer.In this study, we clari ed that Pten negatively regulates NR2F1 to inhibit cilia-related gene expression in EGFR L858R -induced lung cancer, a mechanism that is involved in bronchial epithelial cell hyperplasia.In addition, numerous studies have indicated that NR2F1-AS1 sponges many miRNAs, subsequently inducing the expression of several oncogenes (10)(11)(12).Therefore, Pten may also inhibit NR2F1-AS1 to suppress lung cancer progression.In addition, previous studies have indicated that NR2F1 may regulate dormancy and metastasis in the latestage of lung cancer (16,17).In this study, we used EGFR L858R mice to study the initiation of lung cancer, not the late-stage of lung cancer.Based on previous studies and this study, NR2F1 may have differential effects on cancer in the early and late stages.In addition, although Pten also induces hyperplasia in bronchial epithelial cells of Kras G12D -induced lung cancer mice (15), the molecular mechanism is not yet clari ed.Second, Pten plays different roles in bronchial and alveolar epithelial cells of mice with EGFR L858R -induced lung cancer but not in mice with Kras G12D -induced lung cancer.This is the rst study to identify the dual roles of Pten as a tumour suppressor and an oncogene in bronchial and alveolar epithelial cells, respectively, in EGFR L858R -induced lung cancer.Overexpression of Pten increased cell growth in the EGFR-mutant cell lines PC9 and H1299 L858R but not in the Kras-mutant cell line A549, indicating that EGFR mutation is critical for Pten-mediated cell growth.In analysing the RNA-seq data, we found that Pten knockout increased Akt1 and Akt2 expression but decreased Akt3 expression (data not shown).Previous studies also indicated that, unlike Akt1 and Akt2 phosphorylation, Akt3 phosphorylation inhibits cancer cell proliferation (18,19).However, why the EGFR mutation is required for the enhancement of cell proliferation by Pten in alveolar cells needs to be addressed in the future.Finally, EGFR is one of the most well-known tyrosine kinase receptor domain-containing proteins and is commonly mutated in tumour, with uncontrolled cell growth, proliferation, and migration documented in approximately 33% of NSCLCs (20)(21)(22).The ve-residue deletion (746ELREA750) in exon19 accounts for 47% of EGFR mutations, and the exon 21 (L858R) substitution accounts for 41% (23).Herein, we used mice with EGFR L858R -induced lung cancer and cancer cells to study the role of Pten in bronchial and alveolar cells.Whether other EGFR mutation types also have the same effects needs to be clari ed in the future.
In this study, we found that loss of Pten signi cantly induced the proliferation of ciliated cells among bronchial epithelial cells of mice with EGFR L858R -induced lung cancer.Furthermore, we clearly clari ed the molecular mechanism by which PTEN regulates ciliogenesis in bronchial epithelial cells during lung cancer progression.During lung morphogenesis, the ciliated cell differentiation pathway is activated (24,25).Geminin coiled-coil containing protein 1 (GMNC), the "master regulator" of ciliated cell fate, and the MYB proto-oncogene are induced (24).Forkhead box protein J1 (Foxj1), a transcription factor required for cilia formation and motility, is also expressed (26,27).Chronic obstructive pulmonary disease (COPD) develops in smokers.Patients with COPD have a considerable reduction in the number of ciliated cells (28, 29).Ciliated cell dysfunction not only causes this disorder but is also found in various cancers.A primary cilium is frequently present on cells of human differentiated thyroid tumours.The relationship between primary cilia and thyroid cancer was uncovered by (30,31).In that study, the researchers established a mouse model of thyrocyte-speci c loss of the primary cilium, and the results indicated that the lack of primary cilia resulted in increased apoptosis in thyroid cancer cells, possibly reveling a new therapeutic target for thyroid cancers (31).In addition, in a study on pancreatic cancer, the authors analysed survival and outcome in 100 patients and drew on conclusion that primary cilia can be formed on pancreatic cancer cells and that their presence is strongly correlated with the prognosis of pancreatic ductal adenocarcinoma (32,33).In contrast, primary cilia are also positively regulated in oral squamous cell carcinoma (OSCC).In OSCC, a signi cant reduction in the percentage of ciliated cells was found in oral leukoplakia (OLK), especially in patients with OSCC, and EGFR was a target, suggesting that loss of cilia induced oral tumour growth (34).Because of Pten is a well-known as tumour suppressor that regulates the expression of numerous genes involved in cell biogenesis, the link between Pten and cilia needs to be discussed.However, another study showed the con icting result that Pten negatively regulates dendritogenesis.Loss of Pten is related to autism spectrum disease (ASD) and causes excessive neuronal development, including the formation of lengthened and branched total dendritic spines.Microtubules are an important part of the ciliary structure that are highly polymerized in primary hippocampal culture (35).Ciliated cells are progeny of club cells, such as goblet cells, and a complicated transcriptional network including Notch signalling controls how these cells differentiate (36,37).Mucins are glycoproteins that are secreted by gel-forming mucin-producing goblet cells.
In this study, we found that Pten knockout induced mucin-related genes.Two high-molecular-weight secreted mucins that are expressed mainly in the mucus layer, which contains electrolytes, metabolites, uids, and antimicrobial substances, are MUC5AC and MUC5B (26, 38).The mucus layer serves as the rst line of innate protection in the respiratory tract against inhaled pathogens and particles (39)(40)(41).Ciliated cells facilitate mucin tra cking to trap particles and as components of the ciliary escalator that drives mucus into the oropharynx for eventual removal by expectoration or swallowing.Pten regulates mucin-related genes but not through NR2F1.The detailed mechanism by which Pten regulate mucin gene expression will be addressed in the future.Dynein Axonemal Intermediate Chain 2 (DNAI2) is part of the dynein complex of respiratory cilia and sperm agella (42,43).Mutations in DNAI2 are involved in the development of primary ciliary dyskinesia type 9. Recent studies have also shown that various isoforms encoded by alternatively spliced transcript variants are involved in primary ciliary dyskinesia (PCD), which may be as a new genetic risk factor for PCD (44,45).There is no reported study about DNAI2 in cancer.This is the rst study to determine the role of PTEN-mediated DNAI2 expression in the hyperplasia of bronchial epithelial cells in EGFR L858Rinduced lung cancer.Recent studies have also revealed that PTEN mutations might be involved in mediating drug resistance and immunotherapy e cacy during cancer therapy (46-48).Pten mutation induced ciliogenesis in bronchial epithelial cells might be a critical mechanism.We found that more lymphocytes were recruited to the region with hyperproliferation of bronchial epithelial cells in EGFR L858R mice, implying that Pten mutation regulates not only cancer cells but also other cells in the surrounding environment of cancer cells (data not shown).Several genes (Gsn/Cd24a/Igfbp2/Foxj1/Adam8/Zbtb16/Hsph1/Efnb2/Gpam/Vcam1/Ctla2a) related to positive regulation of lymphocyte activation were upregulated in EGFR L858R *Pten −/− mice.The detailed function of Pten in regulating the tumour microenvironment in lung cancer and the associated mechanism need to be explored in the future.In addition to cilia-related genes, other Pten-regulated genes were also found in our RNA-seq analysis.For example, ACE2, which has been reported to be overexpressed in different cell subsets of NSCLC, was also upregulated in EGFR L858R *Pten −/− mice (49,50).In addition, previous studies indicated that Pten inactivation in mice with Kras G12D -induced lung cancer increased the immune response (15,51,52).In this study, we also found that several genes related to the in ammatory response, lymphocyte activation and innate immune response in the mucosa (Hp/Reg3g/Nupr1/Adam8/Cd55/Cfh/Ednrb) were upregulated in mice with EGFR L858R *Pten −/− induced lung cancer.Finally, many genes related to epithelial cell migration, extracellular matrix assembly and cellular extravasation were upregulated by Pten knockout, implying that Pten may signi cantly promote cancer metastasis.The effect of the Pten-regulated tumour microenvironment on cancer metastasis in vivo might be evaluated in vivo by using models of EGFR L858R -or Kras G12D -induced lung cancer in the future.
Alcian blue staining -The para n embedded sections were incubated in xylene for dewaxing and a graded series of ethanol for hydration.Next, sections were incubated in acetic acid 3% for 5min and stained by Alcian blue (pH = 2.5) (Merk Millipore Corp, Billerica, MA, USA) solution for 30 min at room temperature.To remove exceed Alcian Blue and prevent non-speci c staining, slides were rinsed brie y by acetic acid 3% for 10 second.The tissue samples were rinsed two times with distilled water and stained with Nuclear Fast Red solution (Merk Millipore Corp, Billerica, MA, USA) 0.1% for 5min to visualize histologic changes.All slices were dehydrated through graded alcohols before mounting.
Animal study -Transgenic mice were acquired from Jackson Lab and maintained at the National Laboratory Animal Center in Taiwan.Reverse tetracycline trans-activator (rtTA) protein was expressed

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