Human chorionic gonadotropin β (β-hCG) is a well-known and accurate marker for the diagnosis and monitoring of pregnancy, trophoblastic tumors and ovarian germ cell tumors. Recently, β-hCG has been found to be closely related to poor prognosis and metastasis in various other malignant tumors, while its role and mechanism in ovarian cancer is still unclear.
In the present study, lentiviral-mediated transfection and small interfering RNA (siRNA) were used to alter β-hCG expression in the ovarian cancer cell lines ES-2 and SKOV3, respectively. Then, migration and invasion activity regulated by β-hCG were evaluated by wound-healing and Transwell assays in vitro and in a peritoneal xenograft nude mouse model in vivo. EDTA and trypsin were utilized to investigate the attachment ability of these cells. Moreover, the expression of epithelial mesenchymal transition (EMT) markers (β-catenin, Slug, vimentin, Snail, claudin, E-cadherin and N-cadherin) was assessed by western blotting and immunofluorescence in ES-2 and SKOV3 cells. Furthermore, β-hCG and EMT markers were evaluated in human ovarian cancer specimens by IHC.
The results showed that overexpression of β-hCG clearly promoted migration and invasion in ES-2 and SKOV3 cells (P. Introduction Ovarian carcinoma is the most lethal gynecologic malignancy. To date, there is no effective early screening method, and more than one-half of cases are diagnosed at an advanced stage. Unlike other gynecological malignancies, extensive pelvic and peritoneal dissemination is the most common path of metastasis in ovarian cancer.
Therefore, better understanding of the mechanisms of cell migration and invasion is urgently needed to identify novel therapeutic strategies for ovarian cancer treatment. Human chorionic gonadotropin (hCG) has a physiologically significant role during pregnancy. The hCG family refers to a group of five molecules, each sharing a common amino acid sequence but differing in multimeric structure and carbohydrate side chain structure. Of these five molecules, β-hCG and hyperglycosylated β-hCG were confirmed to be associated with advanced malignancies. Recently, it was shown that elevated levels of β-hCG in serum, urine or tumor tissue correlates with poor prognosis, aggressiveness and resistance to therapy in a variety of nontrophoblastic tumors, such as bladder, colon, lung and testicular cancer. Moreover, it is also ectopically expressed in numerous gynecological malignancies, such as endometrial carcinoma , ovarian cancer and cervical carcinoma (,).
In our previous study, β-hCG was confirmed to facilitate proliferation and cell cycle progression, attenuate apoptosis and promote tumorigenesis in ovarian surface epithelial cells. To test whether this molecule plays a potential role in ovarian epithelial cancer tumorigenesis, a series of experiments were performed. We found that the β-hCG level was significantly elevated in metastatic tissue compared to the level noted in the primary ovarian cancer tissue, which indicated that β-hCG may play a role in ovarian cancer metastasis. To further verify this hypothesis, a series of in vitro and in vivo investigations were performed. Patients and tissue specimens The present study was approved by the Medical Ethics Committee at The First Affiliated Hospital of the Medical College of Shihezi University. Written consent was obtained from all enrolled patients. Paraffin-embedded human ovarian cancer specimens were obtained from 24 patients who underwent surgical resection without prior adjuvant therapy from December 2006 to December 2013 at The First Affiliated Hospital of the Medical College of Shihezi University, and 20 cases of normal ovarian tissue samples were used as a control.
All samples were pathologically diagnosed according to the World Health Organization (WHO) classification guidelines (2004). All procedures were performed in accordance with the Declaration of Helsinki.
Immunohistochemistry (IHC) Following formalin fixation and paraffin-embedding, the 4-µm thick tissue sections were incubated with primary rabbit polyclonal antibodies against β-hCG (1:50; ab53087; Abcam, Cambridge, MA, USA) overnight at 4°C, washed with phosphate-buffered saline (PBS), and then incubated with the secondary antibody for 1 h at 37°C. Finally, the sections were stained with 3,3-diaminobenzidine and then counterstained with hematoxylin. Images were obtained with a Nikon Eclipse TE2000 fluorescence microscope (Nikon, Tokyo, Japan). Stained tissues were classified according to staining intensity by two investigators.
The extent of β-hCG staining in tissue cores was quantified using a four-tier grading system: 0, ≤5% positive staining; 1, 5–20% positive staining; 2, 20–50% positive staining; and 3, ≥50% positive staining. For statistical analysis, we divided cases into two groups: negative expression (with scores of 0) and positive expression (with scores of 1, 2 or 3). Establishment of β-hCG-overexpressing cell lines We established β-hCG-overexpressing ovarian cancer cell lines in ES-2 and SKOV3 cells via lentivirus transfection. A lentiviral vector encoding β-hCG (LV-β-hCG) and a negative control vector (LV-vector) were purchased from Obio Technology (Shanghai, China), carrying an enhanced green fluorescent protein reporter gene, eGFP. For β-hCG exogenous overexpression, lentivirus containing LV-β-hCG or the LV-vector were transfected into ES-2 and SKOV3 cells using Polybrene (5.0 µg/ml) from Obio Technology, following the manufacturer's instructions. Medium containing puromycin (0.2 mg/ml) was used to select stably transduced cells.
The cells were photographed with a fluorescence microscope. Β-hCG upregulation efficiency was assessed using qPCR and a western blot assay.
Wound healing and Transwell assays Wound healing assay: cells were seeded into 24-well plates and allowed to grow to 90–95% confluence. Similar sized wounds were introduced to a monolayer of cells using a sterile white pipette tip. The wounded monolayer of cells was washed three times with PBS to remove cell debris and then cultured. The speed of wound closure was monitored and photographed every 4 h until the wound filled.
Transwell assay: 1.0×10 5 cells in 100 µl of RPMI-1640 with 2% FBS were seeded into Transwell upper chambers (cat. 3422; Corning Inc., Corning, NY, USA) with or without pre-coated Matrigel matrix (cat. 356234; BD Biosciences, Franklin Lakes, NJ, USA), and 500 µl of RPMI-1640 containing 10% FBS was added into the lower chamber to serve as the chemoattractant.
After 16–48 h of incubation, the cells that did not migrate or invade through the pores were carefully removed. Cells on the filters were fixed in 100% methanol followed by hematoxylin staining (BA4025; Baso Diagnostics, Inc., Zhuhai, China). The number of migrated cells were counted with an inverted microscope (magnification, ×200; Nikon Eclipse), in 10 random fields/chamber. All experiments were performed in triplicate. Cell adhesion assay Cells were seeded into 6-well plates until they reached 90–95% confluence, and then, the cell culture medium was removed, and cells were washed twice with PBS.
After that, the mixture of trypsin (cat. 25200072; Gibco) and EDTA (cat. E8008; Sigma-Aldrich, St.
Louis, MO, USA) in a ratio of 1:20 was added into the plates, and cell images were captured at 0, 5, 10 and 20 min, separately. The pre-experimental adhesion assay was firstly performed to determine the optimal time in ES-2 and SKOV3 cells, and the remaining cells were captured and counted at different time points. According to the results of pre-experiment (data not shown), ES-2 and SKOV3 cells were captured at 5 and 20 min after replacing the cell culture medium with the mixture of trypsin and EDTA, respectively. Finally, the cells that remained adherent were counted, the residual cell adhesion proportion was calculated, and the residual cells were divided to initial cells in ES-2 and SKOV3 cells at 5 or 20 min, respectively. Immunofluorescence staining For immunofluorescence microscopy, the cells were seeded on a culture dish (cat.
801001; Nest Biotechnology, Rahway, NJ, USA) and incubated with a primary antibody against E-cadherin, N-cadherin, Snail, vimentin, β-catenin (cat. 9782; Cell Signaling Technology, Beverly, MA, USA), followed by incubation with Alexa 488-conjugated secondary antibody (Sigma, St. Louis, MO, USA). Fluorescence staining for vimentin was visualized by confocal laser-scanning microscopy (FluoView FV1000; Olympus, Japan); DAPI (Sigma) was used to counterstain DNA.
RNA extraction and real-time RT-PCR Total RNA from ES-2 and SKOV3 cells was isolated by TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription reactions were performed with PrimeScript™ RT Master Mix kit (Takara Bio, Inc., Shiga, Japan) according to the protocol. RT-PCR was performed on an Applied Biosystems StepOnePlus™ Real-Time PCR System using a SuperReal PreMix Plus (SYBR-Green) kit (Tiangen Biotech, Beijing, China). The PCR cycling program was run with an initial predenaturation step at 95°C for 15 min, then with 45 cycles for amplification, at 95°C for 10 sec and 60°C for 32 sec. GAPDH was used as an internal reference. Each test was performed in triplicate, and the 2 −ΔΔCt method was used to calculate the expression of mRNA in each of the cell lines.
The primers used were: β-hCG forward, 5′-TCTGTGCCGGCTACTGCCCC-3′ and reverse, 5′-TTGGGACCCCCGCAGTCAGT-3′; GAPDH forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. Western blotting Total protein from cells and tissues was lysed in KeyGen Whole Cell Lysis Assay (cat. KGP250; KeyGen Biotech, Nanjing, China).
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Then, 20–50 µg protein/sample from different cell lines or treatments was separated by SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes that were blocked for 2 h at room temperature with 5% BSA in TBS containing 0.05% Tween-20. The membranes were then incubated overnight at 4°C in BSA in TBS containing 0.05% Tween-20 and probed with mouse antibodies against β-actin (1:4,000; cat. 20010; Abmart, Arlington, MA, USA) or rabbit antibodies against β-hCG (1:1,000; cat. AP13036b, Abgent, San Diego, CA, USA), EMT markers β-catenin, Slug, vimentin, Snail, claudin, N-cadherin and E-cadherin (1;1,000; cat. 9782; Cell Signaling Technology).
Membranes were washed in Tris-buffered saline with Tween-20 (TBST) and peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:10,000; cat. KGAA35; KeyGen Biotech) and peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:10,000; cat.
L3032-2; Signalway Antibody, College Park, MD, USA) were added and incubated at room temperature for 1 h. The membranes were washed with PBST three times, and visualization of the protein bands was achieved using an enhanced chemiluminescence Plus kit (cat. WBKLS0500; Millipore, Billerica, MA, USA) as recommended by the manufacturer. Construction of a peritoneal xenograft model in nude mice All animal procedures were approved by the Institutional Use and Care of Animals Committee. Female nude mice, aged 4–6 weeks (weighing 20 g) were housed and cared for at the Animal Center of Tongji University (Shanghai, China). Abdominopelvic cavity invasion is the most common form of tumor dissemination in human ovarian cancer in the clinic; therefore, direct intraperitoneal implantation in mice is a routine method to simulate an ovarian cancer model to measure metastatic ability in vivo. For xenografts, 1×10 7 cells transfected with LV-vector or LV-β-hCG were intraperitoneally injected into mice (6 mice/group) in the right flank.
Xenograft growth was monitored by NightOWL LB 983 In Vivo Imaging System (cat. LB983 NC100; Berthold Technologies, Bad Wildbad, Germany) every two days.
Mice were sacrificed after 20 days of follow-up, or according to tumor burden. Tumors were removed, fixed in 10% formalin, and subjected to routine histological examination. The ovaries, uterus, omentum, spleen, liver, intestines, colon and kidney were dissected from the mice, fixed in 10% formalin and IHC stained to detect β-hCG. All mouse experiments were conducted according to the approved animal protocol of the Animal Center of Tongji University. Β-hCG is highly elevated in metastatic tissue compared to tumor tissue of the ovary and normal tissue To determine the biological function of β-hCG in human ovarian cancer progression, IHC was performed to examine β-hCG expression in 20 normal ovarian tissue samples, 24 human ovarian cancer tissue samples and 24 metastatic tissues of ovarian cancer samples. The location of the metastatic sample were mainly from omentum and mesenterium, and all the tumor samples used in the present study were epithelial ovarian tumors, including serous ovarian and mucinous ovarian cancer, and clear cell ovarian carcinoma, verified and analyzed by two gynecologic pathologists. Results showed that 5.0% (1/20), 54.2% (13/24) and 83.3% (20/24) of the cases were β-hCG-positive in normal ovarian tissue samples, human ovarian cancer tissue samples and metastatic tissue of ovarian cancer samples, respectively.
Β-hCG was expressed at significant levels in the metastatic tissues of ovarian cancer and at relatively insignificant levels in the primary ovarian cancer tissues, and it was barely expressed in normal ovarian tissue (P. Successful construction of β-hCG-overexpressing and silenced ovarian cancer cell lines To obtain a β-hCG overexpression model in ovarian cancer cells, LV-β-hCG or the LV-vector (both with a GFP gene) were introduced into the ovarian cancer cell lines ES-2 and SKOV3 via a lentiviral expression vector. Sequence information for the LV-β-hCG and LV-vector plasmids were validated by sequencing (data not shown). Stable β-hCG-overexpressing cell lines were selected with puromycin. As shown in, the stable cell lines were successfully constructed, and nearly 90–95% infection efficiency was determined by GFP assay in both ES-2 and SKOV3 cell lines using fluorescence microscopy. Results showed that β-hCG was obviously overexpressed at both the mRNA and protein levels in the LV-β-hCG-transfected ES-2 and SKOV3 cells compared with the control group by RT-PCR and western blotting (P.
Β-hCG regulates ovarian cancer cell migration, morphology and attachment ability in vitro Wound-healing and Transwell assays were performed to validate the effect of β-hCG overexpressing and silencing of ovarian cancer ES-2 and SKOV3 cells on migration and invasion ability. As shown in and the overexpression of β-hCG greatly increased cell migration and invasion abilities compared with the control (P. Β-hCG regulates EMT in ovarian cancer cells To determine the potential molecular mechanisms of β-hCG in EMT, western blot analysis was performed to detect the expression of epithelial and mesenchymal protein markers. The results showed that overexpression of β-hCG upregulated the expression of mesenchymal markers: vimentin, N-cadherin, β-catenin, Slug and Snail, while it downregulated the expression of the epithelial markers E-cadherin and claudin. Conversely, the β-hCG-depleted ES-2 and SKOV3 cells demonstrated increased E-cadherin and claudin expression, but decreased vimentin, N-cadherin, β-catenin, Slug and Snail expression.
Furthermore, the immunofluorescence staining of β-hCG-overexpressing cells pictured by laser scanning confocal microscopy were consistent with the western blot results. Overexpression of β-hCG promotes metastasis in a nude mouse peritoneal xenograft tumor model β-hCG-overexpressing and control cells were intraperitoneally injected into nude mice, and the growth of xenografts was continuously detected by NightOWL LB 983 In Vivo Imaging System. Results showed that β-hCG upregulation promoted tumor burden and spread range.
On the 23rd and 26th day after intraperitoneal inoculation, ES-2 and SKOV3 group nude mice were sacrificed and dissected, respectively. Gross visualization showed that overexpression of β-hCG induced more and larger metastatic nodule formation in omentum, mesentery, peritoneum and the diaphragm compared with the control group.
In order to objectively evaluate the metastasis ability regulated by β-hCG, all the metastatic nodules, diameter 2 mm, were collected and weighted, and the results showed that β-hCG significantly promoted tumor metastasis. Pathology experts confirmed nodular lesions observed with the naked eye to be ovarian cancer tissue by microscopic examination of H&E-stained tissues, and β-hCG was confirmed to be upregulated compared to the control group by IHC. Discussion HCG is an accurate marker that is currently widely used in clinical diagnosis and monitoring of pregnancy, trophoblastic and ovarian germ cell tumors. Of course, the involvement of hCG in the progression of malignancy is intrinsically different from pregnancy-associated hCG. Among the five family members of hCG, sulfated hCG and hCG are hormones produced by placental syncytiotrophoblast cells and pituitary gonadotrope cells, whereas hyperglycosylated hCG is an autocrine factor produced by placental cytotrophoblast cells, which drives malignancy in placental cancers and testicular and ovarian germ cell malignancies. Β-hCG and hyperglycosylated β-hCG are autocrine factors produced by many advanced malignancies. We found that β-hCG facilitated proliferation and tumorigenesis in ovarian epithelial cells and was significantly elevated in malignant ovarian tumors, compared with normal epithelial expression in ovaries, fallopian tubes and endometrium.
In the present study, we observed that the expression of β-hCG in metastases was obviously higher compared to the tumor tissues from the ovary, which indicates that β-hCG may play a role in promoting the spread of ovarian cancer. To verify this speculation, β-hCG was upregulated and downregulated in ovarian cancer cell lines by lentiviral transfection and siRNA interference techniques, respectively, and the modulated effect of β-hCG on migration and invasion of ovarian cancer cells was confirmed by a series of experiments in vitro and in vivo. Some scholars have found that β-hCG may promote ovarian cancer growth and vasculogenic mimicry formation via activation of the luteinizing hormone receptor signal transduction pathway. Various scholars have demonstrated that β-hCG, Erk1/2 and MMP-2 are potential targets with which to block glioblastoma invasion. There are other scholars that have verified that β-hCG has a cystine knot structure, and this structure happens to be similar to that of transforming growth factor β (TGFβ), and thus, can seemingly antagonize a TGFβ receptor, while the pregnancy-related hormone hCG does not appear to expose these sequences and structures (,). TGF-β, a ubiquitously expressed cytokine, is perhaps the best-characterized promoter of EMT. EMT refers to a global cellular and molecular transition by which polarized epithelial cells gain mesenchymal properties allowing them to migrate, which plays a vital role in local invasion and metastatic dissemination during malignancy.
Activating TGFβ ligands initiate signaling, and closely related Smad-dependent pathways, including phosphoinositide 3-kinase (PI3K)-Akt , focal adhesion kinase (FAK) , p38 mitogen-activated protein kinase (p38 MAPK) , and extracellular signal-regulated kinase (Erk) , have been identified as crucial for EMT. During EMT, epithelial cells reorganize their cytoskeleton, resolve cell-cell junctions and switch off the expression of epithelial markers, turning on mesenchymal genes, such as, E-cadherin, vimentin, N-cadherin, β-catenin, Snail, claudin, ZO-1 and others. Accordingly, we found that overexpression of β-hCG induced morphological changes, and the appearance of epithelial ovarian cancer cells changed from closely arranged polygons into a fusiform morphology with a loose arrangement, demonstrating that β-hCG has a role in regulating EMT-associated gene expression.
In combination with the findings from other recent studies, our results shed light on the molecular mechanisms of β-hCG expression and its functional role in promoting ovarian carcinoma metastasis and invasion, and thus may point to a new target for therapeutic intervention.
Idiopathic pulmonary fibrosis (IPF) and idiopathic nonspecific interstitial pneumonia (INSIP) are two related diseases involving varying degrees of pulmonary fibrosis with no effective cure. Bone morphogenetic protein 3 (BMP3) is a member of the transforming growth factor-β (TGF-β) super-family, which has not been implicated in pulmonary fibrosis previously. In this study, we aimed to investigate the potential role of BMP3 playing in pulmonary fibrosis from clinical diagnosis to molecular signaling regulation. RNA sequencing was performed to explore the potential biomarker of IIP patients.
The expression of BMP3 was evaluated in 83 cases of IPF and INSIP by immunohistochemistry. The function of BMP3 was investigated in both fibroblast cells and a bleomycin-induced murine pulmonary fibrosis model. The clinical relevance of BMP3 expression were analyzed in 47 IIP patients, which were included in 83 cases and possess more than five-year follow-up data. Both RNA-sequencing and immunohistochemistry staining revealed that BMP3 was significantly down-regulated in lung tissues of patients with IPF and INSIP. Consistently, lower expression of BMP3 also was found in pulmonary fibrotic tissues of bleomycin-induced mice model. Up-regulation of BMP3 prevented pulmonary fibrosis processing through inhibiting cellular proliferation of fibroblasts as well as TGF-β1 signal transduction. Finally, the relatively higher expression of BMP3 in IPF patients was associated with less/worse mortality.
Intravenous injection of recombinant BMP3. Taken together, our results suggested that the low expression level of BMP3 may indicate the unfavorable prognosis of IPF patients, targeting BMP3 may represent a novel potential therapeutic method for pulmonary fibrosis management. INTRODUCTION Idiopathic interstitial pneumonias (IIPs) are a group of interstitial lung diseases of unknown etiology that are characterized by varying degrees of chronic inflammation and progressive fibrosis of lung parenchyma ,. Idiopathic pulmonary fibrosis (IPF) and idiopathic nonspecific interstitial pneumonia (INSIP) are two major sub-types of IIPs ,.
IPF is histopathologically defined by the presence of the typical form of pulmonary fibrosis and often results in death within 3–5 years of diagnosis. INSIP is universally associated with a more cellular interstitial pneumonia with or without accompanying fibrosis and occurs earlier in life with a better prognosis than IPF –. Although some patients have a certain response to corticosteroid the effect is limited. Therefore, it has been suggested that efforts to combating IPF and INSIP should be aimed at exploring potential anti-fibrotic treatment strategies ,. A number of cytokines, including interleukins (ILs), transforming growth factor-β (TGF-β), and chemokines –, secreted by lung epithelial cells, endothelial cells, stromal cells, and many types of activated inflammatory cells are known to be involved in the progress of pneumonia-related inflammation and pulmonary fibrosis. It is well known that TGF-β not only play important roles in regulating several physiological processes of the lung development, but also associated with a variety of pulmonary diseases, including fibrosis ,. Interestingly, increased evidences shown that Bone morphogenetic proteins (BMPs), as the members of TGF-β superfamily, are endogenous antagonism of TGF-β signaling.
Physiologically, they are required for the maintenance of tissue homeostasis and regeneration after injury. Besides, they also heavily involved in the development of bone, cartilage, lung, and other organs in rodents. Accumulating evidence suggests that BMPs participate in the processes of a variety of organ fibroses ,.
BMP2 significantly attenuates TGF-β–induced renal fibrosis in rodents by modulating epithelial-mesenchymal transition (EMT). BMP6 was recently defined as a key regulator of renal fibrosis.
Augmenting the expression of BMP7 can reverse chronic renal injury via inhibition of TGF-β–mediated EMT. In renal fibrosis mouse models, BMP7 was shown to suppress the fibrotic process by inhibiting the TGF-β/Smad signaling pathway, which plays an essential role in converting fibroblasts into large numbers of myofibroblasts leading to fibrosis –. Although these studies have uncovered important roles of BMPs in organ fibroses, including pulmonary fibrosis in rodent models, the clinical relevance of BMPs in pulmonary fibrosis diseases, including IPF and INSIP, were under investigation. In the present study, RNA sequencing of lung tissue samples from IPF and INSIP patients was performed and the transcriptome of fibrosis lungs was compared to that of normal lungs. The expression of BMP3, which has not been previously reported to regulate fibrosis, was further evaluated in fibrotic lungs in 83 patients including 46 cases of IPF and 37 cases of INSIP by immunohistochemistry. The role of BMP3 in the pathogenesis of pulmonary fibrosis in vivo was determined using a bleomycin–induced murine pulmonary fibrosis model The bleomycin animal model is widely used in the assessment of potential antifibrotic agents. A large number of compounds have been shown to prevent fibrotic progression in this model and have been suggested to qualify for clinical use.
And we used primary mice fibroblasts culture to find the role of BMP3 in vitro. Finally, the clinical relevance of BMP3 was analyzed in 47 patients, which were cohort of 83 cases and have integrated five years of follow-up data. Identification of genes dysregulated in lung tissues from IPF and INSIP patients The illumina mRNA sequencing approach was used to determine the relative abundance of various genes in IIPs.
For each tissue sample, 8.3 ± 1.0 million reads with an average read length of around 50 bp was generated to ensure sufficient and saturating sequencing depth. The reads were aligned with the human reference genome (GrCH37, Ensemble build 74) using Tophat version 2.0.12, yielding an average mapping rate of 94.7 ± 2.5%. Gene expression levels, which were represented as fragments per kilo-base per million mapped reads (FPKM), were obtained for 63, 783 genes/transcripts annotated using the Ensemble GrCH37 database release 74. The global gene expression profiles indicated that the gene expression patterns in diseased lung samples were distinct compared with those of healthy lung samples. Using a cut-off P-value 1.5, a total of 1652 differentially expressed genes were identified by comparing healthy and diseased lung tissue samples.
Overall, 671 genes were up-regulated and 981 were down-regulated in fibrotic lungs. Besides, when 5156 differentially expressed genes were subjected to supervised weighted gene co-expression network analyses (WGCNA) based on FDR. BMP3 was decreased in a bleomycin-induced murine pulmonary fibrosis model To determine whether BMP3 is a novel factor involved in the pathogenesis of IPF and INSIP as well as a potential new therapeutic target, we established a murine pulmonary fibrosis model based on bleomycin treatment.
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First, the expression level of BMP3 in mouse lung tissues was measured at different time points after intratracheal instillation of bleomycin. Hematoxylin (HE) staining demonstrated abundant infiltration of lymphocytes into the alveolar space, deposition of extracellular matrix (ECM) in the interstitial space, and substantial widening of the alveolar septa in lung tissues of bleomycin-instilled mice (Figure ). In contrast, a minimal inflammatory response was observed in the lung tissues of saline-instillation group (Figure ). Importantly, BMP3 expression was markedly reduced in alveolar epithelial and bronchial epithelial cells as well as in interstitial cells in the lungs of bleomycin-treated mice (Figure ). To further validate these results, Western blot analysis was performed and demonstrated significantly reduced BMP3 protein levels in 7, 14, and 21 days bleomycin-induced fibrotic lung tissues compared with murine normal lung tissue and saline-treated lung tissues (saline-instillation group) (Figure ).
In contrast, expression of α-smooth muscle action (α-SMA), a typical marker of myofibroblasts, was significantly increased, demonstrating that bleomycin was rather effective in causing pulmonary fibrosis in mice (Figure ). Quantification of Western blots showed that BMP3 expression and the degree of pulmonary fibrosis were inversely correlated (Figure ).
Enhanced BMP3 expression attenuated the fibrotic process triggered by bleomycin in the murine pulmonary fibrosis model To determine the contribution of reduced BMP3 expression to the pathogenesis of pulmonary fibrosis, a recombinant human (rh) BMP3 was injected via the tail vein of mice 7 days as establishment of the murine pulmonary fibrosis model with bleomycin. Two doses, one low (100 μg/kg) and one high (300 μg/kg) were used. At Day 21, mice were sacrificed, and gross anatomical analyses showed that lung tissues of the bleomycin-only treated group were dark red with evidence of congestion and swelling, whereas lung tissues treated with additional rhBMP3 showed the reduced congestion (Figure ). Microscopic images of HE-stained lung tissues revealed substantial lymphocyte infiltration, formation of lymphoid follicles, and a widened interstitial space for the fibrotic lung (Figure ). With rhBMP3 treatment the number of inflammatory cells and congestion were reduced (Figure ). Thus, the fibrotic pathology was partly reverted in a dose-dependent manner following injection of rhBMP3. The fibrotic progress was reversed upon BMP3 administration in the bleomycin-induced murine model of pulmonary fibrosis Increased α-SMA expression indicated the conversion of fibroblasts into myofibroblasts, which is accompanied by shrinkage of fibroblasts and secretion of large amounts of collagen, fibronectin, laminin, and other ECM components ,.
Western blot analysis for BMP3 and α-SMA expression confirmed that intravenous injections of rhBMP3 dose-dependently reversed α-SMA expression following bleomycin treatment (Figure ). This was indicative of a reversal of the myofibroblasts back to the normal pulmonary fibroblast phenotype. Masson's Trichrome staining demonstrated a substantial increase in collagen deposition in bleomycin-induced mice at Day 21 compared with saline-instillation group. Collagen deposition was significantly decreased with increasing doses of injected rhBMP3. Especially in the high-dose group (300 μg/kg) compared with 0.1% bovine serum albumin (BSA) control-injection group (Figure ).
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In addition, the level of hydroxyproline, a metabolic product of collagen, was measured to indirectly quantify collagen content in lung tissues. Again, rhBMP3 treatment at concentrations of 100 and 300 μg/kg significantly reduced the content of hydroxyproline compared with that in the 0.1% BSA control-injection group at Day 21 following bleomycin-treatment (Figure, P. BMP3 reduced proliferation and activation of primary murine pulmonary fibroblasts To investigate the underlying mechanism by which increasing BMP3 could attenuate the progression of pulmonary fibrosis, we isolated primary pulmonary fibroblasts from 21-day bleomycin-induced mice (21 day's fibroblasts) and saline-instilled mice (Normal fibroblasts) according to the method described in a previous study. Isolated cells from BLM lungs were identified as fibroblasts by immunocytochemical staining for α-SMA, vimentin and cytokeratin. Cellular proliferation test showed that BLM fibroblasts had a higher proliferative ability than normal fibroblasts (Figure, P. BMP3 prevented the fibrotic process via inhibition of the TGF-β1/Smad signaling pathway Previous study demonstrated that TGF-β1 was highly expressed after bleomycin treatment as previously reported.
And in this study, TGF-β1 was increased in both RNA sequencing test and immunochemistry staining. Therefore, BMP3 and TGF-β1 expression appeared to be inversely regulated during pulmonary fibrotic process.
To delineate the relationship between these two important, seemly opposing factors, quantitative realtime-PCR and Western blot analyses were carried out. The mRNA expression of Tgf-β1 and its downstream signaling molecules was measured in lung tissues of BLM-instilled mice with or without rhBMP3 treatment. Tgf-β1, Smad2, and Col1α1 mRNA expressions were increased in BLM-induced pulmonary fibrosis, whereas they were decreased after rhBMP3 administration in a dose-dependent manner (Figure and ). Interestingly, Smad4 expression showed no obvious difference across all conditions (Figure ). As expected, the expressions of Bmp3 as well as its downstream signaling molecules Smad5 and Stat1 were significantly increased with rhBMP3 treatment, indicating that injected rhBMP3 was functional and the BMP3 and TGF-β1 pathways were mutually antagonizing (Figure ). Semi-quantitative RT-PCR revealed the mutually inhibitory relationship between Bmp3 and Tgf-β1 in fibrotic fibroblasts of BLM and NIH-3T3 fibroblasts Given that administration of rhBMP3 reduced TGF-β1 expression, the question arose whether the converse treatment with TGF-β1 would down-regulate BMP3 expression. Therefore, the murine fibroblast cell line NIH3T3 was treated with TGF-β1 with or without its antagonist SB431542, or with rhBMP3 (Figure ).
After TGF-β1 stimulation, NIH3T3 cells became mitotically active , and the expression of BMP3 was significantly reduced, which was accompanied by a decrease in expression of BMP3's downstream signaling factors phospho-Smad1/5/8 (p-Smad1/5/8) and a concurrent increase in α-SMA (Figure ). The TGF-β1 receptor (Alk5)-specific inhibitor SB431542 strongly counteracted the effect of TGF-β1 on BMP3 expression, without affecting TGF-β1 expression. In consistent with the outcome by SB431542 administration, rhBMP3 application also reversed the TGF-β1 effect (Figure ). Taken together, these results suggested that BMP3 may alleviate the fibrotic processing by suppressing the activation of TGF-β1 signaling pathway and that the mutually antagonistic relationship between BMP3 and TGF-β1 plays a crucial role in the pathogenesis of pulmonary fibrosis (Figure ). Reduced BMP3 expression was associated with worse survival rate in IPF patients To further determine the clinical importance of our finding regarding the role of BMP3 in the pathogenesis of pulmonary fibrosis, BMP3 expression was examined using tissue array technology and immunohistochemistry in 47 cases of IPF and INSIP patients with more than 5 years of detailed clinical follow-up data including in 83 IIP cases (, ). A more than 5-year clinical follow-up of 22 INSIP and 25 IPF patients provided a data matrix of eight parameters including gender, age at disease diagnosis, smoking, chronic toxin exposure, survival time, whether the patient was deceased (death), as well as the levels of TGF-β1 and BMP3 proteins in lung tissue biopsy specimens.
The data matrix with these eight parameters plus the disease type (INSIP or IPF) was subjected to hierarchical clustering, and the Pearson's correlation coefficients between any random pairs of these parameters were calculated (Figure ). It is known that INSIP patients survive longer than those with IPF, and accordingly, disease type was correlated positively with death and negatively with survival time (IPF was designated as 1 and INSIP as 0 to digitize the disease type parameter).
Most of the smokers in this patient cohort were male, and thus, gender was negatively correlated with smoking (correlation coefficient: -0.71). Interestingly, smoking was weakly but significantly correlated with TGF-β1 expression (correlation coefficient: -0.37). Therefore, smokers exhibited lower TGF-β1 levels in their diseased lung tissues. TGF-β1 was also weakly correlated with gender, probably because gender and smoking were tightly correlated in this particular data set. Age was weakly correlated with gender, death, disease type, and BMP3, probably due to the fact that the IPF patient pool was older than the INSIP patient pool, which was consistent with the clinical observation that INSIP occurred more frequently in younger people as compared to IPF (5). BMP3 was found to be expressed at low levels in patients with IPF, which presented as a more severe disease with higher lethality as compared to INSIP.
Consequently, BMP3 expression was correlated negatively with death and age, and positively with survival time (Figure and ). High BMP3 expression predicted better survival rate in IIP patients It became obvious that IPF patients who expressed below the average levels of BMP3 survived shorter times than those who expressed above the average levels (Figure and = 0.006). Most INSIP patients had higher BMP3 expression levels and consequently showed overall longer survival times.
This result was consistent with results showing that high BMP3 expression prolonged survival time in INSIP patients. In contrast, TGF-β1 expression did not predict prognosis in either IPF and INSIP patients regardless of low or high, TGF-β1 expression levels. Taken together, these results indicate that BMP3 may be a beneficial factor for predicting prognosis in both IPF and INSIP patients. Particularly in IPF, elevated BMP3 expression can serve as a potential treatment strategies for IIP. DISCUSSION IIPs, either IPF or INSIP, are always associated with fibrosis, but the underlying pathological mechanisms are under investigation. In order to identify genes that are dysregulated in lung tissues from IPF and INSIP patients, RNA-sequencing of patient lung tissues combined with Weighted Gene Co-expression Network Analyses (WGCNA) was employed as a very powerful bioinformatics tool for transcriptome analyses. This approach identified dramatic changes in the expression of not only individual genes but also of functionally relevant gene clusters/modules that were positively and negatively correlated with IPF/INSIP.
TGF-β1, representing positive cell cycle regulation, was found to be upregulated, whereas BMP2, representing negative regulation of cell cycle, and BMP3, representing crucial cell–cell signaling, were downregulated in IPF/INSIP. Since BMP3 has not been previously reported to be involved in IIPs, an extensive series of in vivo and in vitro studies were carried out using the bleomycin-induced pulmonary fibrosis animal model.
These experiments demonstrated that a decrease in BMP3 expression not only served as a biomarker for IPF, but that BMP3 per se was a functionally relevant factor. Manipulation of BMP3 expression significantly attenuated the fibrotic process following BLM. Moreover, the in vitro cell culture studies with normal and fibrotic murine fibroblasts further demonstrated that, like BMP2 , , BMP3 also functionally antagonized TGF-β1 to reduce fibrotic cell proliferation. Balanced TGF-β and BMP signaling has been proposed to be crucial for the progression of fibrosis. Suggested that TGF-β1 is activated upon fibrosis, disrupting the balance between TGF-β1 and BMP7 resulted in the suppression of BMP7 expression and its target genes ,. Similarly, in the present study, a mutually inhibitory relationship between TGF-β1 and BMP3 was observed in both human patients and a murine fibrosis model.
Our results showed that regardless of whether TGF-β1 was up- or down-regulated, it affected the expression of BMP3 signaling in a converse manner. Moreover, BMP3 also affected the TGF-β1 signaling pathway and TGF-β1 expression. One of the strengths of this study was the use of clinical data including more than 5 years of clinical follow-up of 25 IPF and 22 INSIP patients.
Although some BMPs have been discovered as anti-fibrosis factors, the involvement of BMPs in the pathogenesis of pulmonary fibrosis in humans, and the relationship between BMPs and disease progression of IIPs remains largely unknown –. By using patient clinical information together with quantified BMP3 and TGF-β1 levels from lung biopsies, the present study confirmed that BMP3 was negatively correlated with disease severity. Surprisingly, TGF-β1 was not statistically significant associated with patients’ survival time but showed a trend of positive correlation with disease severity. It is likely that analysis with increased patient numbers will show the TGF-β1 is a reliable predictor of disease severity. Nevertheless, when comparing TGF-β1 and BMP3, the latter was significantly associated with survival time of IPF patients in the present study. This is likely because BMP3 is more directly linked to IIPs, whereas TGF-β1 might be linked somewhat more indirectly, as its expression can be regulated by a variety of factors, including lung and immune cells.
BMP3 was expressed at higher levels in INSIP lungs compared with IPF lungs, consistent with its beneficial role, as INSIP is known to be a less severe clinical condition than IPF. However, in INSIP, BMP3 downregulation was a minor event and did not significantly impact the disease outcome. In IPF on the other hand, because patients displayed much more reduced BMP3 levels, variations in BMP3 levels served as a good indicator for survival rates. In the future, larger patient cohorts need to be analyzed for BMP3 expression levels to confirm the role of BMP3 as a clinically relevant predictor of disease prognosis, particularly for IPF. Given that in animal studies BMP3 showed protective effects, it also will be interesting to determine whether enhancing BMP3 protein levels might serve as a therapeutic strategy for IPF. In summary, we identified that BMP3 plays a protective role in fibrosis and is a valuable anti-fibrotic factor for IPF. Although the number of samples used in the initial RNA sequencing analysis was relatively low, we further confirmed the expression of BMP3 in the lung tissues of 83 patients with IIPs confirmed by clinical-radiologic-pathological diagnosis.
Importantly, more than 5 years of follow-up clinical data of 47 cases including in 83 cases were available, which strongly supported that BMP3 might be a novel biomarker in IPF and INSIP, and especially IPF. We also showed that BMP3 could suppress the fibrotic process of pulmonary fibrosis both in vivo and in vitro. Finally, our results uncovered an antagonistic relationship between BMP3 and TGF-β1, which played a critical role in the pathogenesis of pulmonary fibrosis in BLM. Our study highlights the potential clinical value of BMP3 for the treatment of IPF and INSIP patients in the China population. Study design Clinical data of 83 patients including 46 IPF cases and 37 INSIP cases were collected from Tongji Hospital affiliated with the Tongji University School of Medicine (Shanghai) and the Guangzhou Institution of Respiratory Diseases, (Guangzhou, China).
The detail clinical data of these patients are summarized in. All biopsy samples from IIP patients were collected at initial diagnosis and taken by video-assisted thoracoscopic surgery (VATS) or a small incision. Control samples were collected from normal lung tissue adjacent to the benign pulmonary tumors without fibrosis. Among the 83 IIPs cases, the lung biopsy samples of seven most typical cases of IIP patients were used for RNA sequencing, including four cases of IPF and three cases of INSIP. Correspondingly, 5 normal lung tissues were randomly chosen for RNA sequencing. The expression of BMP3 and TGF-β was confirmed by immunohistochemistry in all tissue samples, including 47 specimens (25 cases of IPF and 22 cases of INSIP) with more than five years follow-up data, which were used to analyze the clinical relevance of these genes, the detail information of 47 cases are summarized in.
The criteria for all patients inclusion were: (i) a diagnosis according to the criteria of the American Thoracic Society (ATS)/European Respiratory Society (ERS) classification guidelines on IIPs , –; (ii) availability of integrated clinical, radiologic, and pathologic information; and (iii) a final diagnosis made by pathologists, clinicians and radiologists through multi-disciplinary discussion with all other known causes of ILD excluded. For in vivo experiments in the bleomycin-induced murine pulmonary fibrosis model, at least five mice were randomly selected per group.
All analyses were performed blinded to treatment, and all experiments were repeated independently three times. Library construction and RNA sequencing Total RNA from 10 mg tissue was isolated with depleting genomic DNA for RNA-seq library construction following standard TruSeq RNA sample preparation v2 protocol (Illumina, San Diego, CA, USA). The sequencing libraries were then sequenced using the Illumina HiSeq2000 platform. From reads averaging 50 bp in length, 9.0 ± 1.5 million reads per sample were generated.
After that, the reads were aligned to the human reference genome (GrCH37, Ensembl build 74) using Tophat version 2.0.12, yielding an average mapping rate of 94.8 ± 2.1%. Gene expression levels, which were represented as fragments per kilo-base per million mapped reads (FPKM), were obtained for 63,783 genes/transcripts annotated using Ensemble GrCH37 database release 74. The RNA-sequencing data were deposited to the Gene Expression Omnibus (GEO) repository (accession number ). Weighted gene co-expression analysis (WGCNA) For WGCNA, 5156 transcripts that were significantly differentially expressed between the two groups were selected (Tukey's HSD test with P value.
Hydroxyproline assay The hydroxyproline content per 100 mg of lung tissue was measured as an index of lung fibrosis using a Hydroxyproline Assay Kit (KeyGen Biotech; Nanjing, China). Briefly, Lung tissues were cut into small fragments and hydrolysis in 6N HCL at 95°C by 5 hours. The hydroxyproline was then separated from powdered activated carbon by centrifugation and supernatant of each sample was incubated as the instruction with solutions supplied by the kit. After the incubation, the suspension was centrifuged at 3500rpm for 5 minutes. The supernatant OD was read under 550nm wavelength of light and hydroxyproline concentrations calculated from standard curve. The formula was hydroxyproline content ( ug/mg) = (sample's OD-blank control's OD) / (standard's OD-blank control's OD) x 5ug/ml x total volume of hydrolysis ( 10ml) /weight of tissue ( mg).
Cell proliferation and cell cycle assays Cultured fibroblasts (1×10 3/well) were seeded in 96-well plates for 24 hours and exposed to different concentrations of rhBMP3 for 1–6 days. Cellular proliferation was detected using a tetrazolium salt-based colorimetric assay kit (CCK8 kit, KeyGen Biotech, Nanjing, China), as previously described. The cell cycle was estimated by flow cytometry using a cell cycle and apoptosis analysis kit (Beyotime, China). Briefly, Amount of 1×10 6 cells were digested by trypsin and the suspension was centrifuged at 1000× g for 5 minutes. After washing the sediment with cool PBS, the cells were fixed in cool 70% ethanol at 4°C for 12 hours. Propidium Iodide (PI) and RNase A were added in cell suspension according to instructions and incubated at 37°C for 30 minutes. After staining, samples were tested under 488nm excitation wavelength of light.
Data were analyzed by homologous software Flowjo 7.6 (FlowJo LLC, Oregon, USA). Western blot analysis Total protein was extracted from lung homogenates or cell lysates using radioimmunoprecipitation (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors as previously described. After electrophoresis and membrane transfer, the immunoblots were probed with the following primary antibodies: BMP3 (1:200, Santa Cruz Biotechnology), phosphor-Smad1/5/8, Smad4 (1:1000, Cell Signaling Technology; Beverly, MA, USA), α-SMA (1:200, Dako), GAPDH, and β-actin (1:5000, Bioworld, Dublin, OH, USA). The secondary antibodies were goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated antibody (Cell Signaling Technology). Enhanced chemiluminescence reagent was used for detection and blots were scanned using the Alphaview SA software (Proteinsimple, San Jose, CA, USA). Immunofluorescence staining Cells were cultured on lysine-treated slides in a 6-well chamber prior to immunofluorescent staining. The cells were fixed in 4% paraformaldehyde for 15 min at room temperature.
After washing with PBS, cells were permeabilized with 0.1% Triton X-100 for 30 min and blocked with 10% BSA in phosphate-buffered saline (PBS) for 60 min in a humidified chamber. Cells were incubated with α-SMA antibody (1:200 dilutions, Dako) overnight at 4°C. After washing with PBS, cells were incubated with TRITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI (Beyotime Biotechnology). Phase contrast and fluorescent microscopy was performed using an Olympus IX81 inverted research microscope (Olympus; Tokyo, Japan).
Statistical analysis Statistical analysis was performed using the SPSS software program, version 19.0 (IBM Corp. Released 2010. Statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Tukey's test for qPCR, mean integrity OD, and hydroxyproline measurements. Survival functions were evaluated by Kaplan–Meier survival analysis. The results are expressed as means ± standard deviation (SD). A probability level of P. Contributed by Author contributions Conception and design: XH.Y, AB.L and YY.G; Experimental work, analysis, and interpretation: XT.Y, G.P, X.F, ZL.H, XY.Z, MN.Z, X.L, LC.F, Y.J, Q.L, RC.C, JH.Y and JB.W; Drafting the manuscript and intellectual content: XH.Y and AB.L.
CONFLICTS OF INTEREST All authors declare no conflicts of interest. FUNDING This work was supported by the National Natural Science Foundation of China (81401882, 81570053, 81600043), Shanghai Municipal Natural Science Foundation (16ZR1432100), Key Medical Research of Shanghai (034119868, 0), Key Medical Research Foundation of Health Bureau of Shanghai (20134034) and Shanghai leading talent project (2014054).