Dysregulated lung stroma drives emphysema exacerbation by potentiating resident lymphocytes to suppress an epithelial stem cell reservoir.

Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses Hhip, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of Hhip induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.

MicroRNAs regulate distal region of mandibular development through Hh signaling.

Mandibular anomalies are often seen in various congenital diseases, indicating that mandibular development is under strict molecular control. Therefore, it is crucial to understand the molecular mechanisms involved in mandibular development. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating the level of gene expression. We found that the mesenchymal conditional deletion of miRNAs arising from a lack of Dicer (an essential molecule for miRNA processing, Dicerfl/fl ;Wnt1Cre), led to an abnormal groove formation at the distal end of developing mandibles. At E10.5, when the region forms, inhibitors of Hh signaling, Ptch1 and Hhip1 showed increased expression at the region in Dicer mutant mandibles, while Gli1 (a major mediator of Hh signaling) was significantly downregulated in mutant mandibles. These suggest that Hh signaling was downregulated at the distal end of Dicer mutant mandibles by increased inhibitors. To understand whether the abnormal groove formation inDicer mutant mandibles was caused by the downregulation of Hh signaling, mice with a mesenchymal deletion of Hh signaling activity arising from a lack of Smo (an essential molecule for Hh signaling activation, Smofl/fl ;Wnt1Cre) were examined. Smofl/fl ;Wnt1Cre mice showed a similar phenotype in the distal region of their mandibles to those in Dicerfl/fl ;Wnt1Cre mice. We also found that approximately 400 miRNAs were expressed in wild-type mandibular mesenchymes at E10.5, and six microRNAs were identified as miRNAs with binding potential against both Ptch1 and Hhip1. Their expressions at the distal end of the mandible were confirmed by in situ hybridization. This indicates that microRNAs regulate the distal part of mandibular formation at an early stage of development by involving Hh signaling activity through controlling its inhibitor expression level.

Hhip inhibits proliferation and promotes differentiation of adipocytes through suppressing hedgehog signaling pathway.

Adipogenesis, which directly control body fat mass, plays a crucial role in lipid metabolism and obesity-related diseases. Hedgehog interacting protein (Hhip) belongs to Hedgehog (Hh) signaling pathway. The Hh signaling pathway was already linked with adipogenesis in previous reports, however, the physiological functions of Hhip on lipid deposition are still poorly understood. In this study, the level of Hhip was down-regulated during the development of porcine adipose tissues. Recombinant Hedgehog interacting protein (rHhip) could down-regulate cell cycle related genes and cell numbers in S phage to inhibit cell proliferation. Moreover, rHhip could increase adipocytes differentiation by targeting canonical Hh signaling, indicated by the increase of lipid accumulation and up-regulation of Glut4 and PPARγ expression. Collectively, these findings illustrated the essential role of Hhip in the proliferation and differentiation of adipocytes, and provided a potential novel target for preventing obesity.

A novel long noncoding RNA HHIP-AS1 suppresses hepatocellular carcinoma progression through stabilizing HHIP mRNA.

Aberrant expression of long non-coding RNAs (lncRNAs) has been observed in hepatocellular carcinoma (HCC) and confirmed to participate in the initiation and progression of HCC. In the present study, we identified a novel functional lncRNA, hedgehog-interacting protein antisense RNA 1 (HHIP-AS1). The expression levels of HHIP-AS1 were significantly decreased in HCC tissues. Downregulation of HHIP-AS1 expression correlated with larger tumor size, metastasis, and advanced TNM stage, and also predicted worse overall survival rate of HCC patients. Through performing overexpression and knockdown experiments, the biological function of HHIP-AS1 was identified to suppress HCC cell proliferation, migration and invasion, while promote apoptosis. Further investigation showed that HHIP-AS1 interacted with and positively regulated the stability of HHIP mRNA in a HuR-dependent manner. HHIP-AS1 exerted its suppressive effects through HHIP. Taken together, our findings demonstrate that HHIP-AS1 represses HCC progression by promoting HHIP expression, and indicate that the use of HHIP-AS1 may offer a promising treatment for HCC patients.

Histone methyltransferase SUV39H2 regulates cell growth and chemosensitivity in glioma via regulation of hedgehog signaling.

BACKGROUND: Malignant glioma is one of the essentially incurable tumors with chemoresistance and tumor recurrence. As a histone methyltransferase, SUV39H2 can trimethylate H3K9. SUV39H2 is highly expressed in many types of human tumors, while the function of SUV39H2 in the development and progression of glioma has never been elucidated. METHODS: RT-qPCR and IHC were used to test SUV39H2 levels in glioma tissues and paired normal tissues. The clinical relevance of SUV39H2 in glioma was analyzed in a public database. Colony formation assays, CCK-8 assays, and flow cytometry were conducted to explore the role of SUV39H2 in the growth of glioma cells in vitro. A cell line-derived xenograft model was applied to explore SUV39H2's role in U251 cell proliferation in vivo. Sphere formation assays, RT-qPCR, flow cytometry, and IF were conducted to illustrate the role of SUV39H2 in the stemness and chemosensitivity of glioma. Luciferase reporter assays and WB were applied to determine the function of SUV39H2 in Hh signaling. RESULTS: SUV39H2 was highly expressed in glioma tissues relative to normal tissues. SUV39H2 knockdown inhibited cell proliferation and stemness and promoted the chemosensitivity of glioma cells in vitro. In addition, SUV39H2 knockdown also significantly inhibited glioma cell growth in vivo. Moreover, we further uncovered that SUV39H2 regulated hedgehog signaling by repressing HHIP expression. CONCLUSIONS: Our findings delineate the role of SUV39H2 in glioma cell growth and chemosensitivity as a pivotal regulator of the hedgehog signaling pathway and may support SUV39H2 as a potential target for diagnosis and therapy in glioma management.

Hhip haploinsufficiency sensitizes mice to age-related emphysema.

Genetic variants in Hedgehog interacting protein (HHIP) have consistently been associated with the susceptibility to develop chronic obstructive pulmonary disease and pulmonary function levels, including the forced expiratory volume in 1 s (FEV1), in general population samples by genome-wide association studies. However, in vivo evidence connecting Hhip to age-related FEV1 decline and emphysema development is lacking. Herein, using Hhip heterozygous mice (Hhip(+/-)), we observed increased lung compliance and spontaneous emphysema in Hhip(+/-) mice starting at 10 mo of age. This increase was preceded by increases in oxidative stress levels in the lungs of Hhip(+/-) vs. Hhip(+/+) mice. To our knowledge, these results provide the first line of evidence that HHIP is involved in maintaining normal lung function and alveolar structures. Interestingly, antioxidant N-acetyl cysteine treatment in mice starting at age of 5 mo improved lung function and prevented emphysema development in Hhip(+/-) mice, suggesting that N-acetyl cysteine treatment limits the progression of age-related emphysema in Hhip(+/-) mice. Therefore, reduced lung function and age-related spontaneous emphysema development in Hhip(+/-) mice may be caused by increased oxidative stress levels in murine lungs as a result of haploinsufficiency of Hhip.

Inhibition of HHIP Promoter Methylation Suppresses Human Gastric Cancer Cell Proliferation and Migration.

BACKGROUND/AIMS: Human hedgehog-interacting protein (HHIP) is a negative regulator of the hedgehog (HH) signaling pathway. It is deregulated in gastric cancer. The underlying molecular mechanism of HHIP-induced inhibition of HH signaling remains to be determined. METHODS: A lentiviral HHIP expression vector ("LV-HHIP") was established to exogenously over-express HHIP in gastric cancer cells. HHIP protein and mRNA were tested by Western blotting assay and quantitative real-time PCR assay, respectively. Cell survival was tested by the Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was examined by the BrdU ELISA assay and [H3] Thymidine DNA incorporation assay. Cell invasion and migration were tested by the phagokinetic track assay and the "Transwell" assay. The bisulfite-sequencing PCR was applied to test HHIP promoter methylation. RESULTS: In the established (AGS cell line) and primary human gastric cancer cells, LV-HHIP transfection increased HHIP expression and inhibited cancer cell survival and proliferation as well as cell migration and invasion. Furthermore, LV-HHIP significantly attenuated promoter methylation of the endogenous HHIP gene in AGS cells, causing it upregulation. Inhibition of methylation by 5-aza-dc similarly induced HHIP expression in gastric cancer cells, which inhibited cancer cell proliferation and migration. CONCLUSIONS: Our results suggest that inhibition of HHIP promoter methylation can efficiently inhibit human gastric cancer cell proliferation and migration.

AT2 R deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein (Hhip) gene expression.

Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFß1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expression analysis of Shh signaling members in early stages of chick lung development.

Lung organogenesis is guided by epithelial-mesenchymal interactions that coordinate cellular events responsible for the formation of the respiratory system. Several signaling pathways have been implicated in this process; among them, sonic hedgehog (Shh) signaling has emerged as a crucial regulator of branching morphogenesis in the mammalian lung. Canonical Shh signaling requires the presence of patched (Ptch) and smoothened (Smo) transmembrane receptors in order to induce the activation of glioblastoma (Gli) zinc finger transcription factors that are the true effectors of the pathway. Signal transduction is finely regulated by Ptch1, Gli, and Hhip (hedgehog-interacting protein). The present work characterizes, for the first time, the expression pattern of shh, ptch1, smo, gli1, and hhip in early stages of the embryonic chick lung. In situ hybridization studies revealed that these genes are expressed in the same cellular compartments as their mammalian counterparts, although their proximo-distal distribution is slightly changed. Moreover, the molecular interactions between fibroblast growth factor (FGF) and Shh signaling pathway were assessed, in vitro, by grafting beads soaked in SU5402 (an FGF receptor inhibitor). In the chick lung, Shh signaling seems to have some features that are species specific since shh is not a downstream target of FGF signaling. Nonetheless and despite the observed differences, these findings suggest a role for Shh signaling in the epithelial-mesenchymal interactions that control chick lung morphogenesis.

The hedgehog antagonist HHIP as a favorable prognosticator in glioblastoma.

Inactivation of hedgehog-interacting protein (HHIP) and overexpression of Gli1 play vital roles in the development of diverse human cancers. The aim of this study is to examine the association of HHIP and Gli1 with the clinicopathologic features and prognosis of patients with glioblastoma (GBM). The expression of HHIP and Gli1 in 103 patients with GBM and 32 control patients was investigated by immunohistochemistry. Statistical analysis was utilized to evaluate the association of HHIP as well as Gli1 with clinicopathological characteristics and prognosis of patients. HHIP and Gli1 were dysregulated in GBM. Spearman's rank analysis showed that HHIP and Gli1 had an inverse correlation (r = -0.386, P = 0.000). Expression of HHIP was significantly correlated with age (P = 0.000), gender (P = 0.003), seizure (P = 0.013), resection degree (P = 0.033), adjuvant treatment (P = 0.030), and O(6)-methylguanine-DNA methyltransferase (MGMT) methylation (P = 0.021), while Gli1 expression was significantly correlated with age (P = 0.002), gender (P = 0.033), Karnofsky performance status (KPS) score (P = 0.028), resection degree (P = 0.000), adjuvant treatment (P = 0.014), and MGMT methylation (P = 0.030). Kaplan-Meier method showed that patients with low Gli1 expression had longer overall survival (OS) than those with high Gli1 expression (P = 0.000) and the OS of the patients with HHIP-positive GBM was significantly longer than that of the patients with HHIP-negative GBM (P = 0.000). Univariate and multivariate analyses confirmed that HHIP expression and Gli1 expression were independent prognostic factors. Our data suggested that expression of HHIP could be considered as significant prognostic marker for patients with GBM.

Hydrothermal Hot Isostatic Pressing (HHIP)-Experimental Proof of Concept.

A new hydrothermal hot isostatic pressing (HHIP) approach, involving hydrothermal water conditions and no usage of inert gas, was hypothesized and tested on 3D-printed Al-10%Si-0.3%Mg (%Wt) parts. The aluminum-based metal was practically inert at the applied HHIPing conditions of 300-350 MPa and 250-350 °C, which enabled the employment of a long (6-24 h) HHIP treatment with hardly any loss of material (the overall loss due to corrosion was mostly <0.5% w/w). Applying the new approach on the above-mentioned samples resulted in an 85.7% reduction in the AM micro-pores, along with a 90.8% reduction in the pores' surface area at a temperature of 350 °C, which is much lower than the 500-520 °C applied in common argon-based aluminum HIPing treatments, while practically maintaining the as-recieved microstructure. These results show that better mechanical properties can be expected when using the suggested treatment without affecting the material fatigue resistance due to grain growth. The proof of concept presented in this work can pave the way to applying the new HHIPing approach to other AM metal parts.

LncRNA HHIP-AS1 suppresses lung squamous cell carcinoma by stabilizing HHIP mRNA.

AIMS: Lung squamous cell carcinoma (LUSC) causes over 400,000 deaths annually, yet it lacks targeted therapy. A major antagonist of Hedgehog pathway, HHIP (Hedgehog Interacting Protein) plays an important role in LUSC; however, the regulatory mechanism remains unclear. Long non-coding RNA HHIP-AS1 plays suppressive or promotive roles in different cancers, but its role in LUSC remains unknown. This manuscript is to investigate regulatory mechanism of HHIP and the role of HHIP-AS1 in LUSC. MAIN METHODS: Precision-cut lung slices (PCLS) from human LUSC samples are cultured to mimic LUSC growth. Overexpression and knockdown in multiple LUSC cell lines and PCLS are achieved by lentivirus infection. Transcriptome profile and lung cancer activity are evaluated by RNA-sequencing, immunostaining and CCK8 assay etc. KEY FINDINGS: HHIP is regulated independently of Hh pathway in LUSC. Additionally, downregulation of HHIP-AS1 is associated with poor prognosis. Consistently, HHIP-AS1 inhibits LUSC growth by suppressing cell proliferation and migration. Transcriptome profiling of HHIP-AS1 knockdown (KD) cells uncovered HHIP downregulation. Interestingly, a comparison between the transcriptomes of HHIP-AS1 KD or HHIP KD cells manifested high similarity. Subsequently it's confirmed that HHIP-AS1 regulates HHIP in LUSC cells. Notably, HHIP-AS1 regulation on LUSC growth is achieved through stabilizing HHIP mRNA rather than regulating MIR-153-3P/PCDHGA9 or MIR-425-5P/DNYC1I2. Finally, it's confirmed in PCLS from human LUSC samples that HHIP-AS1 suppresses LUSC via regulating HHIP mRNA. SIGNIFICANCE: This study uncovers HHIP-AS1 as a novel tumor suppressor in LUSC and provides new insights into the molecular regulation of LUSC, which will help developing new therapeutic strategies.

Differential association of hedgehog pathway in development of cervical carcinoma and its chemo-tolerance.

Cervical carcinoma (CACX) is still a dreadful threat to women in developing countries. Available conventional chemo-radiation therapies are not sufficient to restrict the disease recurrence. To unravel the mechanism of the disease recurrence, alteration of hedgehog self-renewal pathway was evaluated during development of CACX and in chemo-tolerance of the tumor. We have analyzed the alterations (expression/methylation/deletion) of some key regulatory genes (HHIP/SUFU/SHH/ SMO/GLI1) of hedgehog self-renewal pathway in cervical lesions at different clinical stages and compared with different datasets, followed by their clinico-pathological correlations. The changes in expression/methylation of the genes were then evaluated in two CACX cell lines (SiHa/HeLa) after treatment with chemotherapeutic drug cisplatin at different concentrations. Down regulation (mRNA/protein) of the antagonists HHIP and SUFU due to promoter methylation and/or deletion along with upregulation (protein) of agonists SHH, SMO and GLI1 was seen in early invasive lesions and subsequent clinical stages. Reduced protein expression of HHIP and SUFU showed significant association with high/intermediate expression of agonists SHH, SMO, GLI1 in the tumors and also poor prognosis of the patients. It was evident that cisplatin could restrict the growth of HeLa and SiHa cells through significant upregulation of antagonists HHIP and SUFU due to their promoter hypomethylation and down regulation of SHH in a concentration dependent manner without any significant changes in expression of SMO and GLI1, leading to the tumor cells in a dormant state. Thus, interplay of the agonists and antagonists has important role in activation of hedgehog pathway during development of CACX, whereas inactivation of the pathway due to upregulation of the antagonists is an important phenomenon in chemo-tolerance of the tumor. This suggests importance of epigenetic modification in chemo-resistance of CACX.

CircFAM114A2 inhibits the progression of hepatocellular carcinoma via miR-630/HHIP axis.

BACKGROUND: Many studies have shown that circular RNAs (circRNAs) are abnormally expressed in various tumor tissues and served as a key regulator in the occurrence and development of cancer. However, in hepatocellular carcinoma (HCC), the molecular mechanism of circRNAs in body fluids remains to be further explored. METHODS: The expression levels of genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Cell counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), wound healing assay, Transwell assays, flow cytometry, and tumor formation models in nude mice were conducted to investigate the effects of circFAM114A2 on HCC cells both in vitro and in vivo. RNA antisense purification (RAP), dual luciferase reporter assays and rescue assays were carried out to verify the interaction between circFAM114A2, miR-630 and HHIP. RESULTS: CircFAM114A2 was significantly downregulated in HCC tissues and was associated with microvascular invasion and lymph node metastasis of HCC patients. We also observed that circFAM114A2 was lowly expressed in HCC plasma, which may serve as an effective biomarker to screen HCC patients from healthy controls (area under curve (AUC)=0.922). In vitro, circFAM114A2 overexpression significantly blunted HCC cell proliferation, migration, invasion, and promoted apoptosis, whereas circFAM114A2 silencing posed opposite effects. In vivo, circFAM114A2 overexpression inhibited the growth of HCC cells. Mechanistically, circFAM114A2 could increase the expression of the tumor suppressor HHIP via acting as a sponge for miR-630. CONCLUSIONS: CircFAM114A2 exerts a tumor suppressor role in HCC through miR-630/HHIP axis, and may be served as a potential diagnostic and therapeutic biomarker for HCC patients.

Circulating HHIP Levels in Women with Insulin Resistance and PCOS: Effects of Physical Activity, Cold Stimulation and Anti-Diabetic Drug Therapy.

Serum human hedgehog-interacting protein (HHIP) concentration is associated with diabetes. However, the relationship between HHIP and polycystic ovary syndrome (PCOS) or abnormal sex hormones remains unknown. This study was an observational cross-sectional study, with additional short-term intervention studies and follow-up studies. Bioinformatics analysis was performed to explore the association of PCOS with metabolic-related genes and signaling pathways. OGTT and EHC were performed on all participants. Lipid infusion, cold exposure, and 45-min treadmill test were performed on all healthy women. A total of 137 women with PCOS were treated with metformin, GLP-1RA, or TZDs for 24 weeks. Serum HHIP levels were higher in insulin resistance (IR) and PCOS women. Circulating HHIP levels were significantly correlated with adiponectin (Adipoq) levels, obesity, IR, and metabolic indicators. A correlation presented between HHIP and DHEA-S, FAI, SHBG, and FSH. Serum HHIP levels were significantly elevated by oral glucose challenge in healthy women, but not affected by EHC. Lipid infusion decreased serum HHIP levels, while cold exposure increased HHIP levels in healthy women. GLP-1RA and TZD treatment reduced serum HHIP levels in PCOS women, while metformin treatment did not affect HHIP levels. HHIP may be a useful biomarker and novel drug target for PCOS and IR individuals.

lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway.

BACKGROUND: lncRNA, a type of non-coding RNA, plays an important role in the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). In this study, lncRNA and mRNA microarrays were performed to study the change of gene expression during osteogenic differentiation of BM-MSCs. We focused on Hedgehog interacting protein (HHIP), because HHIP mRNA and lncRNA HHIP-AS1 were gradually down-regulated on days 0, 7, and 14 during osteogenic differentiation. In addition, the gene coding lncRNA HHIP-AS1 is located on the anti-sense of Hhip gene, implying the potential interaction between lncRNA HHIP-AS1 and HHIP mRNA. METHODS: BM-MSCs with over-expressed or silenced lncRNA HHIP-AS1 were constructed to explore the biological role of HHIP-AS1 in osteogenic differentiation. BM-MSCs were lysed to determine the alkaline phosphatase activity. Fluorescence in situ hybridization and immunofluorescence were performed to analyze HHIP-AS1, HHIP, RUNX2 and osteocalcin. RESULTS: Overexpression of lncRNA HHIP-AS1 increased HHIP expression, which suppressed Hedgehog signaling pathway, as indicated by the reduction of SMO, Gli1 and Gli2. The suppression of Hedgehog signal was associated with the inhibited osteogenesis. HHIP knockdown abolished the suppression of osteogenesis induced by lncRNA HHIP-AS1 overexpression. Through binding to HHIP mRNA, lncRNA HHIP-AS1 recruited ELAVL1 to HHIP mRNA, whereby increasing the mRNA stability and the protein level. CONCLUSIONS: This study revealed that down-regulation of HHIP due to lncRNA HHIP-AS1 reduction promoted the osteogenic differentiation of BM-MSCs though removing the suppression of Hedgehog signal.

Hedgehog pathway and its inhibitors in chronic obstructive pulmonary disease (COPD).

COPD affects millions of people and is now ranked as the third leading cause of death worldwide. This largely untreatable chronic airway disease results in irreversible destruction of lung architecture. The small lung hypothesis is now supported by epidemiological, physiological and clinical studies. Accordingly, the early and severe COPD phenotype carries the most dreadful prognosis and finds its roots during lung growth. Pathophysiological mechanisms remain poorly understood and implicate individual susceptibility (genetics), a large part of environmental factors (viral infections, tobacco consumption, air pollution) and the combined effects of those triggers on gene expression. Genetic susceptibility is most likely involved as the disease is severe and starts early in life. The latter observation led to the identification of Mendelian inheritance via disease-causing variants of SERPINA1 - known as the basis for alpha-1 anti-trypsin deficiency, and TERT. In the last two decades multiple genome wide association studies (GWAS) identified many single nucleotide polymorphisms (SNPs) associated with COPD. High significance SNPs are located in 4q31 near HHIP which encodes an evolutionarily highly conserved physiological inhibitor of the Hedgehog signaling pathway (HH). HHIP is critical to several in utero developmental lung processes. It is also implicated in homeostasis, injury response, epithelial-mesenchymal transition and tumor resistance to apoptosis. A few studies have reported decreased HHIP RNA and protein levels in human adult COPD lungs. HHIP+/- murine models led to emphysema. HH pathway inhibitors, such as vismodegib and sonidegib, are already validated in oncology, whereas other drugs have evidenced in vitro effects. Targeting the Hedgehog pathway could lead to a new therapeutic avenue in COPD. In this review, we focused on the early and severe COPD phenotype and the small lung hypothesis by exploring genetic susceptibility traits that are potentially treatable, thus summarizing promising therapeutics for the future.

Differential promoter usages of PTCH1 and down regulation of HHIP are associated with HNSCC progression.

PURPOSE: The study was aimed to understand the importance of the hedgehog signaling pathway in development of head and neck squamous cell carcinoma (HNSCC). METHODS: The molecular profiles of the key regulatory genes of the pathway were analysed in the adjacent normal epithelium and tumor samples. The findings were validated in HNSCC cell line. RESULTS: In the bioinformatical analysis, severe reduction in the expression of HHIP was evident in the datasets. The protein and mRNA expression studies in our sample pool revealed interplay of various isoforms of PTCH1 gene (PTCH1-1 and 1B) together with high/medium expression of GLI, SHH, SMO and HHIP in the basal/parabasal layers of the normal epithelium. As the disease progressed, severe downregulation of HHIP coupled with upregulation of GLI1 and differential expression pattern of various PTCH1 gene isoform was evident. Promoter methylation analysis of PTCH1 gene revealed the involvement of more than one promoter of PTCH1 in regulating the expression of different isoform of this gene during tumorigenesis. Treating the FaDu cell line with the demethylating agent 5-aza-2'-deoxycytidine reversed the methylation effects of HHIP and PTCH1 and de-activated the pathway. Also, reduced expression of HHIP-AS1 was observed in our sample pool suggesting multiple ways of regulation of the HHIP gene. Lastly, the patients with under expression of HHIP, HHIP-AS1, high expression of GLI1 showed worse five-year over-all survival trend. CONCLUSION: Dynamic promoter switching of PTCH1 and frequent inactivation of HHIP are the key regulatory events of hedgehog pathway activation in HNSCC.

MiR-199b-5p Promotes Gastric Cancer Progression by Regulating HHIP Expression.

OBJECTIVES: Gastric cancer (GC) is one of the most common malignant tumors. More and more evidences support the role of microRNAs (miRNAs) in tumor progression. However, the role of miRNAs in human GC remains largely unknown. METHODS: Based on the published gastric cancer expression profile data, combined with bioinformatics analysis, potential miRNAs in the process of GC were screened. The expression of miR-199b-5p in GC cells and patients' plasma was detected by RT-PCR. The effects of miR-199b-5p on GC in vitro were detected by EdU proliferation assay, colony formation assay, Transwell assay and wound healing assay. Western blot was used to detect epithelial-mesenchymal transition (EMT) related proteins. The subcutaneous tumorigenesis model and metastatic tumor model of mice were used to study its effect in vivo. Bioinformatics and Dual luciferase reporter assay were used to verify the effect of miR-199b-5p and its target gene. RESULTS: Through bioinformatics analysis, we screened a novel miRNA miR-199b-5p that was significantly up-regulated in GC tissue and associated with poor prognosis of GC patients. RT-PCR results showed that its expression was also up-regulated in GC cell lines and patients' plasma. MiR-199b-5p can significantly promote GC cell proliferation and migration in vitro and in vivo. Western blot showed that miR-199b-5p could promote the EMT process of GC. HHIP has been proved to be a target of miR-199b-5p, and the recovery of HHIP can weaken the effect of miR-199b-5p. CONCLUSION: MiR-199b-5p may play an oncogene role in GC by targeting HHIP, suggesting that miR-199b-5p may be a potential therapeutic target for GC.

MicroRNA-221 promotes tumor progression by targeting HHIP in human glioblastoma.

BACKGROUND: MicroRNAs are found to be aberrantly expressed in multiple cancers, including glioblastoma (GBM), and microRNA-221 (miR-221) has been verified as an oncogene in various human cancers. Nevertheless, the role of miR-221 in GBM is unclear. This study aimed to investigate the miR-221 expression level in GBM and to evaluate its function and underlying mechanisms. METHODS: Western blotting and qPCR were used to determine the expression of human hedgehog-interacting protein (HHIP) and miR-221 levels. MiR-221-inhibited cell models were constructed, and siRNA was used for HHIP silencing. Cell proliferation was analyzed by MTT and colony formation assays and a subcutaneous xenograft model. Cell migration and invasion was analyzed by wound healing and Transwell invasion assays. A dual luciferase reporter assay system was used to clarify the relationship between miR-221 and HHIP. RESULTS: The results of this study revealed that miR-221 expression was upregulated in GBM tissues and A172, U251, as well as T98G cells, as detected by real-time PCR analysis. MTT, Transwell, and colony formation assays revealed that miR-221 knockdown could suppress GBM cells from proliferating, migrating, and invading in vitro. Moreover, animal experiments showed that tumor growth in vivo was inhibited when miR-221 expression decreased. Furthermore, HHIP was predicted and verified to be a target of miR-221 by bioinformatics analysis, and luciferase and western blot assays. In addition, HHIP silencing rescued the suppressive effect of a miR-221 inhibitor on the proliferation, migration, and invasion of GBM cells. CONCLUSIONS: Our results indicated that miR-221 is upregulated in GBM and enhances tumor progression by targeting HHIP, which suggests this may be a potential therapeutic target for GBM.