Ciliary Hedgehog Signaling Restricts Injury-Induced Adipogenesis.

Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.

SIRT1 mediates the inflammatory response of macrophages and regulates the TIMP3/ADAM17 pathway in atherosclerosis.

OBJECTIVE: Macrophage polarization and the resulting phenotype have versatile roles in atherosclerosis. The study aims to decipher the role of SIRT1 in regulating macrophage phenotypes and atherosclerosis development. METHODS: Two mouse lines of SIRT1△Mac/ApoE-/- and SIRT1fl/fl/ApoE-/- were fed with high-fat diet to generate atherosclerotic lesion. Mouse peritoneal macrophages were isolated and transfected with SIRT1-overexpressing vector or vector-null. RESULTS: The SIRT1△Mac/ApoE-/- mice exhibited greater atherosclerotic lesions, stronger immunofluorescence staining for M1-like macrophage marker, iNOS, and weaker immunofluorescence staining for M2-like macrophage marker, Arginase-1, than the SIRT1fl/fl/ApoE-/- littermates. The gene expressions of M1 markers (IL-1ß, IL-6, and iNOS) were increased and those of M2 markers (IL-10 and Arg-1) decreased in both aortic roots and peritoneal macrophages from SIRT1△Mac/ApoE-/- mice, whereas SIRT1 overexpression rectified the changes in M1/M2 expression. A declined expression of TIMP3 with an increased expression of ADAM17 was noted in SIRT1△Mac/ApoE-/- macrophages, whereas SIRT1 overexpression rescued TIMP3 expression and inhibited ADAM17 expression. CONCLUSION: Our data suggest that SIRT1 deficiency may promote macrophage M1 polarization and regulate the TIMP3/ADAM17 pathway thus favoring atherosclerosis development, indicating an anti-atherosclerotic role of macrophage SIRT1.

Sesame oil downregulates the expression of ADAMTS-4 in high-fat diet-induced atherosclerosis.

Atherosclerosis is a chronic inflammatory disease forming plaques in medium and large-sized arteries. ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) is an extracellular-matrix remodelling enzyme involved in the degradation of versican in the arterial wall. Recent reports indicated that increased expression of ADAMTS-4 is associated with plaque progression and vulnerability. Bioactive components of dietary oil, like sesame oil, are reported to have anti-inflammatory and antioxidant properties. Here, we studied the effect of sesame oil on regulating ADAMTS-4 in high-fat diet-induced atherosclerosis rat model. Our results indicated that sesame oil supplementation improved the anti-inflammatory and anti-oxidative status of the body. It also reduced atherosclerotic plaque formation in high-fat diet-fed rats. Our results showed that the sesame oil supplementation significantly down-regulated the expression of ADAMTS-4 in serum and aortic samples. The versican, the large proteoglycan substrate of ADAMTS-4 in the aorta, was downregulated to normal control level on sesame oil supplementation. This study, for the first time, reveals that sesame oil could down-regulate the expression of ADAMTS-4 in high-fat diet-induced atherosclerosis, imparting a new therapeutic potential for sesame oil in the management of atherosclerosis.

Disease-Causing TIMP3 Variants and Deep Phenotyping of Two Czech Families with Sorsby Fundus Dystrophy Associated with Novel p.(Tyr152Cys) Mutation.

We aim to report the ocular phenotype and molecular genetic findings in two Czech families with Sorsby fundus dystrophy and to review all the reported TIMP3 pathogenic variants. Two probands with Sorsby fundus dystrophy and three first-degree relatives underwent ocular examination and retinal imaging, including optical coherence tomography angiography. The DNA of the first proband was screened using a targeted ocular gene panel, while, in the second proband, direct sequencing of the TIMP3 coding region was performed. Sanger sequencing was also used for segregation analysis within the families. All the previously reported TIMP3 variants were reviewed using the American College of Medical Genetics and the Association for Molecular Pathology interpretation framework. A novel heterozygous variant, c.455A>G p.(Tyr152Cys), in TIMP3 was identified in both families and potentially de novo in one. Optical coherence tomography angiography documented in one patient the development of a choroidal neovascular membrane at 54 years. Including this study, 23 heterozygous variants in TIMP3 have been reported as disease-causing. Application of gene-specific criteria denoted eleven variants as pathogenic, eleven as likely pathogenic, and one as a variant of unknown significance. Our study expands the spectrum of TIMP3 pathogenic variants and highlights the importance of optical coherence tomography angiography for early detection of choroidal neovascular membranes.

Targeting of HBP1/TIMP3 axis as a novel strategy against breast cancer.

Malignant proliferation and metastasis are the main causes of breast cancer death. The transcription factor high mobility group (HMG) box-containing protein 1 (HBP1) is an important tumor suppressor whose deletion or mutation is closely related to the appearance of tumors. Here, we investigated the role of HBP1 in breast cancer suppression. HBP1 enhances the activity of the tissue inhibitors of metalloproteinases 3 (TIMP3) promoter, thereby increasing protein and mRNA levels of TIMP3. TIMP3 increases the phosphatase and tensin homolog (PTEN) protein level by inhibiting its degradation and acts as a metalloproteinase inhibitor to inhibit the protein levels of MMP2/9. In this study, we demonstrated that the HBP1/TIMP3 axis plays a crucial role in inhibiting the tumorigenesis of breast cancer. HBP1 deletion interferes with the regulation of the axis and induces the occurrence and malignant progression of breast cancer. In addition, the HBP1/TIMP3 axis promotes the sensitivity of breast cancer to radiation therapy and hormone therapy. Our study opens new perspectives on the treatment and prognosis of breast cancer.

Suramin analogues protect cartilage against osteoarthritic breakdown by increasing levels of tissue inhibitor of metalloproteinases 3 (TIMP-3) in the tissue.

Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure-activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para/para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use.

SOMAscan Proteomics Identifies Novel Plasma Proteins in Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex disease characterized by the interplay of genetic and environmental factors for which, despite decades of intense research, diagnosis remains rather delayed, and most therapeutic options fail. Therefore, unravelling other potential pathogenetic mechanisms and searching for reliable markers are high priorities. In the present study, we employ the SOMAscan assay, an aptamer-based proteomic technology, to determine the circulating proteomic profile of ALS patients. The expression levels of ~1300 proteins were assessed in plasma, and 42 proteins with statistically significant differential expression between ALS patients and healthy controls were identified. Among these, four were upregulated proteins, Thymus- and activation-regulated chemokine, metalloproteinase inhibitor 3 and nidogen 1 and 2 were selected and validated by enzyme-linked immunosorbent assays in an overlapping cohort of patients. Following statistical analyses, different expression patterns of these proteins were observed in the familial and sporadic ALS patients. The proteins identified in this study might provide insight into ALS pathogenesis and represent potential candidates to develop novel targeted therapies.

Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells.

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III-IV than stage I-II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion.

CircCSNK1G3 up-regulates miR-181b to promote growth and metastasis via TIMP3-mediated epithelial to mesenchymal transitions in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common form of kidney cancer, with a high recurrence rate and metastasis capacity. Circular RNAs (circRNAs) have been suggested to act as the critical regulator in several diseases. This study is designed to investigate the role of circCSNK1G3 on RCC progression. We observed a highly expression of circCSNK1G3 in RCC tissues compared with normal tissues. The aberrantly circCSNK1G3 promoted the tumour growth and metastasis in RCC. In the subsequent mechanism investigation, we discovered that the tumour-promoting effects of circCSNK1G3 were, at least partly, achieved by up-regulating miR-181b. Increased miR-181b inhibits several tumour suppressor gene, including CYLD, LATS2, NDRG2 and TIMP3. Furthermore, the decreased TIMP3 leads to the enhanced epithelial to mesenchymal transition (EMT) process, thus promoting the cancer metastasis. In conclusion, we identified the oncogenic role of circCSNK1G3 in RCC progression and demonstrated the regulatory role of circCSNK1G3 induced miR-181b expression, which leads to TIMP3-mediated EMT process, thus resulting in tumour growth and metastasis in RCC. This study reveals the promise of circCSNK1G3 to be developed as a potential diagnostic and prognostic biomarker in the clinic. And the roles of circCSNK1G3 in cancer research deserve further investigation.

Circ_NNT suppresses the apoptosis and inflammation in glucose-induced human retinal pigment epithelium by regulating miR-320b/TIMP3 axis in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a frequent complication of diabetes. Recent reports have showed that circular RNAs (circRNAs) play important roles in DR progression. Herein, the aim of this study was to explore the role and molecular mechanism of circ_NNT in DR process. METHODS: Human retinal pigment epithelial cells ARPE-19 were treated with high glucose (HG) in experimental group. The expression of circ_NNT, miR-320b, and TIMP3 (TIMP Metallopeptidase Inhibitor 3) were determined using quantitative real-time polymerase chain reaction and Western blot. In vitro experiments were conducted by 5-ethynyl-2'-deoxyuridine (EdU) assay, MTT assay, flow cytometry, Western blot, and ELISA. The binding interaction was confirmed using dual-luciferase reporter and pull-down assays. RESULTS: HG stimulation led to a decrease of circ_NNT and TIMP3 expression, and an increase of miR-320b expression in ARPE-19 cells. Functionally, circ_NNT up-regulation reversed HG-evoked apoptosis and inflammation in ARPE-19 cells. Mechanistically, circ_NNT acted as a sponge for miR-320b to elevate TIMP3 expression. Further rescue experiments showed that miR-320b elevation attenuated the protective effects of circ_NNT on HG-induced ARPE-19 cells. Moreover, inhibition of miR-320b protected ARPE-19 cells against HG-evoked apoptosis and inflammation, which were abolished by TIMP3 knockdown. CONCLUSION: Circ_NNT protected ARPE-19 cells against HG-evoked apoptosis and inflammation via elevating TIMP3 through sequestering miR-320b, indicating that up-regulation of circ_NNT might contribute to the inhibition of DR process.

Comprehensive analysis of PTEN-related ceRNA network revealing the key pathways WDFY3-AS2 - miR-21-5p/miR-221-3p/miR-222-3p - TIMP3 as potential biomarker in tumorigenesis and prognosis of kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is one of the most common malignancies, and there is still a lack of effective biomarkers for early detection and prognostic prediction. In here, we compared the characteristics of RNA sequencing data sets of KIRC samples based on the tumor suppressor gene phosphatase and tensin homolog (PTEN). The 1016 long noncoding RNAs, 48 microRNAs (miRNAs), and 2104 messenger RNAs associated with PTEN were identified and these genes were differentially expressed between tumor and paracancerous tissues. The most relevant pathway was found to be WDFY3-AS2 - miR-21-5p/miR-221-3p/miR-222-3p - TIMP3 according to the rules of competing endogenous RNA (ceRNA) regulation. WDFY3-AS2 and TIMP3 expression were positively correlated and reduced in KIRC samples, while miR-21-5p, miR-221-3p, and miR-222-3p were relatively highly expressed. The relatively low expression of WDFY3-AS2 and TIMP3 in KIRC were associated with poor prognosis in KIRC patients, while higher expression of miR-21-5p, miR-221-3p, and miR-222-3p predicted reduced survival (p < 0.05). Univariate and multivariate Cox regression analysis showed that lower expression of WDFY3-AS2 and TIMP3 was significantly related to tumor grade, tumor size, lymph node metastasis, distant metastasis, and TNM stage. The expression of TIMP3 in KIRC tissues was also verified by immunohistochemistry, and the results were consistent with our analytical data. In summary, this study constructed a new model with clinical predictive value and identified the WDFY3-AS2/TIMP3 pathway that was closely associated with the prognosis of KIRC, which could serve as a promising biomarker for the diagnosis and treatment of KIRC.

Remifentanil reduces the proliferation, migration and invasion of HCC cells via lncRNA NBR2/miR-650/TIMP3 axis.

Cancer cell hyperproliferation and metastasis are major causes of cancer-associated mortality. Although the use of anaesthetics and analgesics may affect cancer cell metastasis, the underlying molecular mechanism remains unclear. This study aimed to explore the mechanisms of action of remifentanil on hepatocellular carcinoma (HCC) progression. Cell viability was measured by the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide assay. Quantitative real-time polymerase chain reaction and Western blotting were performed to assess the expression levels of long non-coding RNA (lncRNA) neighbour of BRCA1 gene 2 (NBR2), microRNA (miR)-650 and tissue inhibitor of metalloproteinase-3 (TIMP3) in HCC cells. Wound healing and transwell assays were employed to evaluate the migration and invasion of HCC cells respectively. The target relationships between miR-650 and NBR2/TIMP3 were confirmed by dual luciferase reporter assay. Remifentanil reduced the viability of HCC cells in a dose-dependent manner. Remifentanil treatment significantly increased the expression of lncRNA NBR2 and TIMP3, and repressed miR-650 expression in HCC cells. Decreased lncRNA NBR2 or increased miR-650 promoted the proliferation, migration and invasion of remifentanil-treated HCC cells. LncRNA NBR2 targeted miR-650, and miR-650 further targeted TIMP3. Moreover, miR-650 down-regulation or TIMP3 up-regulation reversed the effects of lncRNA NBR2 knockdown that caused an enhancement of cell viability, migration and invasiveness in remifentanil-treated HCC cells. Thus remifentanil reduces the proliferation, migration and invasion of HCC cells via the lncRNA NBR2/miR-650/TIMP3 axis in vitro.

Transcriptomic landscape of TIMP3 oncosuppressor activity in thyroid carcinoma.

BACKGROUND: Papillary thyroid cancer (PTC) is the most frequent thyroid tumor. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene encodes a matrix metalloproteinases inhibitor that exerts a tumor suppressor role in several tumor types. TIMP3 is frequently downregulated in PTC by promoter methylation. We have previously functionally demonstrated that TIMP3 exerts an oncosuppressor role in PTC: TIMP3 restoration in the PTC-derived NIM1 cell line affects in vitro migration, invasion and adhesive capability, while reduces tumor growth, angiogenesis and macrophage recruitment in vivo. To get a deeper insight on the mediators of TIMP3 oncosuppressor activity in thyroid tumors, here we focused on the TIMP3 related transcriptome. METHODS: TCGA database was used for investigating the genes differentially expressed in PTC samples with low and high TIMP3 expression. Genome wide expression analysis of clones NIM1-T23 (expressing a high level of TIMP3 protein) and NIM1-EV (control empty vector) was performed. Gene sets and functional enrichment analysis with clusterProfiler were applied to identify the modulated biological processes and pathways. CIBERSORT was used to evaluate the distribution of different immunological cell types in TCGA-PTC tumor samples with different TIMP3 expression levels. Real time PCR was performed for the validation of selected genes. RESULTS: Thyroid tumors with TIMP3-high expression showed a down-modulation of inflammation-related gene sets, along with a reduced protumoral hematopoietic cells fraction; an enrichment of cell adhesion functions was also identified. Similar results were obtained in the TIMP3-overexpessing NIM1 cells in vitro model, where a down-regulation of immune-related function gene sets, some of which also identified in tumor samples, was observed. Interestingly, through enrichment analysis, were also recognized terms related to cell adhesion, extracellular matrix organization, blood vessel maintenance and vascular process functions that have been found modulated in our previous in vitro and in vivo functional studies. CONCLUSIONS: Our results highlight the correlation of TIMP3 expression levels with the regulation of inflammatory functions and the immune infiltration composition associated with different PTC prognosis, thus providing a broader view on the oncosuppressor role of TIMP3 in PTC.

The novel histone deacetylase inhibitor pracinostat suppresses the malignant phenotype in human glioma.

INTRODUCTION: Glioma is the most common malignant brain tumor in adults. The effects of conventional treatment regimens are still limited to prolonging the survival of patients. Histone deacetylases (HDACs) are potential targets for tumor treatment. Pracinostat is a new type of HDAC inhibitor (HDACi) that has a significant antitumor effect on a variety of tumors. Thus, we aim to investigate the role of pracinostat in human glioma and explored its underlying mechanism. METHODS: Cell viability, proliferation and apoptosis of human glioma cell lines were measured by Cell Counting kit 8 and flow cytometry. Pathway verification and protein interaction were determined by quantitative real-time polymerase chain reaction, Western blotting and immunofluorescence staining. Transwell technology was used to assess the migration and invasion of cells. Clinical significance of TIMP3, MMP9 and MMP2 in glioma was analyzed through The Cancer Genome Atlas (TCGA) database and the Genotype-Tissue Expression (GTEx) database. RESULTS: Functionally, pracinostat not only inhibited proliferation and induced apoptosis but also inhibited migration and invasion in human glioma cell lines. Mechanistically, pracinostat increased the expression of TIMP3 and decreased the expression of MMP2, MMP9 and VEGF in human glioma cells in vitro and in vivo. In addition, pracinostat inhibited both the PI3K/Akt signaling pathway and the STAT3 pathway. CONCLUSIONS: Our results strongly support the potential clinical use of pracinostat as a novel therapy for human glioma in the near future.

CHOP Increases TRIB3-Dependent miR-208 Expression to Potentiate Vascular Smooth Muscle Cell Proliferation and Migration by Downregulating TIMP3 in Atherosclerosis.

BACKGROUND: C/EBP homologous protein (CHOP) has been identified as a suitable therapeutic target to combat atherosclerosis but the mechanism has not been fully studied. Here, we sought to define the role and underlying mechanism of CHOP in atherosclerosis. METHODS: Mouse models of atherosclerosis in ApoE-/- mice were established by high-fat feeding, where miR-208 expression was determined. Then atherosclerotic plaque tissues were isolated from the model mice. Loss- and gain-function assays were performed on trypsinized vascular smooth muscle cells (VSMCs) to test the in vitro effect of CHOP in controlling the tribbles homologue 3 (TRIB3)/microRNA-208 (miR-208)/tissue inhibitor of metalloproteinases-3 (TIMP3) axis in atherosclerosis by determining cell proliferation and migration as well as blood lipid levels. Moreover, expression of α-smooth muscle actin (α-SMA) and type I collagen expression was determined using immunofluorescence staining to assess plaque stability in mice. RESULTS: miR-208 expression was elevated in atherosclerosis samples and miR-208 overexpression promoted proliferation and migration of VSMCs but diminished plaque stability in mice. TIMP3 was targeted by miR-208, which could be abrogated by upregulation of TIMP3. In addition, CHOP increased TRIB3 expression to upregulate miR-208 and to downregulate TIMP3, which potentiated VSMC proliferation and migration in vitro and in vivo. CONCLUSION: Taken together, inhibition of CHOP may inhibit the proliferation and migration of VSMCs as well as reduce the levels of TC, TG, and LDL-C but increase the level of HDL-C through the TRIB3/miR-208/TIMP3 axis, thereby inhibiting the progression of atherosclerosis.

Expression of microRNA-21 and TIMP-3 in paradoxical psoriasiform reactions during treatment with antitumor necrosis factor agents.

BACKGROUND: Expression of microRNA-21 (miR-21) is increased in psoriasis, leading to reduced levels of epidermal tissue inhibitor of matrix metalloproteinase 3 (TIMP-3), a highly potent inhibitor of the tumor necrosis factor alpha (TNFα) sheddase TACE (TNFα-converting enzyme)/ADAM17. We described the profile of miR-21 and TIMP-3 in paradoxical psoriasiform reactions induced by anti-TNFα drugs and in a control group to elucidate the pathogenesis of this reactions. METHODS: We performed an analytic, cross-sectional, prospective, experimental case-control study. We compared our findings with those of non-induced psoriasis. RESULTS: We included 15 patients with a change of morphology (plaque to guttate psoriasis) and 10 patients with induced psoriasis (six palmoplantar pustulosis and four plaque psoriasis). Consecutive patients with different subtypes of non-induced, non-systemically treated psoriasis were included as a control group. We found that most cases with guttate psoriasis and with induced plaque psoriasis cases showed high expression of TIMP-3 expression and decreased or poorly increased levels of miR-21. The expression pattern was not homogeneous in the cases of induced palmoplantar pustulosis. These profiles differ from those of non-induced psoriasis. CONCLUSION: We conclude that various pro-inflammatory cytokine profiles are involved in the pathogenesis of paradoxical psoriasiform reactions and non-induced psoriasis.

Gene networks determine predisposition to AMD.

PURPOSE: AMD genetic studies have revealed various genetic loci as causal to AMD pathology. We have described the genetic complexity of Indian AMD by describing the interaction of genotypes and subsequent changes in protein expression under the influence of environmental factors. This can be utilized to enhance the diagnostic and therapeutic efficacy in AMD patients. DESIGN: Genotype association was studied in 464 participants (AMD =277 & controls = 187) for eight genetic variants and their corresponding protein expression METHODS: SNP analysis and protein expression analysis was carried out in AMD and controls in tandem with longitudinal assessment of protein levels during the course of AMD pathology. ANCOVA and contrast analysis were used to examine the genotypic interactions and corresponding alterations in protein levels. In order to identify the important genetic variants Logistic Regression (LR) modeling was carried out and to authenticate the model Area under the Receiver Operating Characteristic curve (AUROC) were also computed. RESULTS: We have found genetic variants of rs5749482 (TIMP-3), rs11200638 (HTRA1), rs769449 (APOE) and rs6795735 (ADAMTS9) to be associated with AMD, concomitant with significant alterations of studied proteins levels. Analysis also revealed that the genetic interaction between APOE-HTRA1 genotypes and changes in LIPC levels (>6 pg/ug) by one unit change in SNP, play a crucial role in AMD. LR model suggested that the seven factors (including both genetic and environmental) can be utilized to predict the AMD cases with 88% efficacy and 95.6% AUROC. CONCLUSION: Results suggest that diagnostic and therapeutic strategy for Indian AMD must include estimation of genetic interaction and concomitant changes in expression levels of proteins under influence of environmental factors.

Thymoquinone Suppresses Angiogenesis in DEN-Induced Hepatocellular Carcinoma by Targeting miR-1-3p.

Hepatocellular carcinoma (HCC) is characterized by its high vascularity and metastasis. Thymoquinone (TQ), the main bio-active constituent of Nigella sativa, has shown anticancer and hepatoprotective effects. TQ's anticancer effect is mediated through miRNA regulation. miR-1-3p plays a significant role in various cancers but its role in HCC invasiveness remains poorly understood. Bio-informatics analysis predicted that the 3'-UTR of TIMP3 is a target for miR-1-3p; Rats were equally divided into four groups: Group 1, the negative control; Group 2 received TQ; Group 3 received DEN; and Group 4 received DEN after pretreatment with TQ. The expression of TIMP3, MMP2, MMP9, and VEGF in rats' liver was determined immunohistochemically. RT-qPCR was used to measure the miR-1-3p level in rats' liver, and TIMP3, MMP2, MMP9, and VEGF in the HepG2 cells after being transfected with miR-1-3p mimic or inhibitor; In rats pretreated with TQ, a decreased expression of MMP2, MMP9 and VEGF, and increased expression levels of TIMP3 and miR-1-3p were detected. Treating the HepG2 cells with miR-1-3p mimic led to the upregulation of TIMP3 and downregulation of MMP2, MMP9, and VEGF, and showed a significant delay in wound healing; These results suggested that the anti-angiogenic effect of TQ in HCC may be mediated through the regulation of miR-1-3p.

CircRNA-001241 mediates sorafenib resistance of hepatocellular carcinoma cells by sponging miR-21-5p and regulating TIMP3 expression.

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and its incidence is on the rise, closely related to advanced liver disease. Sorafenib chemotherapy is one of the main treatment options for patients with advanced HCC. Despite several reports on HCC multidrug resistance, the underlying regulatory mechanisms are still unclear. In this study, we found circ-001241 was significantly upregulated in HCC tissues and cells. Knockdown of circ-001241 markedly inhibited HCC cell proliferation and decreased sorafenib-resistance. More importantly, circRNA acts as a ceRNA to suppress the expression and activity of miR-21-5p, leading to the increase in TIMP3 expression. In addition, circRNA-001241 facilitated HCC sorafenib-resistance by regulating the miR-21-5p/TIMP3 axis. Taken together, our study elucidated the oncogenic role of circ-001241 in mediating sorafenib resistance in HCC, providing insights and opportunities to overcome sorafenib resistance in patients with advanced hepatocellular carcinoma.

Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces.

KEY POINTS: microRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression. miRs are involved in regulating cellular activities in response to mechanical loading in all physiological systems, although it is largely unknown whether this response differs with increasing magnitudes of load. miR-221, miR-222, miR-21-5p and miR-27a-5p were significantly increased in ex vivo cartilage explants subjected to increasing load magnitude and in in vivo joint cartilage exposed to abnormal loading. TIMP3 and CPEB3 are putative miR targets in chondrocytes Identification of mechanically regulated miRs that have potential to impact on tissue homeostasis provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology, in all physiological systems. ABSTRACT: MicroRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression and are involved in mechano-regulation of cellular activities in all physiological systems. It is unknown whether such epigenetic mechanisms are regulated in response to increasing magnitudes of load. The present study investigated mechano-regulation of miRs in articular cartilage subjected to 'physiological' and 'non-physiological' compressive loads in vitro as a model system and validated findings in an in vivo model of abnormal joint loading. Bovine full-depth articular cartilage explants were loaded to 2.5 MPa (physiological) or 7 MPa (non-physiological) (1 Hz, 15 min) and mechanically-regulated miRs identified using next generation sequencing and verified using a quantitative PCR. Downstream targets were verified using miR-specific mimics or inhibitors in conjunction with 3'-UTR luciferase activity assays. A subset of miRs were mechanically-regulated in ex vivo cartilage explants and in vivo joint cartilage. miR-221, miR-222, miR-21-5p and miR-27a-5p were increased and miR-483 levels decreased with increasing load magnitude. Tissue inhibitor of metalloproteinase 3 (TIMP3) and cytoplasmic polyadenylation element binding protein 3 (CPEB3) were identified as putative downstream targets. Our data confirm miR-221 and -222 mechano-regulation and demonstrates novel mechano-regulation of miR-21-5p and miR-27a-5p in ex vivo and in vivo cartilage loading models. TIMP3 and CPEB3 are putative miR targets in chondrocytes. Identification of specific miRs that are regulated by increasing load magnitude, as well as their potential to impact on tissue homeostasis, has direct relevance to other mechano-sensitive physiological systems and provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology.