In adult mammals such as rats, the blood-testis barrier (BTB) conferred by adjacent Sertoli cells in the seminiferous epithelium segregates post-meiotic germ cell development from the systemic circulation and is one of the tightest blood-tissue barriers. Yet it must "open" transiently at stages VIII to IX of the epithelial cycle to accommodate the migration of preleptotene/leptotene spermatocytes. While this is a vital event of spermatogenesis, the mechanism(s) that regulates BTB dynamics is virtually unknown. Recent studies have suggested that transforming growth factor-beta3 (TGF-beta3) and tumor necrosis factor alpha (TNFalpha) secreted by Sertoli and germ cells into the microenvironment of the BTB are capable of inducing reversible BTB disruption in vivo, apparently by reducing the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB via the p38 mitogen activated protein (MAP) kinase signaling pathway. In this study, local administration of TGF-beta3 (200 ng/testis) to the testis was shown to reversibly perturb the BTB integrity in vivo. We next sought to delineate the mechanism by which these cytokines maintain the steady-state level of integral membrane proteins: occludin, junctional adhesion molecule-A (JAM-A) and N-cadherin at the BTB. Primary Sertoli cells cultured in vitro were shown to establish intact tight junctions and functional BTB within two days when assessed by transepithelial electrical resistance (TER) measurement across the cell epithelium. Sertoli cell integral membrane protein internalization at the BTB was assessed by biotinylation of cell surface proteins, to be followed by tracking the endocytosed/biotinylated proteins by using specific antibodies. Both TGF-beta3 (3 ng/ml) and TNFalpha (10 ng/ml) were shown to significantly accelerate the kinetics of internalization of JAM-A, N-cadherin, and occludin versus controls. Treatment of cells with phenylarsine oxide (PAO) at 10 microM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-beta3-induced protein internalization. This inhibition of TGF-beta3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-beta3 on protein internalization was further confirmed by RNAi using specific TGF-beta receptor I (TbetaR1) siRNA duplexes. When TbetaR1 was knocked down, the TGF-beta3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-beta3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-beta3 and TNFalpha via an enhancement of protein endocytosis at the BTB.

In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.

Sertoli cells play a significant role in regulating fetal testis compartmentalization to generate testis cords and interstitium during development. The Sertoli cell Wilms' tumor 1 (Wt1) gene, which encodes ~24 zinc finger-containing transcription factors, is known to play a crucial role in fetal testis cord assembly and maintenance. However, whether Wt1 regulates fetal testis compartmentalization by modulating the development of peritubular myoid cells (PMCs) and/or fetal Leydig cells (FLCs) remains unknown. Using a Wt1-/flox; Amh-Cre mouse model by deleting Wt1 in Sertoli cells (Wt1SC-cKO) at embryonic day 14.5 (E14.5), Wt1 was found to regulate PMC and FLC development. Wt1 deletion in fetal testis Sertoli cells caused aberrant differentiation and proliferation of PMCs, FLCs and interstitial progenitor cells from embryo to newborn, leading to abnormal fetal testis interstitial development. Specifically, the expression of PMC marker genes α-Sma, Myh11 and Des, and interstitial progenitor cell marker gene Vcam1 were down-regulated, whereas FLC marker genes StAR, Cyp11a1, Cyp17a1 and Hsd3b1 were up-regulated, in neonatal Wt1SC-cKO testes. The ratio of PMC:FLC were also reduced in Wt1SC-cKO testes, concomitant with a down-regulation of Notch signaling molecules Jag 1, Notch 2, Notch 3, and Hes1 in neonatal Wt1SC-cKO testes, illustrating changes in the differentiation status of FLC from their interstitial progenitor cells during fetal testis development. In summary, Wt1 regulates the development of FLC and interstitial progenitor cell lineages through Notch signaling, and it also plays a role in PMC development. Collectively, these effects confer fetal testis compartmentalization.

The newly identified metastasis-associated in colon cancer-1 (MACC1) gene is involved in angiogenesis, epithelial-to-mesenchymal transition (EMT), invasiveness, and metastasis in a variety of malignancies. Overexpression of MACC1 gene is a prognostic marker for poor outcome of hepatocellular carcinoma (HCC) patients. However, the association between genetic polymorphisms of MACC1 gene and poor outcome in HCC has been not been performed. We therefore investigated the correlation of MACC1 single nucleotide polymorphisms (SNPs) with tumor recurrence and overall survival in HCC patients undergoing liver transplantation (LT).

The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen α3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Colα3(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNFα. While recombinant Colα3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Colα3(IV) NC1 recombinant protein produced in mammalian cells and purified to apparent homogeneity by affinity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Colα3(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, α-catenin, β-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. These findings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis.

Rai14 (retinoic acid induced protein 14) is an actin binding protein first identified in the liver, highly expressed in the placenta, the testis, and the eye. In the course of studying actin binding proteins that regulate the organization of actin filament bundles in the ectoplasmic specialization (ES), a testis-specific actin-rich adherens junction (AJ) type, Rai14 was shown to be one of the regulatory proteins at the ES. In the rat testis, Rai14 was found to be expressed by Sertoli and germ cells, structurally associated with actin and an actin cross-linking protein palladin. Its expression was the highest at the ES in the seminiferous epithelium of adult rat testes, most notably at the apical ES at the Sertoli-spermatid interface, and expressed stage-specifically during the epithelial cycle in stage VII-VIII tubules. However, Rai14 was also found at the basal ES near the basement membrane, associated with the blood-testis barrier (BTB) in stage VIII-IX tubules. A knockdown of Rai14 in Sertoli cells cultured in vitro by RNAi was found to perturb the Sertoli cell tight junction-permeability function in vitro, mediated by a disruption of F-actin, which in turn led to protein mis-localization at the Sertoli cell BTB. When Rai14 in the testis in vivo was knockdown by RNAi, defects in spermatid polarity and adhesion, as well as spermatid transport were noted mediated via changes in F-actin organization and mis-localization of proteins at the apical ES. In short, Rai14 is involved in the re-organization of actin filaments in Sertoli cells during the epithelial cycle, participating in conferring spermatid polarity and cell adhesion in the testis.

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.

The biology of transport of spermatids and spermatid adhesion across the seminiferous epithelium during the epithelial cycle remains largely unexplored. Nonetheless, studies have implicated the role of motor proteins in these cellular events. In this article, we report findings to unravel the role of myosin VIIa, an F-actin-based barbed (+)-end-directed motor protein, to support cellular transport and adhesion in the testis. Using RNA interference to knock down myosin VIIa in Sertoli cells cultured in vitro as a study model was shown to perturb the Sertoli cell tight junction permeability barrier, mediated through disorganization of actin- or microtubule (MT)-based cytoskeletons owing to disruptive changes on the spatiotemporal expression of F-actin or MT-regulatory proteins. Consistent with these in vitro findings, knockdown of myosin VIIa in the testis in vivo also induced disorganization of the actin- and MT-based cytoskeletons across the seminiferous epithelium, mediated by disruptive changes in the spatiotemporal expression of actin- and MT-based regulatory proteins. More important, the transport of spermatids and organelles across the epithelium, as well as cell adhesion, was grossly disrupted. For instance, step 19 spermatids failed to be transported to the adluminal compartment near the tubule lumen to undergo spermiation; in this manner, step 19 spermatids were persistently detected in stage IX and XII tubules, intermingling with step 9 and 12 spermatids, respectively. Also, phagosomes were detected near the tubule lumen in stage I to III tubules when they should have been degraded near the base of the seminiferous epithelium via the lysosomal pathway. In summary, myosin VIIa motor protein was crucial to support cellular transport and adhesion during spermatogenesis.

In the mammalian testes, such as in rats, the directional alignment of polarized elongating/elongated spermatids, in particular step 17-19 spermatids, across the plane of seminiferous epithelium resembles planar cell polarity (PCP) found in hair cells of the cochlea. It is obvious that spermatid PCP is necessary to support the simultaneous development of maximal number of elongating/elongated spermatids to sustain the daily production of > 50 million sperm per adult rat. Studies have shown that the testis indeed expresses multiple PCP proteins necessary to support spermatid PCP. Herein, using physiological and biochemical assays, and morphological analysis, and with the technique of RNA interference (RNAi) to knockdown PCP protein Dishevelled (Dvl) 1 (Dvl1), Dvl2, Dvl3, or Dvl1/2/3, Dvl proteins, in particular Dvl3, it was shown that Dvl3 played a crucial role of support Sertoli cell tight junction (TJ)-permeability barrier function through changes in the organization of actin- and microtubule (MT)-based cytoskeletons. More important, an in vivo knockdown of Dvl1/2/3 in the testis, defects of spermatid polarity were remarkably noted across the seminiferous epithelium, concomitant with defects of spermatid adhesion and spermatid transport, leading to considerably defects in spermatogenesis. More important, Dvl1/2/3 triple knockdown in the testis also impeded the organization of actin- and MT-based cytoskeletons owing to disruptive spatial expression of actin- and MT-regulatory proteins. In summary, PCP Dishevelled proteins, in particular, Dvl3 is a regulator of Sertoli cell blood-testis barrier (BTB)  and also spermatid PCP function through its effects on the actin- and MT-based cytoskeletons in Sertoli cells.

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

PFOS (perfluorooctanesulfonate, or perfluorooctane sulfonic acid) is an anthropogenic fluorosurfactant widely used in consumer products. While its use in Europe, Canada and the U.S. has been banned due to its human toxicity, it continues to be used in China and other developing countries as a global pollutant. Herein, using an in vitro model of Sertoli cell blood-testis barrier (BTB), PFOS was found to induce Sertoli cell injury by perturbing actin cytoskeleton through changes in the spatial expression of actin regulatory proteins. Specifically, PFOS caused mis-localization of Arp3 (actin-related protein 3, a branched actin polymerization protein) and palladin (an actin bundling protein). These disruptive changes thus led to a dis-organization of F-actin across Sertoli cell cytosol, causing truncation of actin microfilament, thereby failing to support the Sertoli cell morphology and adhesion protein complexes (e.g., occludin-ZO-1, CAR-ZO-1, and N-cadherin-ß-catenin), through a down-regulation of p-Akt1-S473 and p-Akt2-S474. The use of SC79, an Akt1/2 activator [corrected], was found to block the PFOS-induced Sertoli cell injury by rescuing the PFOS-induced F-actin dis-organization. These findings thus illustrate PFOS exerts its disruptive effects on Sertoli cell function downstream through Akt1/2. As such, PFOS-induced male reproductive dysfunction can possibly be managed through an intervention on Akt1/2 expression.

During meiosis, telomeres attach to the nuclear envelope (NE) to promote homologous chromosome moving, pairing, synapsis, and recombination. The telomere-NE attachment is mediated by SUN1, TERB1-TERB2-MAJIN (TTM complex), and TRF1. The interaction of the TTM complex with shelterin is mediated by TERB1 and TRF1, but how SUN1 interacts with the TTM complex is not yet fully understood. In this study, we found that SUN1 not only interacted with TERB1 but also interacted with MAJIN, and the interaction of SUN1 with MAJIN is stronger than TERB1. We also found that SUN1 interacted with SPDYA, an activator of CDK2. The binding sites of MAJIN and SPDYA at SUN1 were mapped, and both MAJIN and SPDYA bound to the N-terminal domain of SUN1 and the two binding sites were close to each other. Furthermore, SPDYA bound to SUN1 via the Ringo domain and recruited CDK2 to SUN1. Then, we found that the interaction of SUN1 with MAJIN was decreased by the CDK2 inhibitors. Taken together, our results provide the possible mechanism of SUN1, MAJIN, and SPDYA-CDK2 in promoting the telomere-NE attachment during meiosis.

Qishen granules (QSG) are a famous formula with cardioprotective properties to heart failure (HF). The aim of this study was to investigate the underlying mechanism of QSG on apoptosis and fibrosis in the treatment of HF. HF model was induced by left anterior descending artery ligation on Sprague-Dawley rats. Transcriptome analysis was used to investigate the regulatory pathways of QSG on HF. Interestingly, downregulated genes of QSG were significantly enriched in Hippo pathway which plays a crucial role in regulating cell apoptosis and proliferation. We found that QSG inhibited the expressions of proapoptotic key proteins P-53 and fibrosis-related proteins TGF-β1, SMAD3, and CTGF. Further, we conducted research on the key proteins in the Hippo pathway upstream of CTGF and P-53. The results showed that MST1, P-MST1, P-LATS1, and RASSF1A that exert proapoptotic function were downregulated after QSG intervention. Similarly, P-YAP and P-TAZ, mediating self-degradation and apoptosis, were both observably decreased after QSG administration. Taken together, QSG are shown to be likely to exert cardioprotective effects by inhibiting the progression of apoptosis and fibrosis through Hippo pathway.

Laminin-α2 chain is one of the major constituent proteins of the basement membrane in the mammalian testis. The laminin-type globular (LG) domains of LG3, 4 and 5 (LG3/4/5, an 80 kDa fragment) can be cleaved from laminin-α2 chain at the C-terminus via the action of matrix metalloproteinase 9 (MMP-9). This LG3/4/5 is a biologically active fragment, capable of modulating the Sertoli cell blood-testis barrier (BTB) function by tightening the barrier both in vitro and in vivo. Overexpression of LG3/4/5 cloned into a mammalian expression vector pCI-neo in Sertoli cells in a Sertoli cell in vitro model with a functional BTB also protected Sertoli cells from cadmium chloride (CdCl2, an environmental toxicant) mediated cell injury. Importantly, overexpression of LG3/4/5 in the testis in vivo was found to block or rescue cadmium-induced BTB disruption and testis injury. LG3/4/5 was found to exert its BTB and spermatogenesis promoting effects through corrective spatiotemporal expression of actin- and MT-based regulatory proteins by maintaining the cytoskeletons in the testis, illustrating the therapeutic implication of this novel bioactive fragment.

Studies suggest that HIV-1 invades the testis through initial permeation of the blood-testis barrier (BTB). The selectivity of the BTB to antiretroviral drugs makes this site a sanctuary for the virus. Little is known about how HIV-1 crosses the BTB and invades the testis. Herein, we used 2 approaches to examine the underlying mechanism(s) by which HIV-1 permeates the BTB and gains entry into the seminiferous epithelium. First, we examined if recombinant Tat protein was capable of perturbing the BTB and making the barrier leaky, using the primary rat Sertoli cell in vitro model that mimics the BTB in vivo. Second, we used HIV-1-infected Sup-T1 cells to investigate the activity of HIV-1 infection on cocultured Sertoli cells. Using both approaches, we found that the Sertoli cell tight junction permeability barrier was considerably perturbed and that HIV-1 effectively permeates the BTB by inducing actin-, microtubule-, vimentin-, and septin-based cytoskeletal changes in Sertoli cells. These studies suggest that HIV-1 directly perturbs BTB function, potentially through the activity of the Tat protein.

Diabetic retinopathy (DR) represents the most typical complication of type 2 diabetes mellitus and one of the most primary oculopathy causing blindness. However, the mechanism of DR remains unknown. RIPK1/RIPK3, as homologous serine/threonine kinases, are key elements in mediating necroptosis and may have functions in DR development. To clarify the relationship between DR and RIPK1/RIPK3, this study established a model of apoptosis using high-glucose induced RGCs, which were treated with 7.5, 19.5, and 35 mM D-glucose for 12, 24, and 48 h, respectively. Subsequently, the expression of RIPK1/RIPK3 was determined and the protective effect of necrostatin-1 on RGCs injury induced by high glucose was explored. The results demonstrated that the expression of RIPK1 and RIPK3 in the cells was increased markedly following 12 h treatment with 19.5 mM D-glucose. Additionally, following an addition of 100 μM necrostatin-1 in 19.5 mM D-glucose medium for RGCs treatment 12 h, the protein expression of RIPK1 and RIPK3 was decreased markedly, and the number of Nissl bodies in cells was increased substantially. The findings of the present study indicated that high glucose could induce the expression of RIPK1/RIPK3, and necrostatin-1 could effectively protect RGCs from D-glucose-induced cell necrosis.

In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.

Breast cancer is one of the most common malignant tumors in women and is the second leading cause of cancer deaths among women. The tumorigenesis and progression of breast cancer are not well understood. The existing researches have indicated that non-coding RNAs, which mainly include long non-coding RNA (lncRNA) and microRNA (miRNA), have gradually become important regulators of breast cancer. We aimed to screen the differential expression of miRNA and lncRNA in the different breast cancer stages and identify the key non-coding RNA using TCGA data. Based on series test of cluster (STC) analysis, bioinformatics analysis, and negatively correlated relationships, 122 lncRNAs, 67 miRNAs, and 119 mRNAs were selected to construct the regulatory network of lncRNA and miRNA. It was shown that the miR-93/20b/106a/106b family was at the center of the regulatory network. Furthermore, 6 miRNAs, 10 lncRNAs, and 15 mRNAs were significantly associated with the overall survival (OS, log-rank P < 0.05) of patients with breast cancer. Overexpressed miR-93 in MCF-7 breast cancer cells was associated with suppressed expression of multiple lncRNAs, and these downregulated lncRNAs (MESTIT1, LOC100128164, and DNMBP-AS1) were significantly associated with poor overall survival in breast cancer patients. Therefore, the miR-93/20b/106a/106b family at the core of the regulatory network discovered by our analysis above may be extremely important for the regulation of lncRNA expression and the progression of breast cancer. The identified key miRNA and lncRNA will enhance the understanding of molecular mechanisms of breast cancer progression. Targeting these key non-coding RNA may provide new therapeutic strategies for breast cancer treatment and may prevent the progression of breast cancer from an early stage to an advanced stage.

Certain risk alleles associated with autoantibody-positive rheumatoid arthritis (RA) have been linked to poorer prognoses. In patients with autoantibody-positive RA, abatacept shows differential efficacy to tumor necrosis factor inhibitors. Our aim was to investigate the relationship between clinical response to abatacept and to adalimumab and presence of risk alleles encoding human leukocyte antigen (HLA)-DRB1 shared epitope (SE) in RA.

Purpose: Choroidal neovascularization (CNV) is the main pathogenic process and a leading cause of severe vision loss in neovascular age-related macular degeneration (AMD). We investigated the antiangiogenic efficacy of dihydroartemisinin (DHA) in an experimental laser-induced CNV mouse model. Methods: After fluorescein angiography confirmed that CNV was induced by laser photocoagulation in C57BL/6J mice, DHA or vehicle was given by intragastric administration once a day. On day 6 and day 12, fluorescein angiography, optic coherence tomography, and flat-mounting analysis were performed to grade CNV leakage, measure CNV thickness and evaluate CNV areas, respectively. Immunofluorescence staining and Western blot analysis were performed to evaluate the expression of NF-κB, VEGF, and VEGFR2. To confirm the safety of intragastric DHA application, changes in retinal morphology and neural cell apoptosis were tested by histopathological examination and TUNEL assay, and retinal function was determined by electroretinogram (ERG). Results: Intragastric administration of DHA significantly suppressed CNV leakage and CNV formation in both thickness and area. Immunofluorescence showed that DHA suppressed VEGFR2 and NF-κB p65 expression in laser-induced lesions. Compared to the normal group, the protein expression of VEGF, VGFER2, NF-κB p65, and NF-κB1 p50 increased significantly in the vehicle group after laser photocoagulation, while it was profoundly inhibited by DHA treatment. In addition, histopathological examination, TUNEL analysis, and ERG test showed no obvious evidence of retinal toxicity caused by DHA. Conclusion: Systemic administration of DHA can effectively inhibit laser-induced CNV formation in mice, which might be due to the suppression of the classic NF-κB signaling pathway and downregulation of VEGFR2 and VEGF expression. The current results suggest that DHA could be a natural potential alternative therapeutic strategy for neovascular AMD.