A Novel TrxR1 Inhibitor Regulates NK and CD8+ T Cell Infiltration and Cytotoxicity, Enhancing the Efficacy of Anti-PD-1 Immunotherapy against Hepatocarcinoma.

Hepatocellular carcinoma (HCC) has the third highest cancer-related mortality rate globally. The immunosuppressive microenvironment of HCC limits effective treatment options. HCC cells and associated microenvironmental factors suppress NK and T cell infiltration and cytotoxic activities. The abnormal number or function of NK and T cells leads to a lack of immune surveillance. Recently, immunotherapy targeting PD-1 and PD-L1 has been shown to activate functionally exhausted cytotoxic immune cells in some solid tumors. However, the response rate and therapeutic efficacy against solid tumors with little lymphocyte infiltration are limited, especially for HCC. Therefore, new targets and therapeutics that induce tumor cell apoptosis and overcome the problem of depletion of immune cells, thereby inhibiting the immune escape of HCC cells, are urgently required. Butaselen (2-bis[2-(1,2-benzisothiazol-2(2H)-ketone)]butane), an organic molecule containing selenium, is a new type of thioredoxin reductase inhibitor. In this study, we found that butaselen promoted NK and T cell activity and infiltration in the tumor microenvironment in HCC-bearing mice by enhancing the expression of CXCR3, NKG2D, and their respective ligands. When used alone, it can significantly inhibit tumor growth and exert a synergistic effect in combination with PD-1 blockade. We suggested the role of the thioredoxin reductase system in the regulation of the tumor immunosuppressive microenvironment and developed a new effective therapeutic molecule for HCC, revealing the mechanism of butaselen in inhibiting tumor cell immune escape.

Knockdown of miR-361-5p promotes the induced activation of SHF-stem cells through FOXM1 mediated Wnt/ß-catenin pathway in cashmere goats.

The inducing activation event of secondary hair follicle (SHF)-stem cells is considered a key biological process in the SHF regeneration, and the morphogenesis of cashmere fiber in cashmere goats. The miR-361-5p was essentially implicated in the induced activation of SHF-stem cells of cashmere goats, but its functional mechanisms are unclear. Here, we confirmed miR-361-5p was significantly downregulated in anagen SHF bugle of cashmere goats compared with that at telogen, and miR-361-5p expression was significantly lower in SHF-stem cells after activation than its counterpart before activation. Further, we found that miR-361-5p could negatively regulate the induced activation event of SHF-stem cells in cashmere goats. Mechanistically, through dual-luciferase reporter assays, miR-361-5p specifically bound with FOXM1 mRNA in SHF-stem cells of cashmere goats and negatively regulated the expression of FOXM1 gene. Also, through overexpression/knockdown analysis of FOXM1 gene, our results indicated that FOXM1 upregulated the expression of Wnt/ß-catenin pathway related genes in SHF-stem cells. Moreover, based on TOP/FOP-flash Wnt report assays, the knockdown of miR-361-5p promotes the Wnt/ß-catenin pathway activation through upregulating the FOXM1 expression in SHF-stem cells. Finally, we demonstrated that miR-361-5p negatively regulated the induced activation of SHF-stem cells through FOXM1 mediated Wnt/ß-catenin pathway in cashmere goats.

CircRNA-1967 participates in the differentiation of goat SHF-SCs into hair follicle lineage by sponging miR-93-3p to enhance LEF1 expression.

Circular RNAs (circRNAs), a novel class of non-coding RNAs, can interact with miRNAs through a sequence-driven sponge mechanism, thereby regulating the expression of their downstream target genes. CircRNA-1967 was found in secondary hair follicles (SHFs) of cashmere goats, but its functions are not clear. Here, we showed that both circRNA-1967 and its host gene BNC2 had significantly higher expression in SHF bulge at anagen than those at telogen of cashmere goats. Also, circRNA-1967 participates in the differentiation of SHF stem cells (SHF-SCs) into hair follicle lineage in cashmere goats. RNA pull-down assay verified that circRNA-1967 interacts with miR-93-3p. We also indicated that circRNA-1967 promoted LEF1 expression in SHF-SCs of cashmere goats. By dual-luciferase reporter analysis, we found that circRNA-1967 up-regulated LEF1 expression through the miR-93-3p-mediated pathway. The results from this study demonstrated that circRNA-1967 participated in the differentiation of goat SHF-SCs into hair follicle lineage by sponging miR-93-3p to enhance LEF1 expression. Our founding might constitute a novel pathway for revealing the potential mechanism of the differentiation of SHF-SCs into hair follicle lineage in cashmere goats. Also, these results provided a valuable basis for further enhancing the intrinsic regeneration of cashmere goat SHFs with the formation and growth of cashmere fibers.

LncRNA-599547 contributes the inductive property of dermal papilla cells in cashmere goat through miR-15b-5p/Wnt10b axis.

The lncRNA-599547 (619-nt in length) is identified in secondary hair follicle (SHF) of cashmere goat, but its functional roles in regulating the inductive property of dermal papilla cells (DPCs) remains unknown. We found that lncRNA-599547 had significantly higher expression in dermal papilla of cashmere goat SHF at anagen than its counterpart at telogen. The overexpression of lncRNA-599547 led to a significant increase of ALP and LEF1 expression in DPCs (p < 0.05), whereas, the siLncRNA-1 mediated silencing of lncRNA-599547 significantly down-regulated the expression of ALP and LEF1 in DPCs (p < 0.05). Based on biotin-labeled RNA pull-down assay, we found that lncRNA-599547 directly interacted with chi-miR-15b-5p in DPCs. Based on both overexpression and silencing analysis of lncRNA-599547, our results indicate that lncRNA-599547 promotes the expression of Wnt10b in DPCs but without modulating its promoter methylation level. Using the mRNA-3'UTR fragments of goat Wnt10b containing the predicted binding sites of chi-miR-15b-5p in Dual-luciferase Reporter Assays, we show that lncRNA-599547 modulates the expression of Wnt10b at the chi-miR-15b-5p mediated post-transcriptional level. Taken together, our results indicate that lncRNA-599547 sponges miR-15b-5p to positively regulate the expression of Wnt10 gene, and thereby contributes the inductive property of DPCs in cashmere goat.

LncRNA-000133 from secondary hair follicle of Cashmere goat: identification, regulatory network and its effects on inductive property of dermal papilla cells.

Long noncoding RNAs (lncRNAs), a class of non-protein conding RNAs > 200 nt in length, were thought to play critical roles in regulating the expression of protein-coding genes. Here, we identified and characterized a novel lncRNA-000133 from the secondary hair follicle (SHF) of cashmere goat with its ceRNA network analysis, as well as, its potential effects on inductive property of dermal papilla cells were evaluated through overexpression analysis. Expression analysis indicated that lncRNA-000133 had a significantly higher expression at anagen than that at telogen in SHF of Cashmere goat, suggesting that lncRNA-000133 might be involved in the reconstruction of SHF with the formation and growth of cashmere fiber. Taken together with methylation analysis, we showed that 5' regulatory region methylation of the lncRNA-000133 gene might be involved in its expression suppression in SHF of Cashmere goat. The ceRNA regulatory network showed that a rich and complex regulatory relationship between lncRNA-000133 and related miRNAs with their target genes. The overexpression of lncRNA-000133 led to a significant increasing in the relative expression of ET-1, SCF, ALP and LEF1 in dermal papilla cells suggesting that lncRNA-000133 appears to contribute the inductive property of dermal papilla cells.

Histological analysis and identification of spermatogenesis-related genes in 2-, 6-, and 12-month-old sheep testes.

Testis development and spermatogenesis are vital factors that influence male animal fertility. In order to identify spermatogenesis-related genes and further provide a theory basis for finding biomarkers related to male sheep fertility, 2-, 6-, and 12-month-old Small Tail Han Sheep testes were selected to investigate the dynamic changes of sheep testis development. Hematoxylin-eosin routine staining and RNA-Seq technique were used to perform histological and transcriptome analysis for these testes. The results showed that 630, 102, and 322 differentially expressed genes (DEGs) were identified in 2- vs 6-month-old, 6- vs 12-month-old, and 2- vs 12-month-old testes, respectively. GO and KEGG analysis showed the following: DEGs in 2- vs 6-month-old testes were mainly related to the GO terms of sexual maturation and the pathways of multiple metabolism and biosynthesis; in 6- vs 12-month-old testes, most of the GO terms that DEGs involved in were related to metabolism and translation processes; the most significantly enriched pathway is the ribosome pathway. The union of DEGs in 2- vs 6-month-old, 6- vs 12-month-old, and 2- vs 12-month-old testes was categorized into eight profiles by series cluster. Subsequently, the eight profiles were classified into four model profiles and four co-expression networks were constructed based on the DEGs in these model profiles. Finally, 29 key regulatory genes related to spermatogenesis were identified in the four co-expression networks. The expression of 13 DEGs (CA3, APOH, MYOC, CATSPER4, SYT6, SERPINA10, DAZL, ADIPOR2, RAB13, CEP41, SPAG4, ODF1, and FRG1) was validated by RT-PCR.

CircERCC6 Positively Regulates the Induced Activation of SHF Stem Cells in Cashmere Goats via the miR-412-3p/BNC2 Axis in an m6A-Dependent Manner.

The cashmere, a kind of nature protein fiber, is one of the main use of cashmere goats. The induced activation of secondary hair follicle (SHF) stem cells by the dermal papilla cell-derived signals is a key biological process for the morphogenesis and growth of cashmere fiber in cashmere goats. Previously, the circRNA-ERCC6 (circERCC6) was identified from cashmere goat SHFs; however, its biological significance is unclear in the SHF physiology process of cashmere goats. In this study, we found that circERCC6 exhibited significantly higher expression at anagen SHF bulge compared with the counterpart of telogen and harbored three m6A modified sites (named m6A-685, m6A-862, and m6A-995) through methylation immunoprecipitation using a real-time quantitative polymerase chain reaction (Me-RIP-qPCR) technique. The knockdown experiments of circERCC6 in SHF stem cells showed that circERCC6 positively regulates the induced activation of SHF stem cells in cashmere goats. Through a dual-luciferase reporter assay, we demonstrated that m6A-modified circERCC6 (m6A-circERCC6) sponged miR-412-3p to upregulate the expression of BNC2 mRNA in SHFstem cells. Through m6A-deficient mutant assay in circERCC6 knockdown SHF stem cells, we further showed that m6A modification within circERCC6 is required to mediate the miR-412-3p/BNC2 axis to finally promote the proper induced activation of SHF stem cells in cashmere goats.

N6-Methyladenosine modification (m6A) of circRNA-ZNF638 contributes to the induced activation of SHF stem cells through miR-361-5p/Wnt5a axis in cashmere goats.

OBJECTIVE: The objective of this study was to investigate the effects of N6-Methyladenosine modification-circRNA-zinc finger protein 638 (m6A-circRNA-ZNF638) on the induced activation of secondary hair follicle (SHF) stem cells with its potential mechanisms in cashmere goats. METHODS: The m6A modification of ZNF638 was analyzed using methylation immunoprecipitation with real-time quantitative polymerase chain reaction technique in SHF stem cells. The effects of circRNA-ZNF638 on the induced activation of SHF stem cells in m6A dependence were evaluated through the overexpression of circRNA-ZNF638/its m6Adeficient mutants in circRNA-ZNF638 knockdown SHF stem cells. The competitive binding of miR-361-5p to circRNA-ZNF638/Wnt5a 3'- untranslated region was analyzed through Dual-luciferase reporter assay. RESULTS: The m6A-circRNA-ZNF638 had significantly higher transcription at anagen SHF bulge of cashmere goats compared with that at telogen, as well as it positively regulated the induced activation of SHF-stem cells in cashmere goats. Mechanismly, m6A-circRNA-ZNF638 sponged miR-361-5p to heighten the transcriptional expression of Wnt5a gene in SHFstem cells. We further demonstrated that the internal m6A modification within circRNAZNF638 is required for mediating the miR-361-5p/Wnt5a pathway to regulate the induced activation of SHF stem cells through an introducing of m6A-deficient mutant of circRNAZNF638. CONCLUSION: The circRNA-ZNF638 contributes the proper induced activation of SHF-stem cells in cashmere goats in m6A-dependent manner through miR-361-5p/Wnt5a axis.

CircRNA-0100 positively regulates the differentiation of cashmere goat SHF-SCs into hair follicle lineage via sequestering miR-153-3p to heighten the KLF5 expression.

Circular RNAs (circRNAs) have stable structures, being a covalently closed loop without 5 ' and 3 ' free ends. They can function as "miRNA sponges" in regulating the expression of their target genes. It was thought that circRNAs are involved in the development of the secondary hair follicle (SHF) in cashmere goats. In our previous investigation, a new circRNA named circRNA-0100 was identified from the SHF of cashmere goats, but its function is unknown. In this work, we found that circRNA-0100 exhibited significantly higher expression at anagen SHF bulge than its counterpart at telogen in cashmere goats. Based on the use of both overexpression and siRNA interference assays, our data indicated that circRNA-0100 promoted the differentiation of cashmere goat SHF stem cells (SHF-SCs) into hair follicle lineage, which was evaluated by analyzing the transcriptional level changes of six indicator genes in SHF-SCs of cashmere goats. Using the RNA pull-down technique, we showed that circRNA-0100 served as "molecular sponges" of miR-153-3p in SHF-SCs. Through the use of dual-luciferase reporter assays, our data indicated that circRNA-0100 positively regulated the transcriptional expression of the KLF5 gene via the miR-153-3p-mediated pathway. Ultimately, we showed that circRNA-0100 promoted the differentiation of SHF-SCs into hair lineage, which might be achieved via sequestering miR-153-3p to heighten the KLF5 expression in SHF-SCs of cashmere goats. Our results provide novel scientific evidence for revealing the potential molecular regulatory mechanisms on the differentiation of SHF-SCs into hair lineage in cashmere goats.

An Integrated Analysis of Lactation-Related miRNA and mRNA Expression Profiles in Donkey Mammary Glands.

Donkey milk is consumed by humans for its nutritional and therapeutic properties. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been implicated in the regulation of milk component synthesis and mammary gland development. However, the regulatory profile of the miRNAs and mRNAs involved in lactation in donkeys is unclear. We performed mRNA-seq and miRNA-seq and constructed coexpression regulatory networks for the mammary glands during the lactating and nonlactating period of jennies. We identified 3144 differentially expressed (DE) mRNAs (987 upregulated mRNAs and 2157 downregulated mRNAs) and 293 DE miRNAs (231 upregulated miRNAs and 62 downregulated miRNAs) in the lactating group compared to the nonlactating group. The DE miRNA target mRNA were significantly associated with pathways related to RNA polymerase, glycosphingolipid biosynthesis, mRNA surveillance, ribosome biogenesis in eukaryotes, glycerophospholipid metabolism, Ras signaling, and the fly hippo signaling pathway. The mRNA-miRNA coregulation analysis showed that novel-m0032-3p, miR-195, miR-26-5p, miR-23-3p, miR-674-3p, and miR-874-3p are key miRNAs that target mRNAs involved in immunity and milk lipid, protein, and vitamin metabolism in the jenny mammary gland. Our results improve the current knowledge of the molecular mechanisms regulating bioactive milk component metabolism in the mammary glands and could be used to improve milk production in donkeys.

Identification and Molecular Analysis of m6A-circRNAs from Cashmere Goat Reveal Their Integrated Regulatory Network and Putative Functions in Secondary Hair Follicle during Anagen Stage.

N6-methyladenosine (m6A) is the most abundant modification in linear RNA molecules. Over the last few years, interestingly, many circRNA molecules are also found to have extensive m6A modification sites with temporal and spatial specific expression patterns. To date, however, little information is available concerning the expression profiling and functional regulatory characteristics of m6A modified circRNAs (m6A-circRNAs) in secondary hair follicles (SHFs) of cashmere goats. In this study, a total of fifteen m6A-circRNAs were identified and characterized in the skin tissue of cashmere goats. Of these, six m6A-circRNAs were revealed to have significantly higher expression in skin at anagen compared with those at telogen. The constructed ceRNA network indicated a complicated regulatory relationship of the six anagen up-regulated m6A-circRNAs through miRNA mediated pathways. Several signaling pathways implicated in the physiological processes of hair follicles were enriched based on the potential regulatory genes of the six anagen up-regulated m6A-circRNAs, such as TGF-beta, axon guidance, ribosome, and stem cell pluripotency regulatory pathways, suggesting the analyzed m6A-circRNAs might be essentially involved in SHF development and cashmere growth in cashmere goats. Further, we showed that four m6A-circRNAs had highly similar expression trends to their host genes in SHFs of cashmere goats including m6A-circRNA-ZNF638, -TULP4, -DNAJB6, and -CAT. However, the expression patterns of two m6A-circRNAs (m6A-circRNA-STAM2 and -CAAP1) were inconsistent with the linear RNAs from their host genes in the SHFs of cashmere goats. These results provide novel information for eluci-dating the biological function and regulatory characteristics of the m6A-circRNAs in SHF development and cashmere growth in goats.

Therapeutic Effects of an Inhibitor of Thioredoxin Reductase on Liver Fibrosis by Inhibiting the Transforming Growth Factor-ß1/Smads Pathway.

Liver fibrosis is an important stage in the progression of liver injury into cirrhosis or even liver cancer. Hepatic stellate cells (HSCs) are induced by transforming growth factor-ß1 (TGF-ß1) to produce α-smooth muscle actin (α-SMA) and collagens in liver fibrosis. Butaselen (BS), which was previously synthesized by our group, is an organic selenium compound that exerts antioxidant and tumor cell apoptosis-promoting effects by inhibiting the thioredoxin (Trx)/thioredoxin reductase (TrxR) system. The aim of this study was to investigate the potential effects of BS on liver fibrosis and explore the underlying molecular mechanisms of its action. Liver fibrosis models were established using male BALB/c mice through intraperitoneal injection of CCl4. BS was administered orally once daily at a dose of 36, 90, or 180 mg/kg. Silymarin (Si), which is a drug used for patients with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, was administered at a dose of 30 mg/kg per day as a control. The action mechanisms of BS against liver fibrosis progression were examined in HSCs. The study revealed that the activity and expression levels of TrxR were elevated in the mouse liver and serum after CCl4-induced liver fibrosis. Oral administration of BS relieved the pathological state of mice with liver fibrosis, showing significant therapeutic effects against liver fibrosis. Moreover, BS not only induced HSC apoptosis but also inhibited the production of α-SMA and collagens by HSCs by downregulating the TGF-ß1 expression and blocking the TGF-ß1/Smads pathway. The results of the study indicated that BS inhibited liver fibrosis by regulating the TGF-ß1/Smads pathway.

The Value of Magnetic Resonance Imaging Histograms in the Preoperative Differential Diagnosis of Endometrial Stromal Sarcoma and Degenerative Hysteromyoma.

Objective: The present study aimed to explore the application value of magnetic resonance imaging (MRI) histograms with multiple sequences in the preoperative differential diagnosis of endometrial stromal sarcoma (ESS) and degenerative hysteromyoma (DH). Methods: The clinical and preoperative MRI data of 20 patients with pathologically confirmed ESS and 24 patients with pathologically confirmed DH were retrospectively analyzed, forming the two study groups. Mazda software was used to select the MRI layer with the largest tumor diameter in T2WI, the apparent diffusion coefficient (ADC), and enhanced T1WI (T1CE) images. The region of interest (ROI) was outlined for gray-scale histogram analysis. Nine parameters-the mean, variance, kurtosis, skewness, 1st percentile, 10th percentile, 50th percentile, 90th percentile, and 99th percentile-were obtained for intergroup analysis, and the receiver operating curves (ROCs) were plotted to analyze the differential diagnostic efficacy for each parameter. Results: In the T2WI histogram, the differences between the two groups in seven of the parameters (mean, skewness, 1st percentile, 10th percentile, 50th percentile, 90th percentile, and 99th percentile) were statistically significant (P < 0.05). In the ADC histogram, the differences between the two groups in three of the parameters (skewness, 10th percentile, and 50th percentile) were statistically significant (P < 0.05). In the T1CE histogram, no significant differences were found between the two groups in any of the parameters (all P > 0.05). Of the nine parameters, the 50th percentile was found to have the best diagnostic efficacy. In the T2WI histogram, ROC curve analysis of the 50th percentile yielded the best area under the ROC curve (AUC; 0.742), sensitivity of 70%, and specificity of 83.3%. In the ADC histogram, ROC curve analysis of the 50th percentile yielded the best area under the ROC curve (AUC; 0.783), sensitivity of 81%, and specificity of 76.9%. Conclusion: The parameters of the mean, 10th percentile and 50th percentile in the T2WI histogram have good diagnostic efficacy, providing new methods and ideas for clinical diagnosis.

Decreased Intrinsic Neural Timescales in Mesial Temporal Lobe Epilepsy.

It is well established that epilepsy is characterized by the destruction of the information capacity of brain network and the interference with information processing in regions outside the epileptogenic focus. However, the potential mechanism remains poorly understood. In the current study, we applied a recently proposed approach on the basis of resting-state fMRI data to measure altered local neural dynamics in mesial temporal lobe epilepsy (mTLE), which represents how long neural information is stored in a local brain area and reflect an ability of information integration. Using resting-state-fMRI data recorded from 36 subjects with mTLE and 36 healthy controls, we calculated the intrinsic neural timescales (INT) of neural signals by summing the positive magnitude of the autocorrelation of the resting-state brain activity. Compared to healthy controls, the INT values were significantly lower in patients in the right orbitofrontal cortices, right insula, and right posterior lobe of cerebellum. Whereas, we observed no statistically significant changes between patients with long- and short-term epilepsy duration or between left-mTLE and right-mTLE. Our study provides distinct insight into the brain abnormalities of mTLE from the perspective of the dynamics of the brain activity, highlighting the significant role of intrinsic timescale in understanding neurophysiological mechanisms. And we postulate that altered intrinsic timescales of neural signals in specific cortical brain areas may be the neurodynamic basis of cognitive impairment and emotional comorbidities in mTLE patients.

Single-Cell Sequencing Reveals Differential Cell Types in Skin Tissues of Liaoning Cashmere Goats and Key Genes Related Potentially to the Fineness of Cashmere Fiber.

Cashmere fineness is one of the important factors determining cashmere quality; however, our understanding of the regulation of cashmere fineness at the cellular level is limited. Here, we used single-cell RNA sequencing and computational models to identify 13 skin cell types in Liaoning cashmere goats. We also analyzed the molecular changes in the development process by cell trajectory analysis and revealed the maturation process in the gene expression profile in Liaoning cashmere goats. Weighted gene co-expression network analysis explored hub genes in cell clusters related to cashmere formation. Secondary hair follicle dermal papilla cells (SDPCs) play an important role in the growth and density of cashmere. ACTA2, a marker gene of SDPCs, was selected for immunofluorescence (IF) and Western blot (WB) verification. Our results indicate that ACTA2 is mainly expressed in SDPCs, and WB results show different expression levels. COL1A1 is a highly expressed gene in SDPCs, which was verified by IF and WB. We then selected CXCL8 of SDPCs to verify and prove the differential expression in the coarse and fine types of Liaoning cashmere goats. Therefore, the CXCL8 gene may regulate cashmere fineness. These genes may be involved in regulating the fineness of cashmere in goat SDPCs; our research provides new insights into the mechanism of cashmere growth and fineness regulation by cells.

CircRNA-1926 Promotes the Differentiation of Goat SHF Stem Cells into Hair Follicle Lineage by miR-148a/b-3p/CDK19 Axis.

Circular RNAs (CircRNAs) are a type of non-coding RNAs, which contain a covalently closed loop structure without 5' to 3' free ends. CircRNAs play essential roles in the regeneration of secondary hair follicle (SHF) and cashmere growth in goats. CircRNA-1926 was previously identified in SHF of cashmere goats, but its potential roles are unclear. In this study, we confirmed the expression of circRNA-1926 in SHF bulge of nine cashmere goats with a significantly higher level at anagen than that of telogen. Through the use of both overexpression and siRNA interference, we showed that circRNA-1926 promoted the differentiation of SHF stem cell into hair follicle lineage in cashmere goats which was evaluated via indictor genes Keratin 7 and Keratin 17. Using RNA pull-down, we found that circRNA-1926 bound with miR-148a/b-3p. Additionally, our data indicated that circRNA-1926 promoted the expression of the CDK19 gene. Using dual-luciferase reporter assays, it was revealed that circRNA-1926 positively regulated the CDK19 expression through miR-148a/b-3p. The results from this study demonstrated that circRNA-1926 contributes the differentiation of SHF stem cells into hair follicle lineages in cashmere goats via sponging miR-148a/b-3p to enhance CDK19 expression.

Author Correction: Plasma activity of Thioredoxin Reductase as a Novel Biomarker in Gastric Cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The antimetastatic effect and underlying mechanisms of thioredoxin reductase inhibitor ethaselen.

Treating cancer metastasis is of vital importance to prolong patients' survival. Thioredoxin reductase (TrxR) is overexpressed in many cancer types and has been recognized as an anti-cancer target. The organoselenium compound ethaselen (BBSKE) has been proved to be a TrxR inhibitor and inhibit various types of tumor growth. However, whether BBSKE could inhibit tumor metastasis remains unclear. In this study, we aim to explore the antimetastatic effect of BBSKE and underlying mechanisms. BBSKE was found to dose-dependently suppress migration and invasion of MCF-7 and LoVo cells in vitro. The underlying mechanisms may include inhibition of TrxR activity, elevation of reactive oxygen species (ROS), decrease of EGFR activation and HER2 expression. Besides, the epithelial to mesenchymal transition process and expression of CD44, MMP-9, VEGFR2 and PD-L1 were also abrogated. Decreased migration and invasion, lower expression levels of EGFR, HER2, N-cadherin, CD44, MMP-9, VEGFR2 and PD-L1 were also observed in TrxR1-knockdown MCF-7 and LoVo cells. In the mouse breast cancer 4T1 model, BBSKE not only inhibited progression of primary tumor, but also suppressed formation of metastatic lung nodules and liver micro-metastases, indicating that BBSKE could effectively abolish tumor metastasis. In conclusion, our findings show that BBSKE is able to inhibit migration and invasion of cancer cells in vitro and in vivo, and may be used to prevent and treat metastasis.

Integrative microRNA-mRNA Analysis of Muscle Tissues in Qianhua Mutton Merino and Small Tail Han Sheep Reveals Key Roles for oar-miR-655-3p and oar-miR-381-5p.

The Qianhua Mutton Merino (QHMM) is a new variety of sheep (Ovis aries) with improved meat performance compared with the traditional Small Tail Han (STH) sheep variety. We recently reported the transcriptome profiling of longissimus muscle tissues between QHMM and STH sheep. In the present study, we aimed to evaluate key micro (mi)RNA-mRNA networks associated with sheep muscle growth and development. We used miRNA sequencing to obtain longissimus muscle miRNA profiles from QHMM and STH sheep. We identified a total of 153 known sheep miRNAs, of which 4 were differentially expressed (DE) between the 2 sheep varieties. We combined these results with mRNA library data to build an miRNA-mRNA network, including 26 target genes of the 4 DE miRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that 26 target genes were significantly enriched in 86 biological processes, including muscle organogenesis, myoblast migration, cell proliferation, and adipose tissue development, and in 9 metabolic pathways, including carbohydrate, nucleotide, and amino acid metabolic pathways. oar-miR-655-3p and its target gene ACSM3 and oar-miR-381-5p and its target gene ABAT were selected for subsequent analysis based on GO and KEGG analyses. The binding sites of oar-miR-655-3p with ACSM3 and oar-miR-381-5p with ABAT were validated by a dual-luciferase reporter gene detection system. This represents the first integrative analysis of miRNA-mRNA networks in QHMM and STH muscles and suggests that DE miRNAs, especially oar-miR-655-3p and oar-miR-381-5p, play crucial roles in muscle growth and development.

Integrated Analysis of miRNA and mRNA Expression Profiles Reveals Functional miRNA-Targets in Development Testes of Small Tail Han Sheep.

Small Tail Han Sheep is a highly valued local breed in China because of their precocity, perennial estrus, and high fecundity. The average annual lambing rate of ewes is as high as 180-270%, the semen of ram has characteristics of high yield, high density, and good motility. To reveal the key miRNAs and miRNA-targets underlying testis development and spermatogenesis in male Small Tail Han Sheep, integrated analysis of miRNA and mRNA expression profiles in 2-, 6-, and 12-month-old testes was performed by RNA-seq technology and bioinformatics methods. The results showed that total of 153 known sheep miRNAs and 2712 novel miRNAs were obtained in 2-,6 - and 12-month-old Small Tail Han Sheep testes; 5, 1, and 4 differentially expressed (DE) known sheep miRNAs, and 132, 105, and 24 DE novel miRNAs were identified in 2- vs. 6-, 6- vs. 12-, and 2- vs. 12-month-old testes, respectively. We combined miRNA results of this study and the mRNA results obtained in our previous study to predict the target mRNAs of DE known sheep miRNAs; 131, 10, and 15 target mRNAs of DE known sheep miRNAs and 76, 1, and 11 DE miRNA-targets were identified in the three groups, respectively. GO and KEGG analyses showed that: in 2- vs. 6-month-olds, the target genes of DE known sheep miRNAs were involved in 100 biological processes and 11 signaling pathways; in 6- vs. 12-month-olds, the target genes of DE known sheep miRNAs were involved in 4 biological processes; and in 2- vs. 12-month-olds, the target genes of DE known sheep miRNAs were involved in 17 biological processes and 4 signaling pathways. Three miR-target regulatory networks were constructed based on these DE miRNA-targets. The key miRNA-Targets involved in testis development and spermatogenesis were screened. 6 known sheep miRNAs and 6 novel miRNAs were selected to validate the accuracy of miRNA sequencing data by qRT-PCR. The binding sites of oar-miR-379-5p with WNT8A was validated by a dual luciferase reporter gene detection system.