Extracellular Expression of Feruloyl Esterase and Xylanase in Escherichia coli for Ferulic Acid Production from Agricultural Residues.

There is still a large amount of ferulic acid (FA), an outstanding antioxidant, present in agricultural residues. Enzymatic hydrolysis has been regarded as the most effective way to release FA. This present study therefore selected feruloyl esterase (FAE) and xylanase (XYN) from the metagenomes of a cow rumen and a camel rumen, respectively, for their recombinant expression in Escherichia coli BL21 and further application in releasing FA. After screening the candidate signal peptides, the optimal one for each enzyme, which were selected as SP1 and SP4, respectively, was integrated into the vectors pET22b(+) and pETDuet-1. Among the generated E. coli strains SP1-F, SP4-X, and SP1-F-SP4-X that could express extracellular enzymes either separately or simultaneously, the latter one performed the best in relation to degrading the biomass and releasing FA. Under the optimized culture and induction conditions, the strain SP1-F-SP4-X released 90% of FA from 10% of de-starched wheat bran and produced 314.1 mg/L FA, which was deemed to be the highest obtained value to the best of our knowledge. This result could pave a way for the re-utilization of agricultural residues and enhancing their add-value.

Effects of Cadmium Stress on Carbon Sequestration and Oxygen Release Characteristics in A Landscaping Hyperaccumulator-Lonicera japonica Thunb.

The carbon sequestration and oxygen release of landscape plants are dominant ecological service functions, which can play an important role in reducing greenhouse gases, improving the urban heat island effect and achieving carbon peaking and carbon neutrality. In the present study, we are choosing Lonicera japonica Thunb. as a model plant to show the effects of Cd stress on growth, photosynthesis, carbon sequestration and oxygen release characteristics. Under 5 mg kg-1 of Cd treatment, the dry weight of roots and shoots biomass and the net photosynthetic rate (PN) in L. japonica had a significant increase, and with the increase in Cd treatment concentration, the dry weight of roots and shoots biomass and PN in the plant began to decrease. When the Cd treatment concentration was up to 125 mg kg-1, the dry weight of root and shoots biomass and PN in the plant decreased by 5.29%, 1.94% and 2.06%, and they had no significant decrease compared with the control, indicating that the plant still had a good ability for growth and photoenergy utilization even under high concentrations of Cd stress. The carbon sequestration and oxygen release functions in terms of diurnal assimilation amounts (P), carbon sequestration per unit leaf area (WCO2), oxygen release per unit leaf area (WO2), carbon sequestration per unit land area (PCO2) and oxygen release per unit land area (PO2) in L. japonica had a similar change trend with the photosynthesis responses under different concentrations of Cd treatments, which indicated that L. japonica as a landscaping Cd-hyperaccumulator, has a good ability for carbon sequestration and oxygen release even under high concentrations of Cd stress. The present study will provide a useful guideline for effectively developing the ecological service functions of landscaping hyperaccumulators under urban Cd-contaminated environment.

Next-generation sequencing identified somatic alterations that may underlie the etiology of Chinese papillary thyroid carcinoma.

To better understand the etiology of papillary thyroid carcinoma, we did next-generation sequencing for the exomes and transcriptomes of a Chinese cohort of 28 pairs of DNA and RNA samples extracted from papillary thyroid carcinoma tumors and adjacent normal thyroid samples. The Chinese papillary thyroid carcinoma tumors harbored somatic mutations in the known driver genes, such as KRAS, TP53, BRAF, ERBB2 , and MET . In addition, we identified novel papillary thyroid carcinoma candidate genes that had not been well studied before. We also identified a gene mutation signature involving SPTA1, MAP2, SYNE1 , and SLIT3 that is significantly associated with survival of papillary thyroid carcinoma patients. Transcriptome analysis using the initial papillary thyroid carcinoma tumor samples and a new Chinese papillary thyroid carcinoma dataset identified six commonly upregulated oncogenic pathways in both datasets including eukaryotic translation initiation factor 2, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/serine/threonine kinase (AKT), Ephrin Receptor, Rho Family GTPase signaling, nuclear factor, erythroid 2 like 2 (NRF2)-mediated oxidative stress response, and remodeling of epithelial adherens junctions. Overall, we identified novel candidate genes and oncogenic pathways important to the etiology of papillary thyroid carcinoma in Chinese patients and found the association of a gene signature with the survival outcome of the thyroid cancer patients. These findings may help in moving toward the more comprehensive and effective personalized treatment of papillary thyroid carcinoma in Chinese.

Improving the diagnosis of thyroid cancer by machine learning and clinical data.

Thyroid cancer is a common endocrine carcinoma that occurs in the thyroid gland. Much effort has been invested in improving its diagnosis, and thyroidectomy remains the primary treatment method. A successful operation without unnecessary side injuries relies on an accurate preoperative diagnosis. Current human assessment of thyroid nodule malignancy is prone to errors and may not guarantee an accurate preoperative diagnosis. This study proposed a machine learning framework to predict thyroid nodule malignancy based on our collected novel clinical dataset. The ten-fold cross-validation, bootstrap analysis, and permutation predictor importance were applied to estimate and interpret the model performance under uncertainty. The comparison between model prediction and expert assessment shows the advantage of our framework over human judgment in predicting thyroid nodule malignancy. Our method is accurate, interpretable, and thus useable as additional evidence in the preoperative diagnosis of thyroid cancer.

Inhibition of lung tumorigenesis by a small molecule CA170 targeting the immune checkpoint protein VISTA.

Expressed on cells of the myeloid and lymphoid lineages, V-domain Ig Suppressor of T cell Activation (VISTA) is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Using a tripeptide small molecule antagonist of VISTA CA170, we found that it exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. Remarkably, lung tumor development was almost completely suppressed when CA170 was combined with an MHCII-directed KRAS peptide vaccine. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 increased CD8+ T cell infiltration and enhanced their effector functions by decreasing the tumor infiltration of myeloid-derived suppressor cells (MDSCs) and Regulatory T (Treg) cells, while the Kras vaccine primarily induced expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory and regulatory T cells, and drives an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS vaccine.

MicroRNA-219a-2-3p modulates the proliferation of thyroid cancer cells via the HPSE/cyclin D1 pathway.

Heparanase (HPSE) is an endo-ß-D-glucuronidase overexpressed in different types of human cancer, and a predicted target of microRNA (miRNA/miR)-219a-2-3p in thyroid cancer. The present study aimed to investigate the potential role of HPSE and miR-219a-2-3p in thyroid cancer, and the molecular mechanism of miR-219a-2-3p regulating the proliferation of thyroid cancer cells via HPSE was confirmed. Immunohistochemistry analysis was performed to detect HPSE expression in thyroid cancer sections. In addition, reverse transcription-quantitative PCR analysis was performed to detect mRNA and miR-219a-2-3p expression levels in thyroid cancer samples and cell lines. miR-219-2-3p mimic or HPSE plasmid were transfected into B-CPAP and TPC-1 thyroid cancer cells. Furthermore, western blot analysis was performed to detect the protein expression levels of HPSE and cyclin D1. Cell cycle analysis was performed using propidium iodide staining and flow cytometry, and EdU incorporation was performed to detect cell proliferation. The results demonstrated that high HPSE expression was significantly associated with tumor size, extracapsular invasion and lymph node metastasis. Notably, a statistically negative correlation was observed between HPSE mRNA expression and miR-219a-2-3p expression in thyroid cancer tumors, as well as in thyroid cancer cell lines. When exogenously expressed in B-CPAP and TPC-1 cells, miR-219a-2-3p induced cell cycle arrest at the G0/G1 phase and decreased the percentage of proliferating cells. Furthermore, HPSE and cyclin D1 protein expression decreased following transfection with miR-219a-2-3p. Notably, when HPSE was ectopically expressed in miR-219a-2-3p transfected cells, cyclin D1 expression and the number of proliferative cells increased. Taken together, these results suggest that HPSE contributes to the proliferation of thyroid cancer cells. In addition, miR-219a-2-3p was confirmed to target HPSE and inhibit cell proliferation, which was associated with cyclin D1 suppression-mediated cell cycle arrest.

MiR-200c/FUT4 axis prevents the proliferation of colon cancer cells by downregulating the Wnt/ß-catenin pathway.

BACKGROUND: MicroRNA (miR)-200c has been widely reported to be involved in colon cancer progress. However, the mechanisms of miR-200c in regulating tumor metastasis and growth remain to be fully elucidated. This study aimed to investigate the mechanism of miR-200c targets fucosyltransferase 4 (FUT4) on the proliferation of colon cancer. METHODS: The miR-200c and FUT4 mRNA levels in LoVo and SW480 cells were measured by real-time quantitative polymerase chain reaction. Further, miR-200c mimic, FUT4 siRNA and FUT4 mimic were transfected into cells, separately. Cell counting kit-8, plate colony formation and transwell assays were used to analyse the cells biological behaviour.. Immunofluorescence was used to analyse the Ki-67 expression Moreover, the Wnt/ß-catenin pathway-related proteins were detected by western blots. A double luciferase experiment was performed to confirm the relationship between miR-200c and FUT4. In vivo, tumour growth and Wnt/ß-catenin pathway-related proteins were also analysed. RESULTS: In vitro, the expression of miR-200c and FUT4 were negatively correlated in LoVo and SW480 cells (correlation coefficients were - 0.9046 and - 0.9236, respectively). MiR-200c overexpression inhibited the proliferation, migration and invasion of LoVo and SW480 cells by downregulating FUT4. The Ki67-positive cells and Wnt/ß-catenin signalling pathway-related proteins were reduced in the miR-200c overexpression and FUT4 silencing groups. A dual luciferase reporting system identified FUT4 as the target of miR-200c. The results in vivo were further confirmed the foundation of cells study. CONCLUSIONS: In summary, miR-200c overexpression inhibits proliferation of colon cancer targeting FUT4 to downregulate the Wnt/ß-catenin pathway, which promises molecular targets to inhibit metastasis for colon cancer therapy.

Distinct miRNA Gene Expression Profiles Among the Nodule Tissues of Lung Sarcoidosis, Tuberculous Lymphadenitis and Normal Healthy Control Individuals.

Background: Sarcoidosis and tuberculosis share similarities in clinical manifestations and histopathological features. We aimed to identify the microRNA (miRNA) profiles of the lymph nodes of individuals with sarcoidosis and of those with tuberculous lymphadenitis to investigate the value of miRNAs in the differential diagnosis of sarcoidosis and tuberculous lymphadenitis. Methods: The miRNA profiles of the lymph nodes of individuals with sarcoidosis, those with tuberculous lymphadenitis (TBLN) and controls were detected by miRNA microarray analysis in the age- and sex-matched development group of the controls (n = 3), patients with TBLN (n = 3) and patients with sarcoidosis (n = 3), and the results were validated by quantitative real-time polymerase chain reaction in the validation group of the controls (n = 30), TBLN (n = 30) and patients with sarcoidosis (n = 31). The relationship between miRNA expression and the clinical parameters of sarcoidosis was analyzed. Results: miR-145, miR-185-5p, miR-301, miR-425-5P, miR-449b and miR-885-5P were differentially expressed between individuals with sarcoidosis and controls (P < 0.0001, P < 0.0001, P = 0.0008, P = 0.0002, P = 0.0018, and P < 0.0001, respectively), and the same six miRNAs were differentially expressed between individuals with tuberculous lymphadenitis and controls (P = 0.0002, P = 0.0004, P = 0.0238, P = 0.0006, P = 0.0149, and P = 0.0045, respectively). miR-185-5p was differentially expressed between individuals with tuberculous lymphadenitis and those with sarcoidosis (P = 0.0101). The area under the receiver operating characteristic curve calculated for miR-185-5p was 0.6860, and the sensitivity and specificity of miR-185-5p for the differential diagnosis of sarcoidosis from TBLN were 61 and 80%, respectively. The levels of miR-145, miR-301, miR-425-5P, and miR-885-5P were positively correlated with CD4+/CD8+ T lymphocytes in bronchoalveolar lavage fluid. Conclusions: miRNAs in lymph nodes show similar expression patterns between individuals with sarcoidosis and those with tuberculous lymphadenitis, which were experimentally selected. miR-185-5p in the lymph nodes can be used as an auxiliary marker for the differential diagnosis of sarcoidosis and tuberculous lymphadenitis.

miR-22 Suppresses Tumor Invasion and Metastasis in Colorectal Cancer by Targeting NLRP3.

PURPOSE: This study aimed to investigate the effects of microRNA (miR)-22 on biological behaviors of colon cancer cells and to explore the relationship between miR-22 and NLRP3. MATERIALS AND METHODS: First, human colon cancer HCT116 cells were transfected with a miR-22 mimic, miR-22 inhibitor, control mimic, and control inhibitor, respectively. CCK8, colony formation, and transwell assays were performed to observe cell proliferation, migration, and invasion. Western blotting was used to analyze the expression of recombinant NLRP3 (NLR family, pyrin domain-containing protein 3) and epithelial-mesenchymal transformation (EMT)-related proteins. The target relationship between miR-22 and NLRP3 was verified by double luciferase report. Second, an NLRP3 inhibitor and NLRP3 mimic were transfected into HCT116 cells, and the biological behaviors and EMT-related proteins were again observed. Finally, a nude mouse xenograft model was constructed to verify the above results. RESULTS: In vitro, compared with the control group, administration of the miR-22 mimic significantly decreased proliferation, migration, and invasion of HCT116 cells, whereas the miR-22 inhibitor markedly increased their proliferation and invasion (p<0.05). Levels of NLRP3, interleukin-1ß (IL-1ß), matrix metalloproteinase-9 (MMP-9), MMP-2, N-cadherin, and vimentin were significantly reduced after miR-22 mimic transfection (p<0.05). Furthermore, silencing of NLRP3, a downstream gene of miR-22 in HCT116 cells, suppressed proliferation, migration, and invasion of HCT116 cells. However, overexpression of NLRP3 weakened the effects of the miR-22 mimic. In vivo, overexpression of miR-22 slowed the growth rate of tumors and reduced Ki-67 expression in tumor tissues compared with the model group (p<0.05). In tumor tissues, overexpression of miR-22 also decreased expression of NLRP3, IL-1ß, MMP-9, MMP-2, N-cadherin, and vimentin compared with the model group (p<0.05). Overexpression of NLRP3 weakened the role of miR-22 overexpression in vivo. CONCLUSION: miR-22 suppresses cell proliferation, migration, and invasion in colorectal cancer by targeting NLRP3.

Six1 Overexpression Promotes Glucose Metabolism and Invasion Through Regulation of GLUT3, MMP2 and Snail in Thyroid Cancer Cells.

INTRODUCTION: Sineoculis homeobox homolog 1 (Six1) overexpression has been implicated in several human cancers. To date, its clinical significance and potential function in human thyroid cancer remain unclear. METHODS: Immunohistochemistry was used to examine the protein expression of BCAT1 in 89 cases of thyroid cancer tissues. We overexpressed and knockdown Six1 in TPC-1 and B-CPAP thyroid cancer cell lines. Biological roles and potential mechanisms of Six1 were examined using CCK-8, colony formation assay, Matrigel invasion assay, Western blot, PCR, ATP assay, and 2-NBDG uptake assay. RESULTS: We showed that Six1 protein was upregulated in thyroid cancers and was associated with tumor size and nodal metastasis. Analysis of TCGA dataset indicated that Six1 mRNA was higher in thyroid cancers compared with normal thyroid. CCK-8, colony formation and Matrigel invasion assays demonstrated that Six1 overexpression promoted proliferation, colony number and invasion while Six1 siRNA knockdown inhibited the growth rate, colony formation ability and invasive ability in both cell lines. Notably, Six1 upregulated glucose consumption, lactate production level and ATP level. 2-NBDG uptake analysis showed that Six1 overexpression upregulated glucose uptake while Six1 knockdown inhibited glucose uptake. Further analysis revealed that Six1 overexpression upregulated Snail, MMP2 and GLUT3 at both mRNA and protein levels. TCGA analysis demonstrated positive associations between Six1 and Snail, MMP2 and GLUT3 at the mRNA levels. CONCLUSION: Taken together, our data demonstrated that Six1 was upregulated in human thyroid cancers and promoted cell proliferation and invasion. Our data also revealed new roles of Six1 in thyroid cancer development by modulating glucose metabolism and invasion, possibly through regulation of Snail, MMP2 and GLUT3.

Novel somatic alterations underlie Chinese papillary thyroid carcinoma.

To characterize the somatic alterations of papillary thyroid carcinomas (PTC) in Chinese patients, we performed the next-generation-sequencing (NGS) study of the tumor-normal pairs of DNA and RNA samples extracted from 16 Chinese PTC patients. The whole genome sequencing (WGS) and transcriptome sequencing (RNA-seq) were conducted for 6 patients who were either current or former smokers and the whole exome sequencing (WES) and RNA-seq were conducted for another 10 patients who were never smokers. The NGS data were analyzed to identify somatic alteration events that may underlie PTC in Chinese patients. We identified a number of PTC driver genes harboring somatic driver mutations with significant functional impact such as COL11A1, TP53, PLXNA4, UBA1, AHNAK, CSMD2 and TTLL5 etc. Significant driver pathways underlying PTC were found, namely, the metabolic pathway, the pathway in cancer, the olfactory transduction pathway and the calcium signaling pathway. In addition, this study revealed genes with significant somatic copy number aberrations and corresponding somatic gene expression changes in PTC tumors, the most promising ones being BRD9, TRIP13, FZD3, and TFDP1 etc. We also identified several structural variants of PTCs, especially the novel in-frame fusion proteins such as TRNAU1AP-RCC1, RAB3GAP1-R3HDM1, and ENAH-ZSWIM5. Our study provided a list of novel PTC candidate genes with somatic alterations that may function as biomarkers for PTC in Chinese patients. The follow-up mechanism studies may be conducted based on the findings from this study.

NR2F1-AS1 regulated miR-423-5p/SOX12 to promote proliferation and invasion of papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is an aggressive histological subtype of thyroid carcinoma (THCA), whose occurrence rate is high. The participation of long noncoding RNAs in the pathologies of cancers has attracted significant attention during the past decades. The purpose of the current study is to investigate the role of NR2F1 antisense RNA 1 (NR2F1-AS1) in PTC. The expression of NR2F1 in THCA samples was analyzed by bioinformatics tool gene expression profiling interactive analysis. Levels of NR2F1-AS1, microRNA-423-5p (miR-423-5p), and SRY-box 12 (SOX12) were evaluated by a quantitative reverse transcription-polymerase chain reaction and Western blot. The impact of NR2F1-AS1 on PTC cell proliferation and invasion was assessed by Cell Counting Kit-8, EdU, and Transwell invasion assays. The interactions among NR2F1-AS1, miR-423-5p, and SOX12 were determined by RNA immunoprecipitation and luciferase reporter assays. Consequently, we found that NR2F1-AS1 and SOX12 levels were elevated in PTC, whereas miR-423-5p was downregulated in PTC cells. Functionally, NR2F1-AS1 silence led to reduced proliferation and invasion of PTC cells. Mechanistically, NR2F1-AS1 interacted with miR-423-5p to induce SOX12 expression in PTC cells. In conclusion, the present study firstly stated that NR2F1-AS1 regulated miR-423-5p/SOX12 to promote proliferation and invasion of PTC, indicating NR2F1-AS1 as a potential novel target for the molecular-targeted therapy of PTC.

SCC-S2 Facilitates Tumor Proliferation and Invasion via Activating Wnt Signaling and Depressing Hippo Signaling in Colorectal Cancer Cells and Predicts Poor Prognosis of Patients.

SCC-S2 overexpression has been implicated in several human cancers, its correlation with prognosis and the mechanism how it reserved biological roles are still uncertain. The current study demonstrated that, in 142 archived colorectal carcinoma (CRC) tissue samples, SCC-S2 expression was significantly correlated with higher histological grade ( p=0.001), tumor invasion ( p=0.001), advanced Dukes staging ( p=0.002), positive regional lymph node metastasis ( p=0.024), and poor overall survival ( p<0.001). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Transwell assays showed that SCC-S2 significantly promoted the proliferation and invasion. SCC-S2 expression was also accompanied by the overexpression CyclinD1, matrix metalloproteinase-7 (MMP-7), active-ß-catenin, yes-associated protein (YAP), and connective tissue growth factor (CTGF), as well as the depression of p-large tumor suppressor kinase 1 (p-LATS1) and p-YAP. Moreover, SCC-S2 interacted and colocalized with LATS1, the interaction may interrupt Hippo signaling and thereafter activate canonical Wnt signaling. In conclusion, our data suggested that SCC-S2 was associated with the progression and unfavorable prognosis of CRCs. Meanwhile, SCC-S2 facilitated canonical Wnt signaling and its downstream effectors (CyclinD1, MMP-7) and promoted tumor proliferation and invasion, which depended on the inhibition of Hippo signaling induced by SCC-S2-LATS1 interaction. These results indicated that SCC-S2 might be used as a novel target for the prevention and treatment of colorectal cancer.

Genome-wide association study using diversity outcross mice identified candidate genes of pancreatic cancer.

To understand the genetic causes of pancreatic cancer (PC), we conducted a genome-wide association study (GWAS) using the diversity outbred (DO) mice population to identify susceptibility genes underlying 7,12-dimethylbenzanthraene (DMBA) induced PC. The phenotype studied was the percent PC lesion area in the DO mice population. We genotyped 7851 SNP markers specifically designed for DO mice across the whole mouse genome. Four susceptibility genes with P values exceeding the genome-wide threshold for percent PC lesion area (P < 2.37 × 10-6) were identified, i.e., Epha4, Gpc5, Kcnj6, Arid1b. The most significant SNP of Gpc5 (UNC140360310) that is associated with PC lesion area in mice also significantly influences the Gpc5 expression, suggesting that this Gpc5 SNP exerts its role in PC through cis-regulating the gene expression of Gpc5. Together, our data supported that Gpc5 as a tumor suppressor gene involved in the etiology of PC.