The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.

Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.

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.

Elevated asymmetric dimethylarginine level as biomarkers of adverse outcomes in individuals undergoing coronary angiography/percutaneous coronary interventions: a systematic review and meta-analysis.

BACKGROUND: No previous systematic review and meta-analysis have comprehensively evaluated the association of asymmetric dimethylarginine (ADMA) level with adverse prognosis in individuals undergoing percutaneous coronary interventions (PCI)/coronary angiography (CAG). The aim of this systematic review and meta-analysis was to assess the predictive value of the elevated ADMA level in individuals undergoing CAG/PCI. MATERIALS AND METHODS: Two authors independently searched PubMed and Embase databases (up to 31 October 2020) for observational studies investigating the association between circulating ADMA level and adverse outcomes in individuals undergoing CAG/PCI. The predictive value of ADMA was expressed by pooling the multivariable-adjusted risk ratio with 95% confidence intervals (CI) for the highest versus lowest ADMA level. RESULTS: A total of nine prospective studies with 6374 participants were identified. Compared with those with the lowest ADMA level, patients with the highest ADMA level conferred an increased risk of all-cause mortality (risk ratio, 2.11; 95% CI, 1.38-3.21), cardiovascular mortality (risk ratio, 2.95; 95% CI, 1.14-7.68), major adverse cardiovascular events (risk ratio, 2.10; 95% CI, 1.35-3.27) and restenosis (risk ratio, 4.57; 95% CI, 2.52-8.30), respectively. CONCLUSIONS: High level of ADMA level is possibly an independent predictor of mortality and cardiovascular events in individuals undergoing CAG/PCI. Detection of blood ADMA level before CAG/PCI may add valuable clinical prognosis information.

LncRNA MIR99AHG mediated by FOXA1 modulates NOTCH2/Notch signaling pathway to accelerate pancreatic cancer through sponging miR-3129-5p and recruiting ELAVL1.

BACKGROUND: Pancreatic cancer (PCa) is a fatal malignancy with poor prognosis, high recurrence and mortality. Substantial reports have suggested long non-coding RNAs (lncRNAs) are implicated in development of numerous malignant tumors, and PCa is included. However, the correlation between novel lncRNA mir-99a-let-7c cluster host gene (MIR99AHG) and PCa remains elusive and needs to be deeply investigated. METHODS: In this study, we firstly used RT-qPCR to examine MIR99AHG expression. Functional assays were implemented for determination of the role of MIR99AHG in PCa cells. Mechanism experiments were designed and carried out for exploring the regulatory mechanism involving MIR99AHG. RESULTS: MIR99AHG was distinctly overexpressed in PCa cell lines. MIR99AHG deficiency abrogated PCa cell proliferation, migration and invasion. Moreover, MIR99AHG up-regulation was induced by transcription factor forkhead box A1 (FOXA1). Furthermore, MIR99AHG modulated notch receptor 2 (NOTCH2) expression and stimulated Notch signaling pathway through sequestering microRNA-3129-5p (miR-3129-5p) and recruiting ELAV like RNA binding protein 1 (ELAVL1). CONCLUSIONS: Altogether, the exploration of FOXA1/MIR99AHG/miR-3129-5p/ELAVL1/NOTCH2 axis in the progression of PCa might provide a meaningful revelation for PCa diagnosis and treatment.

Effects of Long Noncoding RNA HOTAIR Targeting miR-138 on Inflammatory Response and Oxidative Stress in Rat Cardiomyocytes Induced by Hypoxia and Reoxygenation.

OBJECTIVE: To investigate the effects of HOX transcript antisense RNA (HOTAIR) and miR-138 on inflammatory response and oxidative stress (OS) induced by IRI in rat cardiomyocytes. METHODS: H9C2 cells were divided into the control group, H/R group, H/R+siRNA NC group, H/R+si-HOTAIR group, and H/R+si-HOTAIR+inhibitor group. Expression levels of HOTAIR, miR-138, and inflammatory factors were detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The double luciferase reporter gene assay was used to detect the targeting relationship between HOTAIR and miR-138. RESULTS: Compared with the control group, the level of miR-138 and SOD in the H/R group was obviously reduced, while the expression levels of the HOTAIR, MDA, and NF-κB pathway were obviously increased. Compared with the H/R group, the level of miR-138 and SOD in the H/R+si-HOTAIR group was obviously increased, and the expression levels of the HOTAIR, MDA, and NF-κB pathway were obviously decreased. Compared with the H/R+si-HOTAIR group, the level of SOD in the H/R+si-HOTAIR+inhibitor group decreased; MDA content and the NF-κB pathway expression level increased. In the double luciferase reporter gene assay, compared with the HOTAIR wt+NC group, the luciferase activity of the HOTAIR wt+miR-138 mimic group was obviously decreased. CONCLUSIONS: Silent HOTAIR can promote the expression of miR-138 and inhibit H/R-induced inflammatory response and OS by regulating the NF-κB pathway, thus protecting cardiomyocytes.

Pancreatic Cancer Progression Is Regulated by IPO7/p53/LncRNA MALAT1/MiR-129-5p Positive Feedback Loop.

Background: Pancreatic cancer is a malignancy with poor prognosis. Importin 7 (IPO7) is a soluble nuclear transport factor, which has been linked to the pathogenesis of several human diseases. However, its role and underlying mechanism in pancreatic cancer are still obscure. Methods: Immunohistochemical staining and quantitative real-time polymerase chain reaction (qPCR) were performed to determine IPO7 expression in pancreatic cancer tissues and adjacent tissues. Western blot was used to measure IPO7 expression at the protein level in cell lines. Cell Counting Kit-8 (CCK-8), 5-bromo-2'-deoxyuridine (BrdU), flow cytometry, and Transwell assays were employed to explore the biological functions of IPO7. Subcutaneous xenograft transplanted tumor model and caudal vein injection model in mice were also established to validate the oncogenic role of IPO7. Western blot and qPCR were utilized to detect the regulatory function of IPO7 on p53 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), respectively. Interaction between MALAT1 and miR-129-5p and interaction between miR-129-5p and IPO7 were verified by bioinformatics prediction, qPCR, dual-luciferase reporter gene experiment, RNA immunoprecipitation (RIP), and pull-down assay. Results: Upregulation of IPO7 in pancreatic cancer tissues was associated with adverse prognosis of the patients with pancreatic cancer. Knocking down IPO7 remarkably suppressed cancer cell proliferation and metastasis, while it promoted apoptosis. Overexpression of IPO7 facilitated the malignant phenotypes of pancreatic cancer cells. Mechanistically, IPO7 could repress the expression of p53 and induce the expression of MALAT1 but reduce miR-129-5p expression. Furthermore, miR-129-5p was identified as a posttranscriptional regulator for IPO7, and its inhibition led to IPO7 overexpression in pancreatic cancer cells. Conclusion: IPO7 is a novel oncogene for pancreatic cancer, and IPO7/p53/MALAT1/miR-129-5p positive feedback loop facilitates the progression of this deadly disease.

LncRNA HCG11/miR-579-3p/MDM2 axis modulates malignant biological properties in pancreatic carcinoma via Notch/Hes1 signaling pathway.

BACKGROUND: Increasing reports have revealed that dysregulated expression of long non-coding RNAs (lncRNAs) is involved in pancreatic carcinoma progression. This study intends to explore the function and molecular mechanism of lncRNA HLA complex group 11 (HCG11) in pancreatic carcinoma. METHODS: The expression profiles of HCG11 in pancreatic carcinoma samples were detected by qPCR. Bioinformatics analysis was applied to detect the associations among HCG11/miR-579-3p/MDM2. The malignant properties of pancreatic carcinoma cells were measured by numerous biological assays. Xenograft model was exploited to detect the effect of HCG11 on tumor growth. RESULTS: A significant increase of HCG11 was occurred in pancreatic carcinoma samples. Knockdown of HCG11 suppressed the progression of pancreatic carcinoma cells. Bioinformatics analysis revealed that HCG11 upregulated MDM2 expression by competitively targeting miR-579-3p. The rescue assays showed that miR-579-3p reversed cell behaviors caused by HCG11, and MDM2 reversed cell properties induced by miR-579-3p. The Notch1 intracellular domain (NICD) and Hes1 protein levels were increased by overexpression of HCG11/MDM2. The tumor growth was suppressed after depletion of HCG11, followed by suppressing Ki67, PCNA and Vimentin expression, increasing TUNEL-positive cells and E-cadherin expression. CONCLUSIONS: Our observations highlighted that HCG11 contributed to the progression of pancreatic carcinoma by promoting growth and aggressiveness, and inhibiting apoptosis via miR-579-3p/MDM2/Notch/Hes1 axis.

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.

Clinical analysis of thyroid carcinoma showing thymus-like differentiation: report of 8 cases.

Thyroid carcinoma showing thymus-like differentiation (CASTLE) is a kind of rare neoplasm of the thyroid gland. Because thyroid CASTLE is rare and difficult to diagnose, its clinicopathologic features have not been well defined, and no universally accepted treatment recommendation is available. We analyzed retrospectively the clinicopathologic data of 8 patients with thyroid CASTLE who underwent surgery and radiotherapy at the Shengjing Hospital of China Medical University between December 2008 and June 2012. All patients accepted radical surgery. All patients accepted postoperative radiotherapy, except one 79-year-old patient. There was no evidence of recurrence or metastasis during the follow-up period. The pattern of immunohistochemical staining was similar to that of thymic carcinoma. Six of 8 CASTLE cases expressed CD5. All 8 CASTLE patients were negatively expressed in thyroglobulin, thyroid transcription factor 1, and calcitonin. Patients with thyroid CASTLE have good outcomes after radical resection and postoperative radiotherapy. Positive CD5 immunoreactivity can contribute to diagnosis of this disease.

CYP3A5*3 polymorphism and cancer risk: a meta-analysis and meta-regression.

CYP3A5 is a cytochrome P450 superfamily member which is involved in the metabolism of drugs, steroid hormones, and other xenobiotics. Emerging evidences suggest that CYP3A5*3 (rs776746 A>G) polymorphism may play a role in the etiology of carcinogenesis and affect an individual's susceptibility to cancer in humans, but individually published studies showed inconclusive results. This meta-analysis aimed to derive a more accurate estimation of the correlation between CYP3A5*3 polymorphism and cancer risk. A literature search of PubMed, Embase, Web of Science, and China BioMedicine databases was conducted on articles published before January 1, 2013. Seventeen case-control studies were included with a total of 7,458 cancer patients and 7,166 healthy controls. The meta-analysis results showed that CYP3A5*3 polymorphism may increase the risk of cancer, especially in acute leukemia, chronic leukemia, and colorectal cancer. However, no statistically significant associations were found in prostate cancer, liver cancer, and other cancers. Further subgroup analysis by ethnicity indicated that CYP3A5*3 polymorphism was associated with an increased risk of cancer among Asian and Caucasian populations, but not among African populations. In conclusion, the current meta-analysis suggests that CYP3A5*3 polymorphism may play an important role in the development of acute and chronic leukemia and colorectal cancer, especially among Asian and Caucasian populations.

Expression of stromal cell-derived factor 1 and CXCR7 in papillary thyroid carcinoma.

Stromal cell-derived factor 1 (SDF-1) is a chemokine that is expressed in some cancer cells and is involved in tumor cell migration and metastasis. CXCR7, a novel receptor for SDF-1, has been identified recently. Researches demonstrated that interaction between SDF-1 and CXCR7 could play an important role in cancer progression. In this study, we aimed to investigate the expressions of SDF-1 and CXCR7 and the relationship between their expressions and clinicopathological characteristics in papillary thyroid carcinoma (PTC). Expressions of SDF-1 and CXCR7 in 33 cases of thyroid benign lesion tissue and 79 cases of PTC tissue and peritumoral non-malignant tissue were detected by immunohistochemical staining. Expressions of SDF-1 and CXCR7 were negative in peritumoral non-malignant tissues. Respectively, positive expression rates of SDF-1 and CXCR7 were 69.6 and 65.8 % in PTC, 12.1 and 30.3 % in thyroid benign tissue. The expression of SDF-1 and CXCR7 were positively correlated with lymph node metastasis. SDF-1 and CXCR7 expressions were related with the lymph nodes metastasis of PTC.