Multi-regional sequencing reveals the genetic and immune heterogeneity of non-cancerous tissues in gastric cancer.

Gastric cancer (GC) is one of the most heterogeneous tumors. However, research on normal tissue adjacent to the tumor (NAT) is very limited. We performed multi-regional omics sequencing on 150 samples to assess the genetic basis and immune microenvironment in NAT and matched primary tumor or lymph node metastases. NATs demonstrated different mutated genes compared with GC, and NAT genomes underwent independent evolution with low variant allele frequency. Mutation profiles were predominated by aging and smoking-associated signatures in NAT instead of signatures associated with genetic instability. Although the immune microenvironment within NATs shows substantial intra-patient heterogeneity, the proportion of shared TCR clones among NATs is five times higher than that of tumor regions. These findings support the notion that subclonal expansion is not pronounced in NATs. We also demonstrated remarkable intra-patient heterogeneity of GCs and revealed heterogeneity of focal amplification of CD274 (encoding PD-L1) that leads to differential expression. Finally, we identified that monoclonal seeding is predominant in GC, which is followed by metastasis-to-metastasis dissemination in individual lymph nodes. These results provide novel insights into GC carcinogenesis. © 2024 The Pathological Society of Great Britain and Ireland.

Detection and genetic characteristics of porcine bocavirus in central China.

To investigate the epidemic profile and genetic diversity of porcine bocavirus (PBoV), 281 clinical samples, including 236 intestinal tissue samples and 45 fecal samples were collected from diarrheic piglets on 37 different pig farms in central China, and two SYBR Green I-based quantitative PCR assays were developed to detect PBoV1/2 and PBoV3/4/5, respectively. One hundred forty-eight (52.67%) of the 281 clinical samples were positive for PBoV1/2, 117 (41.63%) were positive for PBoV3/4/5, 55 (19.57%) were positive for both PBoV1/2 and PBoV3/4/5, and 86.49% (32/37) of the pig farms were positive for PBoV. Overall, the prevalence of PBoV was 74.73% (210/281) in central China. Subsequently, nearly full-length genomic sequences of two PBoV strains (designated CH/HNZM and PBoV-TY) from two different farms were determined. Phylogenetic analysis demonstrated that the two PBoV strains obtained in this study belonged to the PBoV G2 group and had a close relationship to 10 other PBoV G2 strains but differed genetically from PBoV G1, PBoV G3, and seven other bocaviruses. CH/HNZM and PBoV-TY were closely related to the PBoV strain GD18 (KJ755666), which may be derived from the PBoV strains 0912/2012 (MH558677) and 57AT-HU (KF206160) through recombination. Compared with reference strain ZJD (HM053694)-China, more amino acid variation was found in the NS1 proteins of CH/HNZM and PBoV-TY. These data extend our understanding of the molecular epidemiology and evolution of PBoV.

Prevalence and pattern of refractive error and visual impairment among schoolchildren: the Lhasa childhood eye study.

BACKGROUND: Early and effective ocular screening may help to eliminate treatable eye disorders. The Lhasa Childhood Eye Study (LCES) revealed the particular prevalence of refractive error and visual impairment in grade one schoolchildren (starting age of 6 years old) in Lhasa. METHODS: This is a cross-sectional part of school-based cohort study. One thousand nine hundred forty-three children were enrolled (median age, 6.78 years, range, 5.89 to 10.32). Each child underwent general and ocular examinations, including logarithm of the minimum angle of resolution (logMAR) visual acuity, cycloplegic autorefraction, and slit-lamp biomicroscopy evaluation. Multivariate and correlation analyses were performed to evaluate the association between refractive error with gender and ethnics. RESULTS: The prevalence of visual impairment (logMAR visual acuity ≥0.3 in the better-seeing eye) of uncorrected, presenting and best-corrected visual acuity (BCVA) was 12.2, 11.7 and 2.7%, respectively. Refractive error presented in 177 (78.0%) out of 227 children with bilateral visual impairment. Myopia (spherical equivalent refractor [SER] ≤ - 0.50 diopter [D] in either eye) was present in 4.7% children when measured after cycloplegic autorefraction. Hyperopia (SER ≥ + 2.00 D) affected 12.1% children. Hyperopia was significantly associated with female gender (P<0.001). Astigmatism (cylinder value ≤ - 0.75 D) was present in 44.8% children. In multivariate regression and correlation analysis, SER had no significant difference between ethnic groups. CONCLUSION: The Lhasa Childhood Eye Study is the first school-based cohort study to reveal the prevalence and pattern of refractive error and visual impairment in Lhasa. Effective strategies such as corrective spectacles should be considered to alleviate treatable visual impairment.

Prevalence and risk factors of refractive error in Qinghai, China: a cross-sectional study in Han and Tibetan adults in Xining and surrounding areas.

BACKGROUND: Our study aimed to explore the prevalence and risk factors of refractive error (RE) in Han and Tibetan population aged 50-79 years in Xining and surrounding areas in Qinghai Province on Qinghai-Tibet Plateau. METHODS: As part of the China National Health Survey, our cross-sectional study compared the age-adjusted prevalence of RE in Han and Tibetan older adults aged 50-79 years in Xining and surrounding areas. A multivariate logistic regression model was used to identify risk factors for myopia and hyperopia. RESULTS: Among 769 Han participants and 476 Tibetan participants, the age-adjusted prevalence of myopia (spherical equivalent (SE) < - 0.5D), hyperopia (SE > + 0.5D), high myopia (SE < -6.0D) and astigmatism (cylindrical equivalent > = 0.5D) is 28.56, 22.82, 2.80, and 69.38%. Han participants have higher age-adjusted prevalence of myopia (32.93% vs 21.64%, p < 0.001), high myopia (3.93% vs 1.02%, p = 0.001) and astigmatism (72.14% vs 64.94%, p = 0.021) compared to Tibetan participants. Being Tibetan is the protective factor of myopia compared to being Han (OR 0.58, 95%CI 0.42-0.79, p < 0.001). Older age (p = 0.032), longer time length in rural area (p = 0.048), undergraduate/graduate education level (p = 0.031), lighter active level (p = 0.007) and lower BMI (p = 0.015) are risk factors for myopia. Older age (all p < 0.001) and pterygium status of the same eye (p = 0.013) also increase the hyperopia risk. CONCLUSIONS: Our study found an overall prevalence of myopia of 28.56% in Xining and surrounding areas in adults older than 50 years. Han population has higher myopia risk than Tibetan population. More medical and social resources should be allocated to improve the vision and life quality of older adults.

Development of a duplex SYBR Greenâ based real-time PCR assay for detection of porcine epidemic diarrhea virus and porcine bocavirus3/4/5.

The duplex real-time PCR assay based on SYBR Green І was developed for detection of porcine epidemic diarrhea virus (PEDV) and porcine bocavirus (PBoV) 3/4/5 genotypes simultaneously. Two pairs of specific primers were designed targeting the N gene sequence of PEDV and VP1 gene sequence of PBoV3/4/5. PEDV and PBoV3/4/5 could be distinguished by their different melting temperatures (Tm) in one sample. The Tm value of PEDV was 83.5 °C, and the Tm value of PBoV3/4/5 was 78.5 °C, while other swine pathogens showed no specific melting peaks. The detection limits of this assay were 10 copies/µL for both PEDV and PBoV3/4/5. A total of sixty-three intestinal tissue samples were collected from piglets suffering from diarrhea, and the viral nucleic acids detected and identified by the real-time PCR assay and conventional PCR assay. The duplex real-time PCR detection results showed that the prevalence of PEDV and PBoV3/4/5 was 85.7% and 46%, respectively, and the co-infection rate of the two viruses was 28.6%. These results indicated that this duplex real-time PCR assay was a sensitive, specific and reproducible method for differentiating PEDV and PBoV3/4/5 or their co-infection.

Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR.

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/µL for CSFV and 36 copies/µL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

Characteristics of the spike and ORF3 genes of porcine epidemic diarrhea virus in Henan and Shanxi provinces of China.

To investigate the epidemic characteristics of porcine epidemic diarrhea virus (PEDV), 135 clinical samples (including intestinal tissues and feces) were collected from diseased piglets during outbreaks of diarrhea from 2015 to 2019 on farms in Henan and Shanxi provinces of China where swine had been immunized with attenuated PEDV (CV777). A total of 86 clinical samples (86/135, 63.7%) were positive for PEDV by RT-PCR, and subsequently, the complete spike (S) and ORF3 genes of 32 PEDV samples were sequenced. Phylogenetic analysis showed that the 32 PEDV strains obtained in this study belonged to group 2 (pandemic variant strains) and had a close relationship to 17 Chinese strains after 2010, two South Korean strains (KNU-1305 and KNU-1807), three American strains (PC22A-P140.BI, USA/Colorado/2013, and USA/OK10240-6/2017) and a Mexican strain (PEDV/MEX/QRO/02/2017), but differed genetically from a South Korean strain (SM98), a European strain (Br1/87), a Chinese strain (LZC), and a vaccine strain (CV777). G2-a subgroup strains were the dominant pandemic variant strains circulating in Henan and Shanxi provinces of China. Furthermore, a cross-recombination event was identified in the S region of the SX/TY2/2017 strain, and the putative parental strains were the epidemic strains CH/GDGZ/2012 and CH/YZ1/2015, identified in China in 2012 and 2015, respectively. These results provide further information about PEDV evolution, which could improve our understanding of the circulation of PEDV in Henan and Shanxi provinces. This information will also be helpful for developing new strategies for prevention and control of variant strains.

Androgen receptor variant 12 promotes migration and invasion by regulating MYLK in gastric cancer.

Androgen receptor (AR) and its variants (AR-Vs) promote tumorigenesis and metastasis in many hormone-related cancers, such as breast, prostate and hepatocellular cancers. However, the expression patterns and underlying molecular mechanisms of AR in gastric cancer (GC) are not fully understood. This study aimed to detect the expression of AR-Vs in GC and explored their role in metastasis of GC. Here, the AR expression form was identified in GC cell lines and tissues by RT-PCR and qPCR. Transwell assays and experimental lung metastasis animal models were used to assess the function of AR in cell migration and invasion. Downstream targets of AR were screened by bioinformatics, and identified by luciferase reporter assays and electrophoretic mobility shift assays. AR-v12 was identified as the main expression form in GC cell lines and tissues. Different from full length of AR, AR-v12 was localized to the nucleus independent of androgen. Upregulation of AR-v12 in primary GC tissues was significantly associated with metastasis. Overexpression of AR-v12 promoted migration and invasion independent of androgen. Knockdown of AR-v12 inhibited migration and invasion in vitro, as well as metastasis in vivo. Furthermore, AR-v12, serving as a transcription factor, promoted metastasis through regulating the promoter activity of MYLK. In AR-v12 overexpressing cells, knockdown of MYLK inhibited cell migration and invasion, while in AR-v12 knocked-down cells, overexpression of MYLK promoted cell migration and invasion. Collectively, our study demonstrates that AR-v12 is highly expressed in GC tissues and promotes migration and invasion through directly regulating MYLK. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Development of a TB green II-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine circovirus 2 and 3.

Porcine circovirus 3 (PCV3), as a newly emerged circovirus, is widely distributed in pig populations worldwide. Co-infection of PCV2 and PCV3 has been reported frequently in clinical samples. In the present study, a TB Green II-based duplex real-time polymerase chain reaction (qPCR) was developed to rapidly and differentially detect PCV2 and PCV3. The assay specifically detected PCV2 and PCV3, with no fluorescence signals being detected for other non-targeted pig pathogens. The duplex qPCR showed a high degree of linearity (R2 > 0.998), and its limits of detection were 10 and 78 copies/µL for PCV2 and PCV3, respectively. The duplex qPCR could detect and differentiate PCV2 (melting peaks at 85.5 °C) and PCV3 (melting peaks at 82.5 °C), and showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 2.0%. Fifty-six tissue samples from 18 pig farms were used to evaluate the duplex qPCR method. The results revealed infection rates of 66.07% (37/56) and 39.28% (22/56) for PCV2 and PCV3, respectively. The PCV2 + PCV3 co-infection rate was 39.28% (22/56). The developed method could be used as an efficient molecular biology tool for epidemiological investigations of PCV2 and PCV3.

Epigenetic downregulation of MUC17 by H. pylori infection facilitates NF-κB-mediated expression of CEACAM1-3S in human gastric cancer.

BACKGROUND AND AIMS: Helicobacter pylori invades the mucosal barrier and infects the mucins of gastric epithelial cells. However, whether gastric carcinogenesis caused by H. pylori infection involves the membrane-bound mucins is unclear. This study explored the role of mucin 17 (MUC17) in gastric cancer (GC) associated with H. pylori infection. METHODS: The expression of MUC17 and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was examined in human GC cells and tissues with H. pylori infection. Gain- and loss-of-function assays were performed to assess the role of MUC17 in regulating CEACAM1 in H. pylori-infected GC cells. RESULTS: MUC17 was downregulated in H. pylori-infected GC cells and tissues in association with poor survival of GC patients. Downregulation of MUC17 was attributable to MUC17 promoter methylation mediated by DNA methyltransferase 1 (DNMT1) H. pylori-enhanced GC cell proliferation and colony formation associated with MUC17 downregulation. Gain- and loss-of-function assays showed that MUC17 inhibited the H. pylori-enhanced GC cell growth by preventing the translocation of H. pylori CagA into GC cells. Moreover, MUC17 downregulated the expression of CEACAM1 variant 3S (CEACAM1-3S) in GC cells and tissues with H. pylori infection. Additionally, MUC17 downregulated CEACAM1 promoter activity via attenuation of NF-κB activation in GC cells. CONCLUSIONS: MUC17 was epigenetically downregulated in GC with H. pylori infection. MUC17 inhibited H. pylori CagA translocation via attenuation of NF-κB-mediated expression of CEACAM1-3S in GC cells. Thus, MUC17 may serve as a valuable prognostic biomarker for H. pylori-associated GC.

Prevalence and risk factors of refractive error: a cross-sectional Study in Han and Yi adults in Yunnan, China.

BACKGROUND: Few studies have investigated the prevalence of refractive error (RE) in older adults in China, and most have focused on East China. Our study determined the prevalence and risk factors of RE in Han and Yi adults aged 40-80 years in rural and urban areas in Yunnan Province, Southwest China. METHODS: Our cross-sectional study is part of the China National Health Survey (CNHS). The age-adjusted prevalence rates of RE in Han and Yi adults aged 40-80 years in Yunnan were compared. We used a multivariate logistic regression model to identify risk factors for myopia and hyperopia. RESULTS: Among 1626 participants, the age-adjusted prevalence rates of myopia, hyperopia, high myopia and astigmatism were 26.35% (95%CI 24.01-28.70%), 19.89% (95%CI 18.16-21.61%), 2.64% (95%CI 1.75-3.53%), and 56.82% (95%CI 54.31-59.34%). Compared to the Yi population, the Han population had higher prevalence of myopia (31.50% vs 16.80%, p < 0.0001), high myopia (3.34% vs 1.31%, p = 0.049) and astigmatism (60.07% vs 50.67%, p = 0.026) but lower prevalence of hyperopia (16.58% vs 27.37%, p < 0.0001). In the multivariate logistic regression, individuals aged 45-49 (p < 0.001), 50-54 (p < 0.001), 55-59 (p = 0.014), and 60-64 years (p = 0.005) had a lower myopia risk than those aged 40-44 years, and individuals aged 50-54 (p = 0.002), 55-59, 60-64 and 65 years and older (all p < 0.001) had a higher hyperopia risk than those aged 40-44 years. Myopia was also associated with height (p = 0.035), time spent in rural areas (p = 0.014), undergraduate/graduate education level (p = 0.001, compared with primary school or lower education level) and diabetes (p = 0.008). The Yi population had a higher risk of hyperopia than the Han population (p = 0.025). Moreover, hyperopia was related to time spent in rural areas (p < 0.001) and pterygium (p = 0.019). CONCLUSIONS: Our study investigated the overall prevalence of RE in older adults in rural and urban areas of Southwest China. Compared to the Yi population, the Han population had a higher prevalence of myopia, high myopia and astigmatism but a lower risk of hyperopia. The prevalence of myopia in the Han population in underdeveloped Southwest China was similar to that of residents in East China or of Chinese Singaporeans under urban or rural settings.

STAT3 signaling drives EZH2 transcriptional activation and mediates poor prognosis in gastric cancer.

BACKGROUND: STAT3 signaling plays the pivotal role in tumorigenesis through EZH2 epigenetic modification, which enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. Here, another possible feedback mechanism and clinical significance of EZH2 and STAT3 were investigated in gastric cancer (GC). METHODS: STAT3, p-STAT3 (Tyr 705) and EZH2 expression were examined in 63 GC specimens with matched normal tissues by IHC staining. EZH2 and STAT3 were also identified in five GC cell lines using RT-PCR and western blot analyses. p-STAT3 protein was detected by western blotting. In order to investigate whether EZH2 expression was directly regulated by STAT3, EZH2 expression was further detected using siRNA for STAT3 or IL-6 stimulation, with dual luciferase reporter analyses, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The clinical significance of STAT3, p-STAT3 and EZH2 expression was evaluated by multi-factor COX regression and Kaplan-Meier analyses. RESULTS: Hyper-activation of STAT3, p-STAT3 and EZH2 expression were observed in GC cells and tissues. STAT3 signaling was correlated with EZH2 expression in GC (R = 0.373, P = 0.003), which was consistent with our data showing that STAT3 as the transcriptional factor enhanced EZH2 transcriptional activity by binding the relative promoter region (-214 ~ -206). STAT3 was an independent signature for poor survival (P = 0.002). Patients with STAT3+/EZH2+ or p-STAT3+/EZH2+ had a worse outcome than others (P < 0.001); Besides, high levels of STAT3 and EZH2 was associated with advanced TNM staging (P = 0.017). Moreover, treatment with a combination of siSTAT3 and EZH2-specific inhibitor, 3-deazaneplanocin A (DZNEP), increased the apoptotic ratio of cells. It is benefit for targeting STAT3-EZH2 interplay in GC treatment. CONCLUSIONS: Our results indicate that STAT3 status mediated EZH2 upregulation, associated with advanced TNM stage and poor prognosis, suggesting that combination with knockdown of STAT3 and EZH2 inhibitor might be a novel therapy in GC treatment. Collectively, STAT3, p-STAT3 and EZH2 expression were provided for the precision medicine in GC patients.

Mitogen-activated protein kinase activator with WD40 repeats (MAWD) and MAWD-binding protein induce cell differentiation in gastric cancer.

BACKGROUND: Our previous proteomic analysis revealed that mitogen-activated protein kinase activator with WD40 repeats (MAWD) and MAWD-binding protein (MAWBP) were downregulated in gastric cancer (GC) tissues. These proteins interacted and formed complexes in GC cells. To investigate the role of MAWD and MAWBP in GC differentiation, we analyzed the relationship between MAWD/MAWBP and clinicopathologic characteristics of GC tissues and examined the expression of E-cadherin and pepsinogen C (PGC)-used as gastric mucosa differentiation markers-in MAWD/MAWBP-overexpressing GC cells and xenografts. METHODS: We measured MAWD, MAWBP, transforming growth factor-beta (TGF-beta), E-cadherin, and PGC expression in 223 GC tissues and matched-adjacent normal tissues using tissue microarray and immunohistochemistry (IHC) analyses, and correlated these expression levels with clinicopathologic features. MAWD and MAWBP were overexpressed alone or together in SGC7901 cells and then E-cadherin, N-cadherin, PGC, Snail, and p-Smad2 levels were determined using western blotting, semiquantitative RT-PCR, and immunofluorescence analysis. Alkaline phosphatase (AKP) activity was measured to investigate the differentiation level of various transfected cells, and the transfected cells were used in tumorigenicity assays and for IHC analysis of protein expression in xenografts. RESULTS: MAWD/MAWBP positive staining was significantly lower in GC tissues than in normal samples (P < 0.001), and the expression of these proteins was closely correlated with GC differentiation grade. Kaplan-Meier survival curves indicated that low MAWD and MAWBP expression was associated with poor patient survival (P < 0.05). The differentiation-related proteins E-cadherin and PGC were expressed in GC tissues at a lower level than in normal tissues (P < 0.001), but were upregulated in MAWD/MAWBP-overexpressing cells. N-cadherin and Snail expression was strongr in vector-expressing cells and comparatively weaker in MAWD/MAWBP co-overexpressing cells. MAWD/MAWBP co-overexpression inhibited Smad2 phosphorylation and nuclear translocation (P < 0.05), and AKP activity was lowest in MAWD/MAWBP coexpressing cells and highest in vector-expressing cells (P < 0.001). TGF-beta, E-cadherin, and PGC expression in xenograft tumors derived from MAWD/MAWBP coexpressing cells was higher than that in control. CONCLUSIONS: MAWD and MAWBP were downregulated and associated with the differentiation grade in GC tissues. MAWD and MAWBP might induce the expression of differentiation-related proteins by modulating TGF-beta signaling in GC cells.

Cell cycle-dependent expression of p42.3 promotes mitotic progression in malignant transformed cells.

In an earlier study, we cloned the p42.3 gene and showed that its expression was specific to tumors in a number of tumor cell lines and primary tumor tissues. However, the biological role and function of this gene remains largely unknown. In this study, p42.3 expression was found to be cell cycle-dependent at both the mRNA and protein levels in several human tumor cell lines. Typically, abundant expression was detected at G1 and M phases compared with S and G2 phases. Expression peaked at early G1 phase then decreased drastically at late G1, S, and G2. Furthermore, transfection of the p42.3 gene into NIH3T3 cells promoted malignant transformation, accompanied by accelerated mitotic progression and altered chromosome segregation. It was also observed that Cyclin B1 was upregulated and Cdc2-Tyr15 was downregulated following p42.3 overexpression in NIH3T3 cells. Combined, these results indicate that p42.3 as a cell cycle-regulated gene contributes to promoting cell cycle progression through disruption of mitotic regulation, and may play important roles in malignant transformation.

MiR-23a in amplified 19p13.13 loci targets metallothionein 2A and promotes growth in gastric cancer cells.

Copy number variation (CNV) and abnormal expression of microRNAs (miRNAs) always lead to deregulation of genes in cancer, including gastric cancer (GC). However, little is known about how CNVs affect the expression of miRNAs. By integrating CNV and miRNA profiles in the same samples, we identified eight miRNAs (miR-1274a, miR-196b, miR-4298, miR-181c, miR-181d, miR-23a, miR-27a and miR-24-2) that were located in the amplified regions and were upregulated in GC. In particular, amplification of miR-23a-27a-24-2 cluster and miR-181c-181d cluster frequently occurred at 19p13.13 and were confirmed by genomic real-time PCR in another 25 paired GC samples. Moreover, in situ hybridization (ISH) experiments represented that mature miR-23a was increased in GCs (75.5%, 40/53) compared with matched normal tissues (28.6%, 14/49, P = 0.001). Knocking down of miR-23a expression inhibited BGC823 cell growth in vitro and in vivo. In addition, the potential target genes of miR-23a were investigated by integration of mRNA profile and miRNA TargetScan predictions, we found that upregulation of miR-23a and downregulation of metallothionein 2A (MT2A) were detected simultaneously in 70% (7/10) of the miRNA and mRNA profiles. Furthermore, an inverse correlation between miR-23a and MT2A expression was detected in GCs and normal tissues. Through combining luciferase assay, we confirmed that MT2A is a potential target of miR-23a. In conclusion, these results suggest that integration of CNV-miRNA-mRNA profiling is a powerful tool for identifying molecular signatures, and that miR-23a might play a role in regulating MT2A expression in GC.

Decreased fructose-1,6-bisphosphatase-2 expression promotes glycolysis and growth in gastric cancer cells.

BACKGROUND: Increasing evidence suggests that cancer is a metabolic disease. Here, we investigated the potential role of fructose-1,6-bisphosphatase-2 (FBP2), the enzyme that catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate in glucose metabolism, in gastric cancer (GC) development. RESULTS: Our data indicated that FBP2 was downregulated in GC tissues (86.2%, 100/116), and absent or low FBP2 expression in GC tissues was correlated with poor survival of GC patients (P = 0.019). Conversely, ectopic expression of FBP2 in GC cells activated AMP-activated protein kinase (AMPK) signalling, inhibited the Akt-mTOR pathway, suppressed glucose metabolism, enhanced apoptosis, and reduced cell proliferation. Bisulphite genomic sequencing (BGS) in gastric cancer cell lines revealed that the FBP2 promoter region was densely methylated, and treatment of GC cells with the demethylation reagent, 5-aza-2-deoxycytidine (5-Aza), led to an increase in FBP2 expression. Importantly, forced expression of FBP2 abrogated tumour formation of these GC cells in nude mice. CONCLUSION: Our results indicate that FBP2 does negatively regulate cell growth, and reduced expression of FBP2 may contribute to carcinogenesis for GC. These findings suggest that restoration of FBP2 expression can be a promising strategy for the target therapy of GC.

Metallothionein 2A inhibits NF-κB pathway activation and predicts clinical outcome segregated with TNM stage in gastric cancer patients following radical resection.

BACKGROUND: Metallothionein 2A (MT2A) as a stress protein, plays a protective role in gastric mucosal barrier. Its role in the development of gastric cancer (GC) is unclear. The mechanism of MT2A will be investigated in gastric tumorigenesis. METHODS: MT2A expression was detected in 973 gastric specimens. The biological function was determined through ectopic expressing MT2A in vitro and in vivo. The possible downstream effectors of MT2A were investigated in NF-κB signaling. The protein levels of MT2A, IκB-α and p-IκB-α (ser32/36) expression were analyzed in a subset of 258 patients by IHC staining. The prognostic effects of MT2A, status of IκB-α and TNM stage were evaluated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: Decreased MT2A expression was detected in cell lines and primary tumors of GC. In clinical data, loss of MT2A (MT2A + in Normal (n =171, 76.0%); Intestinal metaplasia (n = 118, 50.8%); GC (n = 684. 22.4%, P < 0.001)) was associated with poor prognosis (P < 0.001), advanced TNM stage (P = 0.05), and down-regulation of IκB-α expression (P < 0.001). Furthermore, MT2A was the independent prognostic signature segregated from the status of IκB-α and pathological features. In addition, MT2A inhibited cell growth through apoptosis and G2/M arrest, which negatively regulated NF-κB pathway through up-regulation of IκB-α and down-regulation of p-IκB-α and cyclin D1 expression. CONCLUSIONS: MT2A might play a tumor suppressive activity through inhibiting NF-κB signaling and may be a prognostic biomarker and potential target for individual therapy of GC patients.

Gastrokine 1 induces senescence through p16/Rb pathway activation in gastric cancer cells.

BACKGROUND AND AIMS: Gastrokine 1 (GKN1) is a stomach-specific protein that is normally expressed in gastric mucosa but not in primary tumours and cell lines. Based on this evidence, it was presumed that GKN1 might play a role in gastric cancer development; however, its function and molecular mechanism are not clear. A systematic study was initiated that combined multiple approaches to define the molecular mechanism of GKN1 in gastric cancer cells. METHOD: Proteomics, western blotting and immunohistochemistry were used to measure the expression level of GKN1. Western blotting combined with immunofluorescence was used to monitor the secretory process of this protein. Subsequently, the function and molecular mechanism of GKN1 was explored in vitro and in vivo. RESULTS: It was shown that GKN1 is an autocrine/paracrine protein and inhibits cell growth due to senescence, which resulted from activation of p16/Rb and p21(waf) pathways. Furthermore, sustained activation of Ras/Raf/MEK/ERK signalling was characterised in gastric cancer cells and a xenograft nude mouse model following GKN1 treatment. CONCLUSION: These results provide comprehensive molecular evidence of GKN1 in inducing senescence of gastric cancer cells, and indicate that GKN1 might be a potential novel target for gastric cancer therapeutics.

A novel somatic mutation in POLE exonuclease domain associated with ultra-mutational signature and MMR deficiency in endometrial cancer: a case report.

BACKGROUND: Defect in proofreading exonuclease activity of polymerases epsilon and delta (Pols ε and δ) leads to mutagenesis and genomic instability and has been described in several cancer types. Somatic POLE exonuclease domain mutations (EDMs) have been reported in 7-12% endometrial cancers (ECs) and defined a subgroup of endometrial cancers with ultrahigh somatic mutation frequencies, high tumor infiltrated lymphocytes and favorable outcomes. CASE PRESENTATION: Herein, we presented a novel somatic mutation in POLE exonuclease domain associated with ultra-mutational signature and MMR deficiency in endometrial cancer. A novel POLE EDM (p.T278K) was found by a 11-gene NGS panel. The MSS status detected by the MSI test was inconsistent with the dMMR status by IHC. The loss of MSH6 expression in the tumor could be interpreted by the two nonsense mutations (p.E1234* and p.E1322*) of the MSH6 gene which may lead to truncated proteins. The T278K mutation was pathogenic identified by a 602-gene NGS panel with 27.3% of C > A substitution, 0.6% of indels, 0.6% of C > G substitution and a high TMB of 203.8 mut/Mb. CONCLUSIONS: We report an endometrial cancer patient harbored a novel somatic POLE T278K mutation. This mutation was a novel pathogenic POLE EDM should be considered as "POLE (ultramutated)" in clinical practice for the molecular classification of EC.

Phylogenetic analysis of porcine circovirus 4 in Henan Province of China: A retrospective study from 2011 to 2021.

Porcine circovirus 4 (PCV4), a novel circovirus, was first discovered in April 2019 in Hunan Province of China. At present, PCV4 infection has been detected in China and South Korea. However, until 2019, there was little information about its circulating status and genetic characteristics. To further clarify the origin and prevalence of PCV4, a total of 152 clinical samples collected from 49 different swine farms of 15 cities in Henan Province of China from 2011 to 2021 were tested for the presence of PCV4 by qPCR, and the complete genome of PCV4 strains was amplified from the positive samples and sequenced. Among these samples, 45.39% (69/152) were positive for PCV4 and 86.67% (13/15) of the cities and 67.35% (33/49) of the swine farms were positive for PCV4. The genome sequences of 15 PCV4 strains were obtained, of which two PCV4 strains (HN-ZMD-201212 and HN-XX-201212) were achieved from archival samples in 2012, indicating that PCV4 has been circulating for at least 10 years in Henan Province of China. The phylogenetic analysis showed that 15 PCV4 strains in our study together with PCV4 strain HNU-AHG1-2019 were clustered into an identical but separate evolutionary branch, with genomic identity ranging from 98.2% to 98.8%. Our research further provides significant epidemiological information on PCV4 in China, which will help understand the origin and genetic characteristics of this new virus.