The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Small bowel obstruction - A rare complication after endoscopic transcecal appendectomy.

Endoscopic transcecal appendectomy has become an alternative treatment for laparoscopic appendectomy due to its less invasive nature. Here we report a rare complication of small intestinal obstruction after endoscopic transcecal appendectomy.

Identification of ORF1B as a unique nonstructural protein for fowl adenovirus serotype 4.

Fowl adenovirus serotype 4 (FAdV-4), the causative agent of hepatitis-hydropericardium syndrome (HHS), is a double-stranded DNA virus. Although many structural proteins have been deeply studied, the coding potential of some other open reading frames (ORFs) and the biological functions of their products during virus infection have not been fully elucidated. Here, a unique nonstructural protein ORF1B of FAdV-4 was identified and its expression kinetics along infection was analyzed. Except that of FAdV-10, a member of the same genus as FAdV-4, FAdV-4 ORF1B shared as low homologous identity as 29.2% in amino acid sequence with the other ten counterparts. Structurally, ORF1B was mapped on the N-terminal region of the genome between 1485 nt to 1808 nt and predicted to only contain two α-helix. Confocal immunofluorescence assay with homemade rabbit polyclonal antibody demonstrated that ORF1B could be simultaneously observed with structural protein Fiber 1 in FAdV-4-infected cells. Western blot further showed that ORF1B could only be detected in the infected cells but not mature virions, suggesting ORF1B was a nonstructural protein. Subsequently, the expression level of ORF1B detected by qRT-PCR and IFA was gradually decreased along with FAdV-4 infection, suggesting ORF1B was an early gene transcript. These results will lay a solid foundation to further study the biological effect of ORF1B on the replication and pathogenicity of FAdV-4.

Corrigendum: Self-assembling and pH-Responsive protein nanoparticle as potential platform for targeted tumor therapy.

[This corrects the article DOI: 10.3389/fmolb.2023.1172100.].

Isolation and genomic characterization of one novel goose astrovirus causing acute gosling gout in China.

Novel goose astrovirus (NGAstV) is a member of the genus Avain Avastrovirus (AAstV) and the family Astroviridae. NGAstV-associated gout disease has caused huge economic losses to the goose industry worldwide. Since early 2020, NGAstV infections characterized by articular and visceral gout emerged continuously in China. Herein, we isolated a GAstV strain from goslings with fatal gout disease and sequenced its complete genome nucleotide sequence. Then we conducted systematic genetic diversity and evolutionary analysis. The results demonstrated that two genotypic species of GAstV (GAstV-I and GAstV-II) were circulating in China, and GAstV-II sub-genotype IId had become the dominant one. Multiple alignments of amino acid sequences of GAstV capsid protein revealed that several characteristic mutations (E456D, A464N, and L540Q) in GAstV-II d strains, as well as additional residues in the newly identified isolate which varied over time. These findings enrich the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies.

Virus usurps alternative splicing to clear the decks for infection.

Since invasion, there will be a tug-of-war between host and virus to scramble cellular resources, for either restraining or facilitating infection. Alternative splicing (AS) is a conserved and critical mechanism of processing pre-mRNA into mRNAs to increase protein diversity in eukaryotes. Notably, this kind of post-transcriptional regulatory mechanism has gained appreciation since it is widely involved in virus infection. Here, we highlight the important roles of AS in regulating viral protein expression and how virus in turn hijacks AS to antagonize host immune response. This review will widen the understandings of host-virus interactions, be meaningful to innovatively elucidate viral pathogenesis, and provide novel targets for developing antiviral drugs in the future.

Circ_001422 aggravates osteosarcoma progression through targeting miR-497-5p/E2F3 axis.

Circular RNAs exert vital functions in the pathogenesis of osteosarcoma (OS). Circ_001422 has been confirmed to be involved in regulating OS progression, but its specific mechanism has not been clearly studied. This work aimed to analyze circ_001422's role in OS cell biological behaviors and the possible molecular mechanisms. This work carried out reverse transcription-quantitative polymerase chain reaction for detecting circ_001422, E2F3 and miR-497-5p levels, whereas Cell counting kit-8 together with Transwell assays for measuring cell growth, migration as well as invasion abilities. Relation of miR-497-5p with E2F3, as well as circ_001422 with miR-497-5p was analyzed through dual-luciferase reporter gene assay. Protein level was identified by western blot. According to our results, circ_001422 expression within OS tissue significantly increased compared with corresponding healthy samples. Inhibition of circ_001422 significantly decreased OS cell growth, invasion and migration. From mechanism research, miR-497-5p was proved as circ_001422's target, and E2F3 was miR-497-5p's target. Besides, miR-497-5p downregulation or E2F3 overexpression abolished circ_001422 inhibition-mediated inhibition on OS cell proliferation, invasion and migration. Collectively, this study has first suggested circ_001422's role in enhancing OS proliferation, migration as well as invasion via miR-497-5p/E2F3 axis. Our results will offer new ideas and new anti-OS targets.

Self-assembling and pH-responsive protein nanoparticle as potential platform for targeted tumor therapy.

Frequent injections at high concentrations are often required for many therapeutic proteins due to their short in vivo half-life, which usually leads to unsatisfactory therapeutic outcomes, adverse side effects, high cost, and poor patient compliance. Herein we report a supramolecular strategy, self-assembling and pH regulated fusion protein to extend the in vivo half-life and tumor targeting ability of a therapeutically important protein trichosanthin (TCS). TCS was genetically fused to the N-terminus of a self-assembling protein, Sup35p prion domain (Sup35), to form a fusion protein of TCS-Sup35 that self-assembled into uniform spherical TCS-Sup35 nanoparticles (TCS-Sup35 NP) rather than classic nanofibrils. Importantly, due to the pH response ability, TCS-Sup35 NP well retained the bioactivity of TCS and possessed a 21.5-fold longer in vivo half-life than native TCS in a mouse model. As a result, in a tumor-bearing mouse model, TCS-Sup35 NP exhibited significantly improved tumor accumulation and antitumor activity without detectable systemic toxicity as compared with native TCS. These findings suggest that self-assembling and pH responding protein fusion may provide a new, simple, general, and effective solution to remarkably improve the pharmacological performance of therapeutic proteins with short circulation half-lives.

Identification and molecular characterization of novel duck reoviruses in Henan Province, China.

Novel Duck reovirus (NDRV) is an ongoing non-enveloped virus with ten double-stranded RNA genome segments that belong to the genus Orthoreovirus, in the family Reoviridae. NDRV-associated spleen swelling, and necrosis disease have caused considerable economic losses to the waterfowl industry worldwide. Since 2017, a significant number of NDRV outbreaks have emerged in China. Herein, we described two cases of duck spleen necrosis disease among ducklings on duck farms in Henan province, central China. Other potential causative agent, including Muscovy duck reovirus (MDRV), Duck hepatitis A virus type 1 (DHAV-1), Duck hepatitis A virus type 3 (DHAV-3), Newcastle disease virus (NDV), and Duck tembusu virus (DTMUV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and two NDRV strains, HeNXX-1/2021 and HNJZ-2/2021, were isolated. Sequencing and phylogenetic analysis of the σC genes revealed that both newly identified NDRV isolates were closely related to DRV/SDHZ17/Shandong/2017. The results further showed that Chinese NDRVs had formed two distinct clades, with late 2017 as the turning point, suggesting that Chinese NDRVs have been evolving in different directions. This study identified and genetic characteristics of two NDRV strains in Henan province, China, indicating NDRVs have evolved in different directions in China. This study provides an insight into the ongoing emerged duck spleen necrosis disease and enriches our understanding of the genetic diversity and evolution of NDRVs.

Chitosan/Calcium-Coated Ginsenoside Rb1 Phosphate Flower-like Microparticles as an Adjuvant to Enhance Immune Responses.

Infectious bursal disease (IBD) is a highly contagious immunocompromising disorder that caused great economic losses in the poultry industry. The field-level control over IBD is primarily via vaccination. The development of a highly effective IBV vaccine has drawn great attention worldwide. Chitosan/Calcium Phosphate (CS/CaP) nanoparticle was a newly developed effective biological delivery system for drug and antigen. Ginsenoside Rb1 is one of the main bioactive components of ginseng root extract, which has antioxidant, anti-inflammatory and immunological enhancement effects. Until now, the combined effect of CS/CaP and ginsenoside Rb1 on the chicken immune response had remained unknown. In this study, the GRb1 and IL-4 were encapsulated into Calcium phosphate and chitosan core structure nanoparticles microspheres (GRb1/IL-4@CS/CaP), and the effect of a newly developed delivery system on an infectious bursal disease virus (IBDV) attenuated vaccine was further evaluated. The results demonstrated that GRb1/IL-4@CS/CaP treatment could induce the activation of chicken dendritic cells (DCs), with the upregulated expression of MHCII and CD80, and the increased production of IL-1ß and TNF-α. Importantly, GRb1/IL-4@CS/CaP could trigger a higher level of IBDV-specific IgG and a higher ratio of IgG2a/IgG1 than the traditional adjuvant groups, promoting the production of cytokine, including IFN-γ, TNF-α, IL-4, IL-6, IL-1α, and IL-1ß, in chicken serum after 28 d and 42 d post-vaccine. Taken in all, GRb1/IL-4@CS/CaP could elicit prolonged vigorous immune responses for IBDV attenuated vaccine in chicken, which might provide an effective adjuvant system for avian vaccine development.

Cellular protein HSC70 promotes fowl adenovirus serotype 4 replication in LMH cells via interacting with viral 100K protein.

Fowl adenovirus serotype 4 (FAdV-4), the predominant causative agent of hepatitis-hydropericardium syndrome (HHS), has caused severe economic losses to poultry industry since 2015. Although fiber2 and hexon have been confirmed to be the virulence-related factors, the roles of nonstructural viral proteins in pathogenicity of FAdV-4 remain poorly understood. Here, a tandem mass spectrometry (MS) was used to identify host factors interacted with 100K protein of hypervirulent FAdV-4 isolate (CH/HNJZ/2015), and 2595 cellular proteins associated with many biological processes and pathways were identified according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Among the proteins, HSC70 was verified to interact with 100K through co-immunoprecipitation assay. Notably, overexpression of HSC70 promoted the replication of FAdV-4 in LMH cells, whereas blocking HSC70 with inhibitor ver-155008 markedly suppressed viral replication. Collectively, these findings suggested that many cellular proteins involved in FAdV-4 infection through interacting with 100K and HSC70 positively regulated virus replication.

Core antigenic advantage domain-based ELISA to detect antibody against novel goose astrovirus in breeding geese.

Goose astrovirus (GAstV), the major causative agent of visceral and joint gout in goslings, is a novel pathogen greatly threatening waterfowl industry. Importantly, the horizontal and vertical transmissibility of GAstV posed a great challenge for disease prevention and control. Given the absence of commercial vaccine, restricting vertical transmission and protecting susceptible goslings must be a priority. Although many detection methods have been established, there is no serological method to detect GAstV-specific antibody, greatly limiting inspection and elimination of infected breeding bird. In this study, the B-cell epitopes of GAstV capsid protein were predicted, and its core antigenic advantage domain (shCAP) was expressed and purified. After authenticating the antigenicity, the recombinant shCAP protein was taken as the coating antigen, and an easily accessible indirect enzyme-linked immunosorbent assay (ELISA) was established to detect GAstV-specific antibody. The working conditions, including antigen concentration, serum dilution and incubation time, blocking buffer concentration, and color developing time, were gradually optimized by checkerboard titration. The cut-off OD450 value of the indirect ELISA for positive sample was 0.379, and the analytical sensitivity was 1:800. There was no cross-reaction with sera against goose parvovirus (GPV), Tembusu virus (TUMV), H5 and H7 subtype avian influenza virus (AIV H5 + H7), and Newcastle disease virus (NDV). The assay was further applied to examine 73 breeding goose serum samples and shared excellent agreement of 93.5% (68/73) with western blot, which also suggested that GAstV is circulating in the goose population in China. In conclusion, the developed indirect ELISA is simple, specific, and sensitive, which will be greatly useful to screen GAstV infection and block vertical transmission. KEY POINTS: • B-cell epitopes of GAstV capsid protein were predicted and expressed as immunogen • A core antigenic advantage domain-based ELISA was established to detect GAstV-specific antibody • The established ELISA will contribute to inspection and elimination of infected breeding geese and provide a useful tool for large scale serological testing of GAstV in geese.

Identification and genomic characterization of emerging goose astrovirus in central China, 2020.

Astroviruses are a non-enveloped virus with large host range breadth. AstV-associated gastroenteritis in human and animal, nephritis in chicken, gout in gosling and hepatitis in duckling pose great threats to public health and poultry industry. Since early 2020, continuous emergence of fatal goose astrovirus (GAstV) infections characterized by articular and visceral gout was reported in China. Here, we described two outbreaks of emerging gout disease in two different goose farms of central China. Two virulent GAstV strains, designated as HNKF-1/China/2020 and HNSQ-6/China/2020, were isolated, and the fifth passage of the isolates could cause urate crystals accumulated in the allantoic fluid and even deposited around great vessels and embryo bodies. Meanwhile, the source of these GAstV outbreaks was tracked to goose hatcheries. The prevalence of GAstV in the goose embryos with hatch failure was confirmed, and embryo-origin HNXX-6/China/2020 was further isolated. The complete genome of these three newly isolates was then sequenced and analysed. The results showed that Chinese GAstVs have formed two distinct groups, and the three GAstV isolates, as well as most of the Chinese GAstVs, belong to the G-I group. There are several amino acid mutations in the three newly identified GAstVs, such as A520T, S535R, V555I and A782T in ORF1a and Q229P in ORF2, suggesting the field stains, HNKF-1/China/2020 and HNSQ-6/China/2020, might derive from the weak goose embryo via vertical transmission. Moreover, the phylogenetic analysis of the complete viral genome and individual viral proteins revealed that Chinese GAstV strains have been constantly evolving towards more complicated and various directions. Our study reported the recently emerging GAstV outbreaks in central China, and further analysed the genetic characteristics of three virulent G-I GAstV isolates from commercial goose farms and goose hatchery, indicating the diverse transmission of the virus and providing a basis for developing effective preventive measures and control strategies.

The game between host antiviral innate immunity and immune evasion strategies of senecavirus A - A cell biological perspective.

Innate immunity is the first line of the cellular host to defend against viral infection. Upon infection, viruses can be sensed by the cellular host's pattern recognition receptors (PRRs), leading to the activation of the signaling cascade and the robust production of interferons (IFNs) to restrict the infection and replication of the viruses. However, numerous cunning viruses have evolved strategies to evade host innate immunity. The senecavirus A (SVA) is a newly identified member of the Picornaviridae family, causing severe vesicular or ulcerative lesions on the oral mucosa, snout, coronary bands, and hooves of pigs of different ages. During SVA infection, the cellular host will launch the innate immune response and various physiological processes to restrict SVA. In contrast, SVA has evolved several strategies to evade the porcine innate immune responses. This review focus on the underlying mechanisms employed by SVA to evade pattern recognition receptor signaling pathways, type I interferon (IFN-α/ß) receptor (IFNAR) signaling pathway, interferon-stimulated genes (ISGs) and autophagy, and stress granules. Deciphering the antiviral immune evasion mechanisms by SVA will enhance our understanding of SVA's pathogenesis and provide insights into developing antiviral strategies and improving vaccines.

Adenovirus vector-attributed hepatotoxicity blocks clinical application in gene therapy.

Adenoviruses (Ads), common self-limiting pathogens in humans and animals, usually cause conjunctivitis, mild upper respiratory tract infection or gastroenteritis in humans and hepatotoxicity syndrome in chickens and dogs, posing great threats to public health and livestock husbandry. Artificially modified Ads, which wipe out virulence-determining genes, are the most frequently used viral vectors in gene therapy, and some Ad vector (AdV)-related medicines and vaccines have been licensed and applied. Inherent liver tropism enables AdVs to specifically deliver drugs/genes to the liver; however, AdVs are closely associated with acute hepatotoxicity in immunocompromised individuals, and the side effects of AdVs, which stimulate a strong inflammatory reaction in the liver and cause acute hepatotoxicity, have largely limited clinical application. Therefore, this review systematically elucidates the intimate relationship between AdVs and hepatotoxicity in terms of virus and host and precisely illustrates the accumulated understanding in this field over the past decades. This review demonstrates the liver tropism of AdVs and molecular mechanism of AdV-induced hepatotoxicity and looks at the studies on AdV-mediated animal hepatotoxicity, which will undoubtedly deepen the understanding of AdV-caused liver injury and be of benefit in the further safe development of AdVs.

[Lung Cancer Screening Study in Macao Smoking Individuals].

BACKGROUND: Lung cancer incidence in Macao increases gradually, smoking is one of the important high risk factors. The purpose of this study is to observe the detection rate of lung cancer and nodules in long-term smoking Macao individuals. METHODS: We recruited eligible Macao residents by publicity, all subjects were arranged to receive low-dose computed tomography screening. Image features of lung nodules were analyzed by radiologist. For suspicious lung cancer, multiple disciplinary team (MDT) was arranged. RESULTS: A total of 291 were adopted, 10 lung cancers were detected, the detection rate of lung cancer was 3.44% (95%CI: 2.78%-4.01%), all were males. There were 5 adenocarcinoma patients, each 2 squamous-cell carcinoma and small cell lung carcinoma patients; 1 adenosquamous cancer patient. Among 10 lung cancers, 40% had stage 1 disease. The detection rate of lung nodules was 72.9% (95%CI: 67.8%-78.0%); The number of suspicious lung nodules were 44, and the detection rate was 15.1% (95%CI: 11.0%-19.2%). There was no significant differences in the lung cancer detection rate between the single and multiple lung nodule groups (P>0.05). There were 168 subjects in the <6 mm solid lung nodule (SN) and <5 mm no-solid lung nodule (NSN) group and no lung cancer was found, 44 subjects in the ≥6 mm SN and ≥5 mm NSN group. All 9 lung cancer patients were detected in this group. The detection rate of lung cancer was higher than that of the <6 mm SN and <5 mm NSN group (P<0.05). CONCLUSIONS: There are high detection rate of lung cancer and lung nodule in the long-term smoking individuals. The lung cancer rate increases when the lung nodule size is larger than 6 mm in SN and 5 mm in NSN. Adenocarcinoma is the major type in the smokers' lung cancers. We suggest long-term smokers should join in the future lung cancer screening trial in Macao. Female lung cancer screening should be established different standard.

Combined effect of chrysin and apigenin on inhibiting the development and progression of colorectal cancer by suppressing the activity of P38-MAPK/AKT pathway.

Either apigenin or chrysin alone has been found to exert anti-inflammatory and tumor suppressive effect. However, the combined effect of apigenin and chrysin on colorectal cancer (CRC) has not been fully clarified. We attempted to explore the effect of chrysin and apigenin on CRC and its related mechanism. SW480 and HCT-116 cells were treated with either apigenin or chrysin alone or two-drug combination at different doses of 5, 25, 50, 100 µM for optimal concentration determination. Then, we focused on the individual and combined effect of apigenin and chrysin on clonogenicity, apoptosis, metastasis-related behaviors of CRC cells by colony formation assay, cell scratch assay, flow cytometry, and transwell assay. The changes of the activation of P38-MAPK/AKT pathway were evaluated underlying apigenin and chrysin intervention, further after co-treated with P38-MAPK agonist anisomycin. Apigenin (25 µM) combined with chrysin (25 µM) were determined to be optimal. Treatment with the combination of apigenin (25 µM) and chrysin (25 µM) significantly reduced cell clone numbers, migration, and invasion ability, while increased the cell apoptosis in both CRC cell lines. The combined effect was higher than chrysin or apigenin alone. Meanwhile, p-P38 and p-AKT were significantly downregulated by chrysin and apigenin treatment. The tumor inhibitive effect of apigenin combined with chrysin was obviously reversed by adding P38 agonist, anisomycin. Apigenin (25 µM) combined with chrysin (25 µM) showed synergetic effect in inhibiting the growth and metastasis of CRC cells by suppressing the activity of P38-MAPK/AKT pathway.

Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A.

Senecavirus A (SVA), a recently emerging picornavirus, poses a great threat to the swine industry because it causes swine idiopathic vesicular disease and epidemic transient neonatal losses. Thus far, the progress in SVA viral pathogenesis studies and vaccine development remains sluggish, and an available and convenient reverse genetics system would undoubtedly promote relevant research. Herein, we established an improved universal dual-promoter reverse genetics system with an SVA-specific hammerhead ribozyme and hepatitis delta virus ribozyme at both terminals of the viral genome; this system could be applied to rescue all SVA strains by both eukaryotic and prokaryotic RNA polymerase systems. The genome of the clone-derived Chinese field strain CH/HeN-2018 was assembled into the universal vector pcDNA-rSVAuni through the Gibson assembly technique. Moreover, two silent mutations, G6848C and C7163 G, were separately engineered into the full-length cDNA clone with one step site-directed mutagenesis to create a KpnI restriction enzyme site, which served as a unique genetic marker. The viruses, designated rCH/HeN-2018-T7, rCH/HeN-2018-CMV, rCH/HeN-2018-6484 m and rCH/HeN-2018-7163 m, were successfully rescued through both CMV- and T7-dependent pathways, and their biological properties were further evaluated. The results showed that all four viruses grew rapidly in PK-15 cells and exhibited viral titers and growth kinetics similar to those of parental wtCH/HeN-2018. The established reverse genetics system is easily operated and can be applied to rescue all SVA strains in a short time, which will be helpful for studying SVA biology, including viral pathogenesis, antiviral therapies and vaccine development.

First report and genetic characterization of bovine torovirus in diarrhoeic calves in China.

BACKGROUND: Coronaviruses are notorious pathogens that cause diarrheic and respiratory diseases in humans and animals. Although the epidemiology and pathogenicity of coronaviruses have gained substantial attention, little is known about bovine coronavirus in cattle, which possesses a close relationship with human coronavirus. Bovine torovirus (BToV) is a newly identified relevant pathogen associated with cattle diarrhoea and respiratory diseases, and its epidemiology in the Chinese cattle industry remains unknown. RESULTS: In this study, a total of 461 diarrhoeic faecal samples were collected from 38 different farms in three intensive cattle farming regions and analysed. Our results demonstrated that BToV is present in China, with a low prevalence rate of 1.74% (8/461). The full-length spike genes were further cloned from eight clinical samples (five farms in Henan Province). Phylogenetic analysis showed that two different subclades of BToV strains are circulating in China. Meanwhile, the three BToV strains identified from dairy calves, 18,307, 2YY and 5YY, all contained the amino acid variants R614Q, I801T, N841S and Q885E. CONCLUSIONS: This is the first report to confirm the presence of BToV in beef and dairy calves in China with diarrhea, which extend our understanding of the epidemiology of BToVs worldwide.

lncRNA PCAT18 inhibits proliferation, migration and invasion of gastric cancer cells through miR-135b suppression to promote CLDN11 expression.

BACKGROUND: Gastric cancer is a severe disease with a high occurrence rate worldwide. And lncRNAs are demonstrated to be responsible for cancer growth and metastasis. So, it is of great importance to explore the lncRNAs involved mechanism of gastric cancer occurrence and development deeply. METHODS: Transfection was conducted to build over-expression and down-expression models. Moreover, RT-qPCR and western blot were used to detect the transcriptional and translational levels. The biological functions such as proliferation, migration and invasion of AGS cells were evaluated by MTT analysis, colony formation assay, scarification detection and transwell assay, respectively. The potential binding of miR-135b and its downstream and upstream molecules was validated by dual luciferase reporter gene assay or RIP. Also, the in-vivo mice model was further used to demonstrate the role of lncRNA PCAT18 in gastric cancer. RESULTS: PCAT18 down-expression promoted proliferation, migration and invasion of gastric cancer cells. Furtherly, over-expression of miR-135b also promoted these biological characteristics of AGS cells. Importantly, we found that PCAT18 could bind miR-135b which also was bound with CLDN11. We found that miR-135b is negatively correlated with CLDN11; PCAT18 and CLDN11 are positively correlated. Moreover, miR-135b mimics could down-regulate protein level of CLDN11, whereas CLDN11 could reverse this effect. In in-vivo experiment, PCAT18 over-expression restrained tumor growth and metastasis. CONCLUSIONS: Over-expressed lncRNA PCAT18 inhibits proliferation, migration and invasion of gastric cancer cells through regulation of miR-135b/CLDN11.