Rabies vaccination adherence and associated factors among rabies-exposed patients in Shenzhen, China: a hospital-based cross-sectional study.

Adherence to post-exposure prophylaxis and post-exposure vaccination (PEV) is an important measure to prevent rabies. The purpose of this study was to explore the adherence to the vaccination protocol and its influencing factors among rabies-exposed patients in Shenzhen, China. A cross-sectional survey was conducted in a tertiary hospital in Shenzhen, China, to obtain epidemiological characteristics of patients; knowledge, attitude, and practice scores of rabies prevention; and medical records. A total of 326 patients requiring full rabies PEV were included in this study, and only 62% (202) completed the full course of vaccination according to the norms of the vaccination guidelines. After multifactor logistic regression, the factors influencing adherence to vaccination were as follows: age 31 to 40 years, time spent to reach the nearest rabies prevention clinic was >60 min, the time of injury was at night to early morning, the place of injury was a school/laboratory, the animal causing injury was a cat, the health status of the animal causing injury could not be determined, and patients with higher practice scores (all p<0.05). Understanding the factors influencing rabies vaccination adherence among rabies-exposed patients in urban areas of China and promote changes in patients' practice toward rabies prevention is essential for rabies elimination by 2030.

Dietary intake of total vegetable, fruit, cereal, soluble and insoluble fiber and risk of all-cause, cardiovascular, and cancer mortality: systematic review and dose-response meta-analysis of prospective cohort studies.

Objectives: To conduct a systematic review and meta-analysis of prospective cohort studies to investigate the association between total, vegetable, fruit, cereal, soluble and insoluble fiber intake and risk of all causes, cardiovascular disease (CVD), and cancer mortality and quantitatively assess the dose-response relation. Methods: Eligible studies were identified by searching PubMed, Embase and Web of science before August 2023. Random effects models were used to calculate summary relative risk (RR) and 95% confidence intervals (CI) and restricted cubic splines to model the linear/non-linear association. Results: The summary RR for all-cause, CVD and cancer mortality of dietary fiber was 0.90 (95% CI: 0.86,0.93), 0.87 (0.84,0.91), 0.91 (0.88,0.93), respectively. Significant association was observed for all-cause and CVD mortality with fruit, vegetable cereal and soluble fiber intake and cancer mortality with cereal fiber intake. No significant association was found for insoluble fiber, vegetable or fruit fiber intake and cancer mortality. Dose-response analysis showed a significant non-linear relation of dietary fiber intake with all-cause mortality, and linear relation for others. Conclusions: Higher dietary fiber including different type and food sources of fiber intake were associated with lower risk of mortality. Our findings provide more comprehensive evidence on dietary fiber intake with mortality. Systematic review registration: https://www.crd.york.ac.uk/prospero, identifier: CRD42022338837.

IGF2BP3 drives gallbladder cancer progression by m6A-modified CLDN4 and inducing macrophage immunosuppressive polarization.

INTRODUCTION: N6-methyladenosine (m6A) is an emerging epigenetic modification, which plays a crucial role in the development of cancer. Nevertheless, the underlying mechanism of m6A-associated proteins and m6A modification in gallbladder cancer remains largely unknown. MATERIALS AND METHODS: The Gene Expression Omnibus database and tissue microarray were used to identify the key m6A-related gene in gallbladder cancer. The function and mechanism of IGF2BP3 were further investigated by knockdown and overexpression techniques in vitro and in vivo. RESULTS: We found that IGF2BP3 was elevated and correlated with poor prognosis in gallbladder cancer, which can be used as an independent prognostic factor for gallbladder cancer. IGF2BP3 accelerated the proliferation, invasion and migration of gallbladder cancer cells in vitro and in vivo. Mechanistically, IGF2BP3 interacted with and augmented the stability of CLDN4 mRNA by m6A modification. Enhancement of CLDN4 reversed the inhibitory effect of IGF2BP3 deficiency on gallbladder cancer. Furthermore, we demonstrated that IGF2BP3 promotes the activation of NF-κB signaling pathway by up-regulation of CLDN4. Overexpression of IGF2BP3 in gallbladder cancer cells obviously promoted the polarization of immunosuppressive phenotype in macrophages. Besides, Gallbladder cancer cells-derived IGF2BP3 up-regulated the levels of STAT3 in M2 macrophages, and promoted M2 polarization. CONCLUSIONS: We manifested IGF2BP3 promotes the aggressive phenotype of gallbladder cancer by stabilizing CLDN4 mRNA in an m6A-dependent manner and induces macrophage immunosuppressive polarization, which might offer a new theoretical basis for against gallbladder cancer.

Burden of stroke attributable to high ambient temperature from 1990 to 2019: A global analysis.

AIMS: To determine the global and regional burden of stroke due to high temperature and the spatiotemporal trends in 204 countries and territories from 1990 to 2019. METHODS: Based on Global Burden of Disease Study 2019, deaths, disability-adjusted life years (DALYs), and age-standardized mortality rate (ASMR) and age-standardized DALY rate (ASDR) for stroke attributable to high temperature (i.e. a daily mean temperature warmer than the theoretical minimum-risk exposure level (TMREL)) were calculated in global, geographical location, and country and analyzed by age, sex, subtypes, and socio-demographic index (SDI) from 1990 to 2019. The trends in ASMR and ASDR from 1990 to 2019 were estimated by linear regression model. The regression coefficients (ß) referred to a mean change of per year for ASMR or ASDR attributable to high temperature. RESULTS: The global burden of stroke attributable to high temperature had an increase trend from 1990 to 2019 (ß = 0.005, 95% uncertainty interval (UI) = 0.003-0.007 for ASMR and ß = 0.104, 95% UI = 0.066-0.142 for ASDR, respectively). Globally, in 2019, an estimated 0.048 million deaths and 1.01 million DALYs of stroke were attributable to high temperature, and the global ASMR and ASDR of stroke attributable to high temperature were 0.60 (95% UI = 0.07-1.30) and 13.31 (1.40-28.97) per 100,000 population, respectively. The largest burden occurred in Western Sub-Saharan Africa, followed by South Asia, Southeast Asia, and North Africa and the Middle East. ASMR and ASDR increased with age and were higher in males and for intracerebral hemorrhage, and were the highest in the low SDI regions. In 2019, the region with the largest percentage increase in ASMR and ASDR attributable to high temperature was Eastern Sub-Saharan Africa from 1990 to 2019. CONCLUSIONS: Stroke burden due to high temperature has been increasing, and a higher burden was observed in people aged 65-75 years, males, and countries with a low SDI. Stroke burden attributable to high temperature constitutes a major global public health concern in the context of global warming.

METTL3 promotes glycolysis and cholangiocarcinoma progression by mediating the m6A modification of AKR1B10.

OBJECTIVE: N6-methyladenosine (m6A) RNA methylation is involved in governing the mechanism of tumor progression. We aimed to excavate the biological role and mechanism of the m6A methyltransferase METTL3 in cholangiocarcinoma (CCA). METHODS: METTL3 expression was determined by database and tissue microarray analyses. The role of METTL3 in CCA was explored by loss- and gain-of-function experiments. The m6A target of METTL3 was detected by RNA sequencing. The role of AKR1B10 in CCA was explored, and the association between METTL3 and AKR1B10 was confirmed by rescue experiments. RESULT: METTL3 expression was upregulated in CCA tissue, and higher METTL3 expression was implicated in poor prognoses in CCA patients. Overexpression of METTL3 facilitated proliferation, migration, invasion, glucose uptake, and lactate production in CCA cells, whereas knockdown of METTL3 had the opposite effects. We further found that METTL3 deficiency inhibited CCA tumor growth in vivo. RNA sequencing and MeRIP-qPCR confirmed that METTL3 enhanced AKR1B10 expression and m6A modification levels. Furthermore, METTL3 directly binds with AKR1B10 at an m6A modification site. A CCA tissue microarray showed that AKR1B10 expression was upregulated in CCA tissue and that silencing AKR1B10 suppressed the malignant phenotype mentioned above in CCA. Notably, knockdown of AKR1B10 rescued the tumor-promoting effects induced by METTL3 overexpression. CONCLUSION: Elevated METTL3 expression promotes tumor growth and glycolysis in CCA through m6A modification of AKR1B10, indicating that METTL3 is a potential target for blocking glycolysis for application in CCA therapy.

Acetoin production from lignocellulosic biomass hydrolysates with a modular metabolic engineering system in Bacillus subtilis.

BACKGROUND: Acetoin (AC) is a vital platform chemical widely used in food, pharmaceutical and chemical industries. With increasing concern over non-renewable resources and environmental issues, using low-cost biomass for acetoin production by microbial fermentation is undoubtedly a promising strategy. RESULTS: This work reduces the disadvantages of Bacillus subtilis during fermentation by regulating genes involved in spore formation and autolysis. Then, optimizing intracellular redox homeostasis through Rex protein mitigated the detrimental effects of NADH produced by the glycolytic metabolic pathway on the process of AC production. Subsequently, multiple pathways that compete with AC production are blocked to optimize carbon flux allocation. Finally, the population cell density-induced promoter was used to enhance the AC synthesis pathway. Fermentation was carried out in a 5-L bioreactor using bagasse lignocellulosic hydrolysate, resulting in a final titer of 64.3 g/L, which was 89.5% of the theoretical yield. CONCLUSIONS: The recombinant strain BSMAY-4-PsrfA provides an economical and efficient strategy for large-scale industrial production of acetoin.

Predicting patient experience of Invisalign treatment: An analysis using artificial neural network.

Objective: Poor experience with Invisalign treatment affects patient compliance and, thus, treatment outcome. Knowing the potential discomfort level in advance can help orthodontists better prepare the patient to overcome the difficult stage. This study aimed to construct artificial neural networks (ANNs) to predict patient experience in the early stages of Invisalign treatment. Methods: In total, 196 patients were enrolled. Data collection included questionnaires on pain, anxiety, and quality of life (QoL). A four-layer fully connected multilayer perception with three backpropagations was constructed to predict patient experience of the treatment. The input data comprised 17 clinical features. The partial derivative method was used to calculate the relative contributions of each input in the ANNs. Results: The predictive success rates for pain, anxiety, and QoL were 87.7%, 93.4%, and 92.4%, respectively. ANNs for predicting pain, anxiety, and QoL yielded areas under the curve of 0.963, 0.992, and 0.982, respectively. The number of teeth with lingual attachments was the most important factor affecting the outcome of negative experience, followed by the number of lingual buttons and upper incisors with attachments. Conclusions: The constructed ANNs in this preliminary study show good accuracy in predicting patient experience (i.e., pain, anxiety, and QoL) of Invisalign treatment. Artificial intelligence system developed for predicting patient comfort has potential for clinical application to enhance patient compliance.

Production of d-Tagatose by Whole-Cell Conversion of Recombinant Bacillus subtilis in the Absence of Antibiotics.

d-tagatose is a popular functional monosaccharide produced from lactose by ß-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for ß-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing ß-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.

Free fatty acids and triglyceride change in the gallbladder bile of gallstone patients with pancreaticobiliary reflux.

BACKGROUND: Pancreaticobiliary reflux (PBR) causes chronic inflammation of the gallbladder mucosa and changes in the bile components, which are known to promote gallstone formation. This study aimed to investigate the bile biochemistry changes in gallstone patients with PBR and provide new clues for research on the involvement of PBR in gallstone formation. METHODS: Patients undergoing surgery for gallstones between December 2020 and May 2021 were eligible for inclusion. The bile biochemistry (including amylase, lipase, triglyceride, cholesterol, free fatty acids [FFAs], alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], and γ-glutamyl transferase [γ-GT]) of the included gallstone patients was analysed to determine correlations with PBR. RESULTS: In this study, 144 gallstone patients who underwent surgery were enrolled. Overall, 15.97 % of the patients had an increased bile amylase level, which was associated with older age and significantly higher bile levels of ALP, lipase, triglyceride, and FFAs. Positive correlations were observed between amylase and lipase, triglyceride, FFAs levels in the gallbladder bile. However, the bile levels of triglyceride, FFAs, and lipase were positively correlated with each other only in the PBR group and showed no significant correlation in the control (N) group. In addition, elevated bile FFAs levels were found to be an independent risk factor for gallbladder wall thickening. CONCLUSIONS: In conclusion, PBR-induced increase in FFAs and triglyceride in the gallbladder bile is a cause of gallstone formation, and an increase in bile ALP suggests the presence of cholestasis in PBR.

Alteration of Intestinal Microbiota Composition in Oral Sensitized C3H/HeJ Mice Is Associated With Changes in Dendritic Cells and T Cells in Mesenteric Lymph Nodes.

This research aimed to investigate the allergic reaction of C3H/HeJ mice after sensitization with ovalbumin (OVA) without any adjuvant and to analyze the association between intestinal microbiota and allergy-related immune cells in mesenteric lymph nodes (MLN). The allergic responses of C3H/HeJ mice orally sensitized with OVA were evaluated, and immune cell subsets in spleen and MLN and cytokines were also detected. The intestinal bacterial community structure was analyzed, followed by Spearman correlation analysis between changed gut microbiota species and allergic parameters. Sensitization induced a noticeable allergic response to the gavage of OVA without adjuvant. Increased levels of Th2, IL-4, CD103+CD86+ DC, and MHCII+CD86+ DC and decreased levels of Th1, Treg, IFN-γ, TGF-ß1, and CD11C+CD103+ DC were observed in allergic mice. Furthermore, families of Lachnospiraceae, Clostridiaceae_1, Ruminococcaceae, and peprostreptococcaceae, all of which belonging to the order Clostridiales, were positively related to Treg and CD11C+CD103+ DC, while they were negatively related to an allergic reaction, levels of Th2, CD103+CD86+ DC, and MHCII+CD86+ DC in MLN. The family of norank_o_Mollicutes_RF39 belonging to the order Mollicutes_RF39 was similarly correlated with allergic reaction and immune cells in MLN of mice. To sum up, allergic reactions and intestinal flora disturbances could be induced by OVA oral administration alone. The orders of Clostridiales and Mollicutes_RF39 in intestinal flora are positively correlated with levels of Treg and CD11C+CD103+ DC in MLN of mice.

Inhibition of CD133 Overcomes Cisplatin Resistance Through Inhibiting PI3K/AKT/mTOR Signaling Pathway and Autophagy in CD133-Positive Gastric Cancer Cells.

Cisplatin is widely used as the standard gastric cancer treatment, but the relapse and metastasis are common as intrinsic or acquired drug resistance. CD133 has been widely known to be associated with chemoresistance in various cancer cells. In this study, we focused on investigating the function and mechanism of CD133 underlying cisplatin resistance in gastric cancer cell line KATO-III. We detected CD133 expression by using quantitative real-time polymerase chain reaction and Western blot and found that expression of CD133 was upregulated in cisplatin resistance of KATO-III cells (Cis-KATO-III) compared with KATO-III cells, indicating the role of CD133 in regulating cisplatin resistance of KATO-III cells. Then we sorted the Cis-KATO-III cells into CD133-positive (CD133+) pools and measured the proliferation and apoptosis after the cell is transfected with pc-CD133 and sh-CD133 by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and flow cytometry. The results showed that the inhibition of CD133 inhibited the cell viability and promoted the cell apoptosis after cisplatin treatment. Furthermore, we found that inhibition of CD133 downregulated the expression of PI3K/AKT and promoted the expression of mammalian target of rapamycin, thus inhibited the autophagic activity in the Cis-KATO-III cells after cisplatin treatment. Besides, we also verified the effects of CD133 in vivo. The results indicated that inhibition of CD133 enhanced the Cis-KATO-III cell sensitivity to cisplatin by regulating PI3K/AKT/mTOR signaling pathway. In summary, our data provide new insight that CD133 activates the PI3K/AKT/mTOR signaling transduction pathway, resulting in activation of autophagy and cisplatin resistance of Cis-KATO-III cells. These results may offer a novel therapeutic target in cisplatin-resistant gastric cancer.

Long noncoding RNA HOTAIRM1 inhibits cell progression by regulating miR-17-5p/ PTEN axis in gastric cancer.

OBJECTIVES: This study was conducted to identify the significantly altered long noncoding RNAs (lncRNAs), messenger RNA (mRNA) and pathways in gastric cancer (GC). METHODS: We used microarray analysis to identify differentially expressed lncRNAs and mRNAs, whereas the obviously changed pathways were found by gene set enrichment analysis. The coexpression network of lncRNA and mRNA was constructed by Cytoscape, and their target relationships with miRNAs were predicted by miRcode and TargetScan. qRT-PCR and Western blot were performed to determine the expression levels of mRNAs and proteins in tissues and cell lines. Dual-luciferase reporter assay was applied to achieve the determination of the specific target relationships. Cell viability, migration, and apoptosis were detected by MTT assay, wound healing assay and flow cytometry, respectively. Through the xenograft assay, the gastric tumor was implanted into nude mice to investigate the influence of HOTAIRM1 in vivo. RESULTS: HOTAIRM1 and phosphatase and tensin homolog (PTEN) were both downregulated in GC, whereas miR-17-5p was upregulated. Moreover, the PI3K/AKT pathway was found activated in GC. HOTAIRM1 targeted miR-17-5p, whereas PTEN was the downstream target gene of miR-17-5p. HOTAIRM1 suppressed proliferation and migration of GC cell line and induced their apoptosis, whereas miR-17-5p played the opposite role on GC cell line. HOTAIRM1 also postponed tumor growth in vivo and inhibited the PI3K/AKT pathway in GC. CONCLUSIONS: LncRNA HORAIRM1 suppressed the PI3K/AKT pathway in GC and inhibited the progression of GC by serving as a competing endogenous RNA of miR-17-5p, mediating the expression of PTEN.

Experimental use of a novel single-port gasless laparoendoscopic operative field formation device.

INTRODUCTION: Traditional laparaendoscopic surgery using CO2 pneumoperitoneum is associated with complications and the existing gasless laparaendoscopic surgery has shortcomings such as poor visibility in the operation field. To overcome the disadvantages of the current lifting gasless laparaendoscopic operation platforms, we developed an inflatable device for gasless laparoscopic operation field formation (LOFF) that can be internally installed and applied in practice. METHODS: We initially designed operation platforms for gasless laparaendoscopic single-port (GLESP) surgery. Subsequently, a triangular prismatic LOFF device was selected and applied successfully to GLESP cholecystectomy of five pigs. Ultimately, using pigs as a model, three surgical approaches (LOFF-assisted laparaendoscopic single-site (LOFF-LESS), LESS surgery, and traditional lifting (GLESP) were compared, and the advantages and drawbacks of inflatable devices for gasless laparoscopic operation field assessed. RESULTS: The use of the LOFF device in GLESP cholecystectomy was first evaluated. The time for surgical space formation (4.4 ± 1.2 and 4.8 ± 1.0), the operating time for gallbladder removal (25.2 ± 4.8 and 25.4 ± 2.7), and the loss of blood (9.4 ± 3.1and 9.2 ± 2.4) was similar between LESS and LOFF, respectively (Table 2). In contrast these parameters were higher in GLESP (6.6 ± 1.0, 30.3 ± 4.4 and 10.1 ± 2.0, respectively. The LOFF-LESS surgery exhibited a clearer exposure of the surgical field and shorter operating time than the GLESP surgery. LESS technology showed less postoperation pain, fast recovery, and extremely high cosmetic satisfaction. CONCLUSION: The LOFF device provides a safe, effective, and feasible operation platform that can be internally installed and inflated for GLESP surgery during cholescytectomy in animal models.

Overexpression of CD133 enhances chemoresistance to 5-fluorouracil by activating the PI3K/Akt/p70S6K pathway in gastric cancer cells.

CD133 has been reported to be associated with chemoresistance in various cancer cells. The efficacy of 5-fluorouracil (5-FU), an important chemotherapeutic agent for advanced gastric cancer (GC), is limited by 5-FU resistance. Hence, the present study investigated the function of CD133 in 5-FU resistance in human GC cells. We isolated CD133+ GC cells by immunomagnetic cell sorting and CD133 expression was modulated by transfection of CD133 gene or CD133 small interfering ribonucleic acid. To assess the 5-FU cytotoxicity, Cell Counting Kit-8 was used. Expression of CD133, P-glycoprotein (P-gp), B-cell lymphoma 2 (Bcl­2), Bcl-2-associated X protein (Bax), phospho-Akt (p-Akt) and phospho-p70S6 kinase (p-p70S6K) were analyzed by western blotting. CD133, P-gp, Bcl-2 and Bax messenger ribonucleic acids were evaluated using semi-quantitative reverse transcriptase-polymerase chain reaction. Cell apoptosis was assessed by Hoechst 33258 staining. CD133+ cells were more resistant to 5-FU than CD133- cells, and showed higher expression of P-gp and Bcl-2 with lower expression of Bax. Furthermore, CD133 silencing enhanced 5-FU cytotoxicity and apoptotic characteristics, whereas CD133 overexpression increased 5-FU resistance. CD133 silencing and activation directly decreased and increased the expression of P-gp, Bcl­2, p-Akt and p-p70S6K, respectively. Notably, Akt inhibition by LY294002 restored the 5-FU cytotoxicity suppressed by CD133 overexpression, while Akt activation by epidermal growth factor reversed the 5-FU cytotoxicity enhanced by CD133 silencing. Therefore, CD133 may inhibit 5-FU-induced apoptosis by regulating the expression of P-gp and Bcl-2 family mediated by phosphoinositide 3-kinase/Akt/p70S6K pathway in GC cells.

Study on the Biological Characteristics of CD133 (+) Cells Interfered by RNA Interference in Gastric Cancer.

Background. To detect the changes of biological characteristics in gastric cancer cells interfered by CD133-specific small interfering RNA (siRNA). Methods. First to select the siRNA which has the strongest interference effect among 3 siRNAs (i.e., siRNA1, siRNA2, and siRNA3) in KATO-III cells by RT-PCR and Western blotting assays. Then, CD133(+) cells were sorted out from KATO-III cells using an immunomagnetic bead sorting method and transfected with the selected siRNA. Furthermore, the proliferating characteristics, the antichemotherapeutic assessment, Transwell invasion assay, monoclonal sphere formation assay, and subcutaneous transplanted tumor formation assay in nude mice were investigated. Results. siRNA3 showed the strongest interference effect in KATO-III cells. As compared to the uninterfered control group, the CD133(+) cells treated by siRNA3 showed significant decreases in the abilities of proliferation, invasion, clone sphere formation, and resistance to antitumour drugs as well as the weight and size of the transplanted tumor, which was nearly similar to that of CD133(-) cells. Additionally, the protein expression level of the EMT factor E-cadherin increased while those of EMT-related Snail and N-cadherin decreased in CD133(+) cells interfered by siRNA3. Conclusion. Inhibition of CD133 gene expression reduces the abilities of gastric cancer cells in proliferation, invasion, clonal sphere formation, and chemoresistance as well as tumor formation in nude mice.

Preliminary Study on the Expression and the Clinical Significance of CD133 in Peripheral Blood of Patients with Gastric Adenocarcinoma.

Background. Significances of CD133 mRNA in peripheral blood mononuclear cells (PBMCs) of gastric adenocarcinoma (GC) patients were investigated. Methods. Correlations of CD133 mRNA expression in PBMCs on clinicopathological parameters or CD133 protein expression were analyzed. Receiver operating characteristic curve according to bright scale value (BSV) of CD133 mRNA was used to group patients for prognosis analysis. Results. BSV of preoperative CD133 mRNA in PBMCs in GC was significantly higher than that in volunteers or in GU. Invasive depth or metastatic lymph node number for higher BSV of preoperative CD133 mRNA and invasive depth or lymphatic vessel invasion for higher BSV of postoperative CD133 mRNA in the PBMCs were identified. Patients with CD133(+) expression in primary lesion had a significantly higher expression of preoperative CD133 mRNA in the PBMCs. The expression of preoperative or postoperative CD133 mRNA in PBMCs related positively to CD133 mRNA expression in primary lesion. Patients with higher expression of preoperative or postoperative CD133 mRNA shared significantly shorter survival compared with that in lower expression group. Conclusion. Higher levels of preoperative or postoperative CD133 mRNA in PBMCs of GC correlated positively to the lymphatic metastasis and the BSV of CD133 mRNA in primary lesion, indicating the poorer survival.

[Effect of RNA interference inhibition to expression of CD133 on tumor cell biological characteristics in KATO-III CD133(+) cells of human gastric cancer].

OBJECTIVE: To investigate the changes in proliferation, invasiveness, clone sphere formation and chemosensitivity of human gastric cancer cell lines of KATO-III CD133(+) cells transfected with small interfering RNA (siRNA) against CD133 gene. METHODS: CD133(+) cells of KATO-III cell lines were isolated by magnetic activated cell sorting (MACS). CD133 siRNA was designed and synthesized, and then transfected into KATO-III CD133(+) cells. Cell fluorescence counting under confocal laser scanning microscope was used to determine the transfection efficiency after transfection with the CD133 FITC-siRNA. The knock-down effect of the CD133 gene and expression of epithelial-mesenchymal transition (EMT)-related factors were detected by RT-PCR and Western blotting. Cell counting kit-8 assay (CCK-8), transwell chamber and colony sphere forming assay were performed to measure the variation of cell proliferative, invasive, colony formation viability and chemosensitivity to 5-FU after the above-mentioned treatment. RESULTS: The transfection efficiency was (87.7±8.1)%. The CD133 mRNA and protein expression levels in the interference group were lower than those in negative control group. Twenty-four, 48 and 72 hours after transfection, cells proliferation activity was significantly inhibited in the interference group compared with negative control group, (all P<0.01). Seventy-two hours after transfection, compared with negative control group, cells proliferation activity was reduced by (52.1±8.0)%. The invasive cell number reduced (41.7±6.0 vs. 130.3±11.0, P<0.05) and clone formation rate decreased significantly [(24.3±4.3)% vs. (45.1±6.4)%, P<0.01] in the interference group. EMT-related gene E-cadherin protein expression increased, while the Snail and N-cadherin protein expression reduced in the interference group (all P<0.01). The cells sensitivity to 5-FU was significantly enhanced in the interference group, and the cell inhibition rate of 5-Fu was (62.4±3.3)%, higher than that in negative control group [(21.5±2.2)%, P<0.01]. CONCLUSIONS: The expression of CD133 gene plays an important role in cell proliferation, invasiveness, colony formation and resistance to chemotherapy of KATO-III CD133(+) gastric cancer cells. It suggests that CD133 can be used as one of surface markers for detection of gastric cancer stem cells. Inhibition of CD133 expression may be a promising way for gastric cancer biotherapy.

[CD133 promotes the invasion and metastasis of gastric cancer via epithelial-mesenchymal transition].

OBJECTIVE: To examine the association between CD133 expression and invasion of gastric cancer, and to elucidate whether CD133 can promote the invasion and metastasis of gastric cancer via epithelial-mesenchymal transition (EMT). METHODS: The CD133(+) and CD133(-) KATO-III( cells were sorted by magnetic activated cell sorting (MACS). The invasion ability was detected by Transwell method. RT-PCR and Western blot were used to detect the expression of EMT-related factors in KATO-III( cells before and after CD133 was knocked out by siRNA method. The expressions of CD133 and EMT-related proteins of cancer and adjacent normal tissues in 50 patients with gastric cancer were detected by Western blot, and correlations among protein expressions were also analyzed. RESULTS: As compared to CD133(-) cells, the number of broken-membrane cells was significantly higher (67.7±10.5 vs. 13.3±6.8, P=0.001) and the invasion ability was stronger (P<0.05) in CD133(+) cells, while the mRNA expression levels of Snail and N-cadherin were significantly higher in CD133(+) cells (0.311±0.015 vs. 0.223±0.016, P=0.040; 0.581±0.020 vs. 0.270±0.018,P=0.004), and the protein expression levels of Snail and N-cadherin were significantly higher in CD133(+) cells as well (0.513±0.015 vs. 0.179±0.023, P=0.030; 0.538±0.028 vs. 0.202±0.032, P=0.020), but E-cadherin mRNA and protein levels were significantly lower in CD133(+) cells (0.231±0.009 vs. 0.460±0.015, P=0.040; 0.426±0.030 vs. 0.748±0.027, P=0.040). After CD133 knock-out, the expressions of Snail and N-cadherin were down-regulated (P<0.05) and the expression of E-cadherin was up-regulated (P<0.05). As compared to normal mucosal tissues, the protein expression levels of Snail, N-cadherin and CD133 in gastric cancer tissues were significantly higher(0.635±0.119 vs. 0.485±0.116, P=0.029; 0.599±0.114 vs. 0.259±0.108, P=0.020; 0.754±0.154 vs. 0.329±0.134, P=0.001), while the protein expression of E-cadherin in gastric cancer tissues was lower (0.378±0.123 vs. 0.752±0.156, P=0.003). The protein expressions of Snail and N-cadherin were positively correlated with CD133 expression (r=0.278, P=0.048; r=0.406, P=0.003) and the protein expression of E-cadherin was negatively correlated with CD133 expression (r=-0.504, P=0.000). CONCLUSION: CD133(+) cells in primary lesion of gastric cancer have relatively higher invasion ability, which may promote the metastasis of gastric cancer via up-regulation of EMT-related factors.

Expression and significances of contactin-1 in human gastric cancer.

Background. This study aimed at determining the relationship between vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), and contactin-1 (CNTN-1) expression in gastric cancer (GC). Methods. The expression level of CNTN-1 mRNA and CNTN-1 protein of 33 cases was determined using RT-PCR and Western Blot. And 105 cases were immunohistochemically examined for VEGF-C, VEGFR-3, and CNTN-1 expressions. Assessment of lymphatic vessel density (LVD) was also performed by D2-40 immunostaining. Then we analyzed the relationships between VEGF-C, VEGFR-3, and CNTN-1, as well as their correlations with clinicopathologic features, LVD, and survival time. Results. The positivity rate of VEGF-C, VEGFR-3, and CNTN-1 in primary tumor was 56.19%, 64.76%, and 58.09%. The expression of CNTN-1 significantly correlated with VEGF-C (P < 0.001) and VEGFR-3 (P < 0.001). All of them were closely related to TNM stage, lymphatic invasion, and lymph node involvement (P < 0.05). LVD was significantly correlated with VEGF-C (P = 0.001), VEGFR-3 (P = 0.011), and CNTN-1 expression (P < 0.001). VEGF-C, VEGFR-3, and CNTN-1 expression significantly associated with poorer prognosis (P < 0.001, P = 0.034, P = 0.012, resp.). Conclusion. CNTN-1 associated with VEGF-C and VEGFR-3 expression in GC. All of them correlated with lymphatic metastasis, which might play an important role in the lymphatic invasion via lymphangiogenesis pathway in GC.

[Sorting of CD133(+) subset cells in human gastric cancer and the identification of their tumor initiating cell-like properties].

OBJECTIVE: To sort CD133(+) subset cells in human gastric cancer (GC) and to identify their tumor initiating cell-like properties. METHODS: The tissues of GC and normal tissues adjacent to GC were obtained from 50 patients. Samples were stained for CD133 by immunohistochemistry. Likewise, assessments of CD133 were undertaken by Western blot. Flow cytometry was used to determine the proportion of CD133(+) cells in four GC cell lines therein the KATO-III was sorted by magnetic activated cell sorting (MACS) method. The growing characteristics and the tumorigenic ability of CD133(+) cells were evaluated in vitro and in vivo. Meanwhile, the growth of single cells in suspension culture was observed and expression of stem cell-specific marker were determined using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of CD133 was demonstrated on the cell membranes in the mucosa and submucosa of primary GC, which were higher than those in the normal gastric tissues adjacent to cancer (P<0.05). Four GC cell lines including KATO-III, SGC-7901, AGS and MKN-45 were found to contain (28 ± 2)%, (17 ± 2)%, (6 ± 2)%, and (4 ± 2)% of CD133(+) cells respectively. In addition, the purity of CD133(+) cells isolated from KATO-III by MACS was (91 ± 3)% and up to(95 ± 2)% after 1-week culture. CCK-8 detection showed that population doubling time of the CD133(+) cells was (21 ± 3)h, significantly shorter than that of the CD133(-) cells[(40 ± 8)h, P<0.05]. Notably, there was a remarkable difference of tumor formation rate between CD133(+) cells (100%), non-sorted cells (80%), and CD133(-) cells(0). The average mass and volume of tumor in group of CD133(+) cells was larger and heavier than those in non-sorted cells (P<0.05, P<0.05). Furthermore, the single cell proliferated well, formed the big sphere and semi-quantitative RT-PCR showed expression of stem cell markers such as Oct-4, Nanog, Sox-2, Musashi-1 and EGFR. CONCLUSIONS: CD133 protein expression in primary lesions is higher than those in the normal gastric tissues. CD133(+) subset cells can be isolated, purified, and amplified in human GC, and possess some properties including the ability of self-renewal, proliferation, and higher tumorigenic ability in vivo and can express some stem cell markers.