Construction and Validation of a m7G-Related Gene-Based Prognostic Model for Gastric Cancer.

Background: Gastric cancer (GC) is one of the most common malignant tumors of the digestive system. Chinese cases of GC account for about 40% of the global rate, with approximately 1.66 million people succumbing to the disease each year. Despite the progress made in the treatment of GC, most patients are diagnosed at an advanced stage due to the lack of obvious clinical symptoms in the early stages of GC, and their prognosis is still very poor. The m7G modification is one of the most common forms of base modification in post-transcriptional regulation, and it is widely distributed in the 5' cap region of tRNA, rRNA, and eukaryotic mRNA. Methods: RNA sequencing data of GC were downloaded from The Cancer Genome Atlas. The differentially expressed m7G-related genes in normal and tumour tissues were determined, and the expression and prognostic value of m7G-related genes were systematically analysed. We then built models using the selected m7G-related genes with the help of machine learning methods.The model was then validated for prognostic value by combining the receiver operating characteristic curve (ROC) and forest plots. The model was then validated on an external dataset. Finally, quantitative real-time PCR (qPCR) was performed to detect gene expression levels in clinical gastric cancer and paraneoplastic tissue. Results: The model is able to determine the prognosis of GC samples quantitatively and accurately. The ROC analysis of model has an AUC of 0.761 and 0.714 for the 3-year overall survival (OS) in the training and validation sets, respectively. We determined a correlation between risk scores and immune cell infiltration and concluded that immune cell infiltration affects the prognosis of GC patients. NUDT10, METTL1, NUDT4, GEMIN5, EIF4E1B, and DCPS were identified as prognostic hub genes and potential therapeutic agents were identified based on these genes. Conclusion: The m7G-related gene-based prognostic model showed good prognostic discrimination. Understanding how m7G modification affect the infiltration of the tumor microenvironment (TME) cells will enable us to better understand the TME's anti-tumor immune response, and hopefully guide more effective immunotherapy methods.

Large Wolffian adnexal tumor of the ovary: A case report and literature review.

Female Wolffian adnexal tumor (WAT) is a rare neoplasm arising from the remnants of the mesonephric duct and <100 cases have been reported globally. The present case report describes a 73-year-old female patient with WAT in the left ovary which, to the best of our knowledge, is the largest benign WAT tumor to be reported. In addition, the present case report reviewed previous studies on the clinical characteristics and therapy for WAT and the surgery methods for female WAT of ovary were summarized. WATs are typically benign; however, a number factors may increase the risk of malignancy.

Bone marrow-derived mesenchymal stem cells increase drug resistance in CD133-expressing gastric cancer cells by regulating the PI3K/AKT pathway.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited to primary tumours to compose the tumour microenvironment. In various cancers, CD133-positive cells have been shown to possess cancer stem cell properties that confer chemoresistance. This study aimed to investigate the role of BM-MSCs in the anti-tumour drug resistance of CD133-expressing gastric cancer cells and explore the underlying mechanisms that governing this role. We found that CD133+ gastric cancer cells displayed more resistance to chemotherapeutics than CD133- cells. In addition, BM-MSCs increased the anti-apoptotic abilities and chemoresistance of CD133+ cells via upregulation of Bcl-2 and downregulation of BAX. Mechanistically, BM-MSCs triggered activation of the PI3K/Akt signalling cascade in CD133+ cells. Blocking the PI3K/Akt pathway inhibited the promotion of chemoresistance. Furthermore, BM-MSCs enhanced the drug resistance of CD133-overexpressing cells in vitro and in vivo, but not that of CD133-knockdown cells, which demonstrated the contribution of CD133 to this process. In conclusion, we demonstrated that BM-MSCs increased the anti-apoptotic abilities and drug resistance of CD133-expressing cells via activation of the PI3K/Akt pathway following Bcl-2 upregulation and BAX downregulation, in which CD133 played a significant role. Targeting this route may help improve the efficacy of chemotherapy in gastric cancer.

Significances of contactin-1 expression in human gastric cancer and knockdown of contactin-1 expression inhibits invasion and metastasis of MKN45 gastric cancer cells.

PURPOSE: Contactin-1 (CNTN-1) has been shown to promote cancer metastasis. Previously, we have reported that the expression of CNTN-1 was upregulated in gastric cancer tissues compared with adjacent normal tissues. Here, we investigated the significance of CNTN-1 expression and its underlying mechanism of metastasis mediated by epithelial-mesenchymal transition (EMT) in gastric cancer. METHODS: The expressions of CNTN-1 and EMT-related proteins were assayed through immunohistochemical staining of pathological specimens from patients with gastric cancer. Other methods including reverse transcriptase polymerase chain reaction, Western blotting, stably transfected against CNTN-1 into MKN45 cells, migration and invasion assays in vitro and nude mouse tumorigenicity in vivo were also utilized. RESULTS: The results revealed that CNTN-1 expression was elevated and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with gastric cancer. Moreover, CNTN-1 expression might associate with invasive ability to some extent in gastric cancer cell lines KATO-Ш, SGC7901 and MKN45. Knockdown of CNTN-1 expression in MKN45 cells using short hairpin RNA (shRNA) had notable effects on cell migration and invasion, rather than proliferation in vitro and in vivo. Furthermore, suppression of CNTN-1 expression altered EMT through inhibition of transcription factor Slug, rather than Snail. CONCLUSION: Our study demonstrated that the elevated CNTN-1 expression closely correlated with cancer metastasis and patient survival, and its functions seemed to be important in migration and invasion of gastric cancer cells via EMT alteration probably mediated by inhibition of Slug. CNTN-1 may be a potential therapeutic target for gastric cancer.

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.

Diagnostic accuracy of intracellular mycobacterium tuberculosis detection for tuberculous meningitis.

RATIONALE: Early diagnosis and treatment of tuberculous meningitis saves lives, but current laboratory diagnostic tests lack sensitivity. OBJECTIVES: We investigated whether the detection of intracellular bacteria by a modified Ziehl-Neelsen stain and early secretory antigen target (ESAT)-6 in cerebrospinal fluid leukocytes improves tuberculous meningitis diagnosis. METHODS: Cerebrospinal fluid specimens from patients with suspected tuberculous meningitis were stained by conventional Ziehl-Neelsen stain, a modified Ziehl-Neelsen stain involving cytospin slides with Triton processing, and an ESAT-6 immunocytochemical stain. Acid-fast bacteria and ESAT-6-expressing leukocytes were detected by microscopy. All tests were performed prospectively in a central laboratory by experienced technicians masked to the patients' final diagnosis. MEASUREMENTS AND MAIN RESULTS: Two hundred and eighty patients with suspected tuberculous meningitis were enrolled. Thirty-seven had Mycobacterium tuberculosis cultured from cerebrospinal fluid; 40 had a microbiologically confirmed alternative diagnosis; the rest had probable or possible tuberculous meningitis according to published criteria. Against a clinical diagnostic gold standard the sensitivity of conventional Ziehl-Neelsen stain was 3.3% (95% confidence interval, 1.6-6.7%), compared with 82.9% (95% confidence interval, 77.4-87.3%) for modified Ziehl-Neelsen stain and 75.1% (95% confidence interval, 68.8-80.6%) for ESAT-6 immunostain. Intracellular bacteria were seen in 87.8% of the slides positive by the modified Ziehl-Neelsen stain. The specificity of modified Ziehl-Neelsen and ESAT-6 stain was 85.0% (95% confidence interval, 69.4-93.8%) and 90.0% (95% confidence interval, 75.4-96.7%), respectively. CONCLUSIONS: Enhanced bacterial detection by simple modification of the Ziehl-Neelsen stain and an ESAT-6 intracellular stain improve the laboratory diagnosis of tuberculous meningitis.

[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.