Objective To investigate the prognostic value of mesenchymal circulating tumor cells ( ^M CTCs) in colon cancer (CC). Methods A total of 115 CC patients hospitalized in the Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University from January 2015 to June 2016 were selected as the research objects. Within the 24 h after admission, 2. 5 mL of peripheral venous blood was administered through peripheral veins into EDTA-containing tube. The number of ^M CTCs in blood sample was detected by using CTCBIOPSY ^® which was previously developed by our team combined with the immunofluorescence ( IF) staining, and the correlation between the number of ^M CTCs and clinicopathologic factors was analyzed. Furthermore, Kaplan-Meier survival curve and COX regression analysis were used to explore the relationship between ^M CTCs and patients’ prognosis. Results There was no significant difference in the number of ^M CTCs in peripheral blood of patients with different tumor locations, but the number of ^M CTCs in patients with low and middle tumor differentiation was significantly higher than that of patients with high tumor differentiation. Moreover, the number of ^M CTCs in peripheral blood of patients with stage Ⅲ was significantly higher than that of patients with stage Ⅱ and stage Ⅰ. Further analysis showed that ^M CTCs positive were correlated with tumor differentiation grade ( P = 0. 013 ), depth of tumor invasion (P = 0. 010), lymph node metastasis (P = 0. 043), TNM stage (P < 0. 001), vascular infiltration (P< 0. 001), nerve invasion ( P = 0. 004) and CEA level ( P < 0. 001). Survival analysis showed that the overall survival time of patients with ^M CTCs positive was significantly lower than that of patients with ^M CTCs negative (χ² = 9. 726, P = 0. 002), and ^M CTCs positive was an independent risk factor for overall survival of patients with CC (HR = 1. 752, 95 % CI: 1. 104 ~ 3. 871, P = 0. 015). Conclusion Mesenchymal CTCs in the peripheral blood of patients with CC are closely related to the malignant progression of tumors and poor prognosis of patients, which is expected to be a novel biomarker for the clinical prognosis evaluation. 

Objective To investigate the effect of PD-1 inhibitors on tumor-associated macrophage (TAM) polarization and proliferation and invasion of prostate cancer, and the related mechanisms. Methods The tumor and adjacent tissues of 25 patients with prostate cancer were collected. The expression levels of programmed death factor 1 ( PD-1), macrophage mannose receptor (CD206) and signal transduction and transcription activator 6 ( STAT6) were detected by realtime quantitative fluorescent PCR ( qRT-PCR), and Spearman correlation analysis was then performed. Human monocytic leukemia cells (THP-1) were induced to TAM, during which the PD-1 inhibitor Camrelizumab or the STAT6 inhibitor AS1517499 was added. The above cells were divided into 4 groups of M0, M0 + Camrelizumab, TAM and TAM + Camrelizumab, or 4 groups of M0, M0 + AS1517499, TAM and TAM + AS1517499. The expression levels of STAT6 and CD206 were detected by qRT-PCR and Western blotting, and the concentration of interleukin-13 ( IL-13 ), transforming growth factor-β ( TGF-β) and nitric oxide synthase ( iNOS) were detected by enzyme-linked immunosorbent assay ( ELISA). Each group of cells were co-cultured with prostate cancer cells (DU145). The proliferation level of DU145 cells were detected by CCK8, and the invasion level were detected by Transwell assay. Results The expression levels of PD-1, CD206 and STAT6 in the prostate tumor tissues were elevated when compared with those in the adjacent tissues, and the expressions of PD-1, STAT6 were positively correlated with the expression of CD206 in tumor tissues respectively (all P< 0. 05). The expression levels of CD206 and STAT6 and the secretion levels of IL-13 and TGF-β were increased, while the level of iNOS was decreased in the TAM group, when compared with those in the M0 group, and the proliferation and invasion ability of DU145 cells co-cultured with the TAM were increased ( all P < 0. 05). The expression levels of CD206 and STAT6 were reduced in the TAM + Camrelizumab group when compared with the TAM group, in addition, the secretion levels of IL-13 and TGF-β were also decreased, the secretion levels of iNOS was increased. Meanwhile, the proliferation and invasion ability of DU145 cells cocultured with cells in the TAM + Camrelizumab group were decreased (all P < 0. 05). The cells in the TAM + AS1517499 group also had the reduced expression level of CD206, decreased secretion levels of IL-13 and TGF-β and increased secretion levels of iNOS when compared with those in the TAM group. Meanwhile, the proliferation and invasion ability of DU145 cells co-cultured with cells in the TAM + AS1517499 group were decreased (all P < 0. 05). Conclusion PD-1 inhibitors reduce the proliferation and invasion of prostate cancer cells by inhibiting the polarization of macrophages to TAM, which may be achieved by inhibiting the STAT6 signaling pathway in macrophages

Objective To investigate the key mechanisms of HIF-1α on the Angiogenesis in malignant meningioma. Methods The mRNA and protein expression levels of HIF-1α and IGF1R in normal placenta tissues, benign meningioma tissues (WHO grade Ⅰ) and recurrent meningioma tissues (WHO grade Ⅱ-Ⅲ) were determined by qPCR and immunohistochemistry. Dual-luciferase gene reporter assay and ChIP assay were used to verify the direct interaction between HIF-1α and IGF1 gene regulatory region. Knockdown of HIF-1α expression in anaplastic meningioma CRL3370 cells were performed by using HIF-1α shRNA. ELISA was used to determine the secretion of IGF1 and VEGF in the CRL-3370 cells after HIF-1α knock-down. Western blotting was used to determine the expression levels of p-IGF1Rβ, IGF1Rβ and VEGFR2 in CRL-3370 cells. A co-cultured system of CRL-3370 cells and HUVEC2 was established, and the expression of HIF-1α in CRL-3370 cells was induced by hypoxia condition and followed by the shRNA knocking-down. CCK8 assay was used to determine the cell proliferation ability of HUVEC2. qPCR assay was used to determine the VEGF, ANGP1 and MMP2 mRNA expression levels in HUVEC2 cells. Wound-healin assay and transwell assay were used to determine the migration and invasion ability of HUVEC2 cells. Results The mRNA and protein expression levels of HIF-1α and IGF1R in meningioma tissues increased as the meningioma progressed from benign (WHO grade Ⅰ) to reccurrent (WHO grade Ⅱ-Ⅲ) (P < 0. 01). The luciferase activity in the IGF1 group was significantly up-regulated when compared with that in the Control group. The luciferase activity in the deletion site-4 group was significantly inhibited when compared with that in the IGF1 group (P < 0. 05). ChIP experiment showed that the DNA content of site-4 in the HIF-1α group was significantly higher than that in the corresponding IgG group (P< 0. 05). The levels of secreted IGF1 and VEGF in the HIF-1α shRNA group were significantly reduced when compared with those in the shRNA NT group. The intracellular expression levels of p-IGF1Rβ, IGF1Rβ and VEGFR2 were down regulated. The cell proliferation ability of HUVEC2 in the co-cultured system was reduced and the mRNA expression levels of VEGF, ANGP1 and MMP2 were decreased. Cell migration and invasion abilities were reduced (P< 0. 05). Conclusion HIF-1α is up-regulated and directly enhances the IGF1 / IGF1R signaling to promote the angiogenesis in HUVEC2 and metastasis of recurrent meningioma. 

Objective Aldo-keto reductase family 1 member B10 (AKR1B10) plays an important role in the proliferation and metastasis of malignant tumors including hepatocellular carcinoma (HCC). However, the role of AKR1B10 in HCC glycolysis is not fully understood. Therefore, this study aims to explore the function of AKR1B10 in HCC glycolysis and investigate its potential mechanism. Methods The expression of AKR1B10 in HCC was analyzed by bioinformatics. The expression of AKR1B10 was detected by Real-time reverse transcription PCR (qRT-PCR) and Western blotting. Cell Counting Kit 8 (CCK-8), Transwell assay and flow cytometry were used to detect the cell proliferation, migration, invasion and apoptosis, respectively. The expression levels of Myelocytomatosis (MYC), hexokinase 2 (HK2) and phosphoglycerate kinase 1 (PGK1) were detected by qRT-PCR. Seahorse XP96 was used to analyze the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). The levels of pyruvate, lactic acid, citric acid and malic acid were detected by kits. Results The bioinformatic analysis showed that AKR1B10 was highly expressed in HCC tissues and cells, and GSEA analysis showed that glycolytic pathway was enriched. Overexpression of AKR1B10 could promote the proliferation, migration and invasion of HCC cells, and inhibit the cell apoptosis. In addition, the overexpression of AKR1B10 could promote the expression of MYC, HK2 and PGK1 in HCC cells, and improve the level of glycolysis. Glycolysis inhibitors (2-Deoxy-D-Glucose, 2-DG) could reverse the effect of AKR1B10 on the proliferation, migration, and invasion in HCC cells. Conclusion AKR1B10 can promote the HCC proliferation by activating glycolytic pathway, suggesting that AKR1B10 may be a new diagnostic marker and therapeutic target for HCC.

Objective To investigate the expression of eosinophil chemotactic factor 1 (Eotaxin-1) and sphingosin-1-phosphate receptor 1 (S1PR1) in serum of patients with acute respiratory distress syndrome (ARDS) and their relationship with prognosis. Methods A total of 140 ARDS patients admitted to Nanjing Brain Hospital from February 2019 to February 2022 were selected. According to the oxygenation index (PaO2 / FiO2 ), they were divided into 3 groups: mild group (201 ~ 300 mmHg) (n = 43), moderate group (101 ~ 200 mmHg) (n = 40) and severe group (≤100 mmHg) (n = 57). Another 50 healthy patients who came to our hospital for physical examination during the same period were selected as the control group. According to the 28-day prognosis, the patients were divided into survival group (95 cases) and death group (45 cases). APACHE-Ⅱ was used to score the ARDS patients. The levels of serum Eotaxin-1 and S1PR1 were detected by enzyme-linked immunosorbent assay (ELISA). Pearson correlation was used to analyze the correlation between the levels of serum Eotaxin-1 and S1PR1 and the APACHE Ⅱ score, lung injury score and PaO2 / FiO2 in ARDS patients. ROC curve was used to analyze the predictive value of Eotaxin-1 and S1PR1 for poor prognosis of ARDS patients. Logistic regression was used to analyze the risk factors for the poor prognosis of ARDS patients. Results The level of serum Eotaxin-1 in the ARDS group was increased and that of S1PR1 was decreased when compared with those in the control group (P < 0. 05). The level of serum Eotaxin-1 level in the moderate and severe ARDS groups was significantly increased (P< 0. 05), and the level of serum S1PR1 level was significantly decreased compared with those in the mild group. The level of serum Eotaxin-1 was significantly increased and that of S1PR1 was significantly decreased in the severe ARDS group when compared with those in the moderate group (P< 0. 05). The level of serum Eotaxin-1 in the death group was significantly higher than that in the survival group, and the level of serum S1PR1 was significantly lower than that in the survival group (P< 0. 05). The level of serum Eotaxin-1 was positively correlated with the APACHE Ⅱ score and the lung injury score, and was negatively correlated with the value of PaO2 / FiO2 (P< 0. 05). The level of S1PR1 was negatively correlated with the APACHE Ⅱ score and the lung injury score, and was positively correlated with the value of PaO2 / FiO2 (P< 0. 05). ROC analysis showed that the area under the curve (AUC) of Eotaxin-1 for the prediction of poor prognosis in ARDS patients was 0. 867, the cut-off value was 104. 348 pg / mL, the sensitivity was 87. 9 % , and the specificity was 82. 0 % . The prediction of S1PR1 for poor prognosis in ARDS patients was 0. 849 in AUC, the truncation value was 71. 099 pg / mL, with an 89. 3 % sensitivity and an 80. 0 % specificity. Logistic regression analysis showed that high Eotaxin-1 level and low S1PR1 level were risk factors for poor prognosis in ARDS patients (P < 0. 05). Conclusion The increase of serum Eotaxin-1 level and the decrease of S1PR1 level in ARDS patients are related to the severity of the disease and the poor prognosis, which may be used as auxiliary indicators for the assessment of the disease and prognosis in ARDS patients.

Objective To investigate the effect of miRNA-34a-5p / fucosyltransferase ( Fut8) axis on the mobility of macrophage-derived foam cells. Methods Firstly, bioinformatics and RT-PCR were used to find and detect the upstream regulatory miRNA of Fut8 gene. Secondly, the correlation between levels of Fut8 and miRNA in serum of the patients with plaque was observed by Pearson correlation analysis. Finally, the in vitro model of foam cell was used to observe the effect of miRNA on cell mobility and the important role Fut8 played in. Results Both miRNA-34a-5p and miRNA-449b-5p were significantly increased in serum of patients with plaque, but miRNA-34a-5p had a stronger correlation with Fut8. The expression level of miRNA-34a-5p was significantly increased during foam cell formation, and inhibition of miRNA-34a-5p increased the expression level of Fut8. Overexpression of miRNA-34a-5p led to a decrease in the number of cells penetrated into the lower compartment of the transwell chamber. The number of cells penetrated into the lower compartment was increased after the overexpression of Fut8 in cells. Conclusion As one of the upstream miRNAs of Fut8, miRNA-34a-5p inhibits the mobility of macrophage-derived foam cells by down-regulation of Fut8.

Objective Death of retinal ganglion cells(RGCs) during acute glaucoma leads to progressive degeneration of retinal nerves and irreversible blindness. Puerarin has been shown to have antioxidant and anti-neuroinflammatory properties that protect the retina from acute glaucoma. However,the mechanism behind this process remains unclear. Methods In this study,rat model of acute glaucoma was established,and rats were randomly divided into five groups:normal control group, SnPP group, I/ R group, puerarin ( 100 μmol / L ) group and puerarin + SnPP group. Hematoxylin and eosin staining were used to evaluate the retinal conditions in the I/ R group, puerarin group and puerarin + SnPP group. Levels of TNF-α and IL-1β were detected by ELISA and qRT-PCR. Expression levels of cleaved caspase-8,Nrf2,and HO-1 were detected by Western blotting. The expression levels of Brn-3a, β3-tubulin and RBPMS were detected by immunofluorescence. Cell apoptosis and intracellular reactive oxygen species were detected by flow cytometry. Results Puerarin treatment significantly reduced retinal damage and RGC death in acute glaucoma rats,and significantly reduced the production of reactive oxygen species and inflammatory cytokines in the retina. In addition,the reduction of ROS production by puerarin had re-balanced the intracellular redox status,thus contributing to the survival of RGCs in vitro. Puerarin treatment also reduced the production of inflammatory cytokines in vitro. Puerarin inhibited the cell apoptosis and the production of inflammatory cytokine in RGCs through regulation of Nrf2 / HO-1 pathway. Moreover, the neuroprotective effect of puerarin on acute glaucoma was eliminated when SnPP,an HO-1 inhibitor,was administered. Conclusion Our findings identify the underlying mechanism of RGC apoptosis and the neuroprotective effect of a novel therapeutic agent puerarin on acute glaucoma.

Objective To investigate the effect of LncRNA MALAT1 on lipopolysaccharide (LPS) -induced survival and expression of inflammatory factors in kupffer cells ( KCs) and its possible mechanism. Methods LPS-induced KCs were used to establish a cell injury model and were randomly divided into 8 groups: Con group, LPS group, LPS + si-NC group, LPS + si-MALAT1 group, LPS + miR-NC group, LPS + miR-181d-5p group, LPS + si-MALAT1 + anti-miR-NC group, LPS + si-MALAT1 + anti-miR-181d-5p group. The expression levels of LncRNA MALAT1 and miR-181d-5p were detected by qRT-PCR. Cell viability was detected by CCK-8 method. The levels of IL-6, IL-12 and TNF-α were detected by ELISA. The targeting relationship between LncRNA MALAT1 and miR-181d-5p was detected by dual luciferase gene reporter assay. Results The expression of LncRNA MALAT1 in the LPS group was increased (P< 0. 05), the levels of IL-6, IL12 and TNF-α were increased (P < 0. 05), while the expression of miR-181d-5p was decreased (P< 0. 05), and the cell viability was decreased (P < 0. 05), when compared with those in the Con group. The cell survival rate in the LPS + si-MALAT1 group was increased (P < 0. 05), while the levels of IL-6, IL-12 and TNF-α were decreased (P< 0. 05), when compared with those in the LPS + si-NC group. The LncRNA MALAT1 negatively regulated the expression of miR-181d-5p. The cell survival rate in the LPS + miR-181d-5p group was increased (P < 0. 05), while the levels of IL-6, IL-12 and TNF-α were decreased (P< 0. 05), when compared with those in the LPS + miR-NC group. The survival rate in the LPS + si-MALAT1 + anti-miR-181d-5p group was decreased (P< 0. 05), while the levels of IL-6, IL-12, TNF-α were increased (P< 0. 05), when compared with those in the LPS + si-MALAT1 + anti-miR-NC group. Conclusion Interfering with the expression of LncRNA MALAT1 could promote the survival of LPS-induced KCs and inhibit the expression of inflammatory factors by promoting the expression of miR-181d-5p.

Objective To investigate the effect of exosomal miR-34c-5p derived from cancerassociated fibroblasts (CAFs) on the stem-like properties of gastric cancer (GC) cells. Methods Exosomes from primary cultured CAFs and paired normal fibroblasts (NFs) were collected and identified, and the differential expression of exosomal miR-34c-5p between CAFs and NFs was detected. The viability of GC cells was detected by CCK8 assay. The proliferation of GC cells was detected by colony formation assay. The invasive ability of GC cells was detected by Transwell assay. The sphere formation capability of GC cells was evaluated by sphere formation assay. The expression levels of stemness-related proteins were detected by Western blotting (WB) assay. Results The expression level of miR-34c-5p in CAFs derived exosomes was significantly decreased. miR-34c-5p from the CAFs-exo inhibited the proliferation, invasion and sphere formation of GC cells, and reduced the expression levels of SOX2, OCT4 and NANOG proteins, when compared with those in the CAFs-exo group (P< 0. 05). Conclusion The exosomal miR-34c-5p from CAFs inhibited the stem-like properties of GC cells.

Objective To analyze the mechanism of blinin in improving acute lung injury (ALI). Methods A total of 60 neonatal rats were randomly divided into 6 groups, the sham operation group, the model group, the blinin low-, medium- and high-dose groups, and the dexamethasone group. The pulmonary edema was assessed by the blood partial pressure of oxygen (PaO2 ) and the lung wet / dry weight ratio (W/ D). The pathological damage of lung tissues was detected by HE staining. The levels of oxidative stress and inflammation factors in the bronchoalveolar lavage fluid (BALF) were detected by ELISA. The expression levels of the high mobility group box 1 protein (HMGB1) / nuclear factor kappa-B (NF-κB) related proteins in lung tissues were detected by Western blotting. Results The PaO2 was significantly increased in the blinin high-dose group when compared with that in the model group, while the W/ D and lung injury scores, the levels of inflammation and oxidative stress fators in BALF, and the expression levels of HMGB1, TLR4 and p-NF-κB p65 / NF-κB p65 in lung tissues were significantly decreased (P < 0. 05). Conclusion Blinin can improve ALI by enhancing anti-oxidation and anti-inflammatory activity and blocking the HMGB1 / NF-κB signaling pathways.

Objective To explore the effect of LncRNA BCYRN1 on the proliferation and motility of MCF-7 breast cancer cells and the growth of xenograft in mice by using short hairpin RNA (shRNA) interfering. Methods The expression level of BCYRN1 in breast cancer and para-cancerous tissues was detected by real-time quantitative PCR (RT-PCR). MCF-7 cells were divided into 3 groups, the blank control group (Control), the negative control group (shRNA-NC) and the interference group (sh-BCYRN1). The target fragments were transfected, their effect on expressions of the target genes were then verified by RT-PCR. The cell proliferation, invasion, migration ability and the expression levels of related proteins were detected by Edu, transwell assay, wound-healing assay and Western blotting, respectively. Vimentin level was detected by immunofluorescence method. The nude mice were given subcutaneous injection of MCF-7 cells to construct xenograft models. After tumor formation, nude mice were divided into two groups, the blank control group (negative control ) and the interference group ( sh-BCYRN1 ), and there were 20 cases in each group. The tumor weight and the expression levels of BCYRN1, Ki67 and N-cadherin in the transplanted tumors were detected 4 weeks later. Results The expression level of BCYRN1 was higher in breast cancer tissues than in para-cancerous tissues ( P < 0. 05). The percentage of Edu positive cells, the number of migrated and invasion cells, the positive rate of Vimentin, and the expression levels of Ki67 and N-cadherin were decreased in the interference group when compared with the blank control group ( P < 0. 05), while the expression level of E-cadherin was increased ( P < 0. 05). In the interference group, the expression level of BCYRN1 was decreased, and the tumor mass, the expression levels of Ki67 and N-cadherin were lower than those in the blank control group (P< 0. 05). Conclusion Interfering of LncRNA BCYRN1 can inhibit the proliferation and motility of MCF-7 breast cancer cells and the growth of xenograft.

Objective To investigate the effect of β-catenin H36P mutation on the proliferation of hepatocellular carcinoma cells. Methods The CTNNB1genes (WT-CTNNB1, G34R-CTNNB1, H36P-CTNNB1) were amplified by PCR amplification. It was cloned into the p-CMV5 plasmid vector with a double digestion method, and the corresponding plasmids were transiently transferred into the Huh7 and HepG2 cells. The protein expression levels were then detected by Western blotting. Cells transfected with the target genes were treated with CHX in a time gradient, the degradation time of the H36P-β-catenin, WT-β-catenin and G34R-β-catenin were compared and their stabilities were analyzed. The nuclear translocation ability of H36P-β-catenin was examined after separation of the nuclear and cytoplasmic fractions. Finally, the effect of H36P-β-catenin on the proliferation of hepatocellular carcinoma cells was examined by CCK-8 method. Results The protein expression levels of β-catenin mutants (G34R and H36P) were not significantly different from that of the wild-type β-catenin. In HepG2 cell line, WT-β-catenin was almost completely degraded by CHX in 8 hours, while the mutants only degraded 75 % and 60 % in the same time period. Similarly, in the Huh7 cell line, WT-β-catenin was almost completely degraded in 6 hours, while the mutants were only degraded about 40 % in the same time period. The differences in the above results were statistically significant (P< 0. 05). Compared with the WT-β-catenin, mutant β-catenin promoted the proliferation of HepG2 cell. The nuclear translocation ability of H36P mutant β-catenin was increased compared with those of WT and G34R mutant β-catenin in the hepatocellular carcinoma cells. Conclusion β-catenin H36P mutant protein blocks the protein ubiquitination degradation process, promotes the proliferation of HepG2 and Huh7 hepatocellular carcinoma cells, and has an increased nuclear translocation.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of head and neck, whose development is related to the abnormal gene expression. Aberrant DNA methylation is a mechanism that affects gene expression and has been proven to play an important role in OSCC initiation and development. This review mainly discusses the research progress and detection techniques of DNA methylation in OSCC, including sample types, methylation levels of genes related to OSCC, their biological functions and clinical interpretations in tumor progression. The future research will continue to explore the relationship between abnormal DNA methylation and OSCC initiation, development, treatment and prognosis, in order to find more effective treatment strategies and prognosis assessment methods

ransfer RNA-derived small RNA ( tsRNA) is a novel non-coding RNA stably expressed in human tissues. The tRNA halves and the tRNA-derived RNA fragment are known as the two tsRNA subtypes. The development and application of high-throughput sequencing technology have provided more and more evidence for tsRNA dysregulation in tumors. Abnormal expression of tsRNA in tumor tissues has been found to be related to the tumor proliferation, invasion and progression. Therefore, these newly discovered tsRNAs have great potential as novel biomarkers and targets for the treatment of cancer. This article reviews the latest research progress of tsRNA in tumors and discusses its potential clinical values.