Objective:To investigate the value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma.Methods:The mRNA sequencing data of 471 lung adenocarcinoma patients and corresponding methylation data of 413 cases were downloaded from The Cancer Genome Atlas (TCGA) database, the methylation levels of SHOX2 and RASSF1A gene promoter regions were calculated, and the difference in methy lation level between normal lung tissues and tumor tissues was analyzed. The clinical data of 54 patients with early-stage lung adenocarcinoma and 31 patients with benign lung tumors who underwent surgery at Drum Tower Hospital Affiliated to Nanjing University Medical School from January 2018 to January 2019 were retrospectively analyzed. The methylation status of SHOX2 and RASSF1A in tumor tissues and normal lung tissues (>5 cm from the edge of the tumor foci) (called clinical samples) was detect, and a positive methylation in the promoter region of either gene was considered as a combination of two genes methylation positivity. Using pathological diagnosis as the gold standard, the efficacy of gene methylation positivity in diagnosing early-stage lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve. Patients with >80% of tumor cells in paraffin samples were screened, and mRNA high-throughput sequencing was performed in their tumor tissues and normal lung tissues. The relationship between positive methylation of the two genes and clinicopathological features was analyzed, and the correlation between the promoter region methylation level of the two genes and mRNA expression levels in clinical samples and TCGA database samples was analyzed by Spearman method. Gene set variance analysis (GSVA) method was used to analyze the differences in Kyoto Encyclopedia of Genes and Genomes enrichment pathways between two-gene methylation-positive clinical lung adenocarcinoma samples and corresponding methylation-negative lung adenocarcinoma.Results:In TCGA database, the SHOX2 promoter region methylation island contained 6 sequenced methylation sites, of which sites cg04532033 and cg01557547 methylation levels were higher in lung adenocarcinoma tissues than in normal lung tissues (both P < 0.05); the RASSF1A gene promoter region methylation island contained 11 sequenced methylation sites, and the methylation levels of 6 of these sites in lung adenocarcinoma tissues were higher than those in normal lung tissues (all P < 0.05). Compared with normal lung tissues, the methylation level of SHOX2 promoter region was higher in stage Ⅰ and Ⅱ lung adenocarcinoma tissues (both P < 0.05); the methylation level of RASSF1A promoter region was higher in all stages of lung adenocarcinoma ( P < 0.001). Among 54 patients with early-stage lung adenocarcinoma, 28 were positive for SHOX2 promoter region methylation in tumor tissues, 21 were positive for RASSF1A promoter region methylation, and 40 were positive for combined methylation of both genes; 31 benign lung nodules were negative for SHOX2 and RASSF1A methylation. ROC curve analysis showed that the sensitivity of positive SHOX2 promoter region methylation for diagnosing early-stage lung adenocarcinoma was higher than that of RASSF1A promoter region methylation positivity (51.8% vs. 38.9%), and the area under the curve (AUC) for diagnosis by two-gene methylation positivity was larger than that for diagnosis by SHOX2 or RASSF1A gene methylation positivity alone (0.870 vs. 0.759 and 0.694). The circulating thresholds (Ct) of SHOX2 and RASSF1A methylation tested by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in stage Ⅰ and Ⅱ lung adenocarcinoma were lower than those in normal lung tissues (all P < 0.05); patients with two-gene methylation positivity were characterized by older age, longer tumor longest diameter and more advanced pathological stage compared with patients with two-gene methylation negativity (all P < 0.05). In clinical stage Ⅰ-Ⅱ lung adenocarcinoma samples, the Ct of SHOX2 and RASSF1A promoter region methylation tested by qRT-PCR was negatively correlated with their mRNA relative expression levels ( r=-0.43, P = 0.003; r = -0.48, P = 0.001); in TCGA database stage Ⅰ-Ⅱ lung adenocarcinoma samples, the level of SHOX2 promoter region methylation was negatively correlated with its mRNA relative expression level ( r = -0.23, P < 0.001), and the level of RASSF1A promoter region methylation was also negatively correlated with its mRNA relative expression level, but without statistical difference ( r = -0.05, P = 0.310). In two-gene promoter methylation-positive lung adenocarcinoma samples, the pathways related to folate metabolism and DNA stability were upregulated, and the pathways related to vasoconstriction and cell growth and differentiation were downregulated. Conclusions:The combined detection of SHOX2 and RASSF1A promoter region methylation can be used as an indicator for screening and diagnosis of early-stage lung adenocarcinoma. Abnormal promoter region methylation of the two genes may affect multiple tumor-related pathways and promote the occurrence and progression of early-stage lung adenocarcinoma.

Objective:To analyze the difference in immune microenvironment between primary tumor tissues and metastatic tumor tissues of metastatic colorectal cancer, and to screen specific immune-related differentially expressed genes (DEG) related to prognosis of metastatic colorectal cancer via bioinformatics methods.Methods:The GSE131418 microarray dataset of colorectal cancer and metastases was downloaded from gene expression omnibus (GEO) database, including 517 samples from the MCC cohort and 618 samples from the Consortium cohort in Moffitt Cancer Center. Immune-related gene sets were downloaded from immunology database and analysis portal IMMPORT, including 2 483 immune-related genes. A total of 695 cases of RNA sequencing data and 627 cases of clinical information of colorectal cancer tumors and adjacent tissues were downloaded from Cancer Genome Atlas (TCGA) data. The stroma cell score, immune cell score and stromal immune total score of metastatic tumor tissues and primary tumor tissues were calculated by using ESTIMATE algorithm, and 22 kinds of immune cell infiltration in primary tumor and metastatic tumor tissues of colorectal cancer were compared and analyzed by using CIBERSORT deconvolution algorithm. Immune-related DEG were screened to make Kyoto Encyclopedia of Genes and Gnomes (KEGG) signaling pathway enrichment analysis. The patients were divided into high and low expression groups according to the median expression levels of immune-related DEG. The Kaplan-Meier method and Cox regression risk model were used to analyze immune-related DEG, and the genes significantly related to prognosis in the results of the two methods were screened (all P < 0.01), and multivariate analysis was performed by using Cox regression method. The expression differences of each gene in tumor tissues, adjacent tissues, primary tumor tissues and metastatic tissues in GSE131418 data sets of TCGA database and GEO database were compared, and survival analysis was also performed. Results:The stroma cell score, immune cell score and stromal immune total score of colorectal cancer metastatic tissues were lower than those of primary tumor tissues (all P < 0.001). Compared with primary tumor tissues, the proportion of activated natural killer (NK) cells, monocytes, CD8 + T cells, T cells, activated dendritic cells in metastatic colorectal cancer tissues was increased, while the proportion of inactive mast cells, inactive dendritic cells, inactive NK cells, activated memory CD4 + T cells, M1 macrophages, and neutrophils was decreased. There were 289 immune-related DEG in metastatic tissues and primary tumor tissues of metastatic colorectal cancer, including 101 up-regulated genes and 188 down-regulated genes. KEGG signaling pathway enrichment analysis showed that in the immune microenvironment of metastatic tissues in metastatic colorectal cancer, vascular endothelial growth factor (VEGF) signaling pathway, programmed death ligand 1 (PD-L1) expression and programmed death 1 (PD-1) checkpoint pathway, T helper cell (Th) 1, Th2 and Th17 cell differentiation, NF-kappa B signaling pathway, interleukin 17 (IL-17) signaling pathway, chemokine signaling pathway, T cell receptor signaling pathway, MAPK signaling pathway, and NK cell-mediated cytotoxicity pathways enrichment were detected. Immune-related DEG related to prognosis including ANGPTL5, FPR1, HSPA8, NR2E3, PSMD2, PSMD8 and SBDS were screened out. Cox regression multivariate analysis showed that immune-related DEG ANGPTL5 ( HR = 2.69, 95% CI 1.22-5.92, P < 0.05), HSPA8 ( HR = 0.57, 95% CI 0.33-0.97, P < 0.05), and SBDS ( HR = 2.23, 95% CI 1.18-4.21, P < 0.05) were independent prognostic factors for metastatic colorectal cancer. The expression of ANGPTL5 in tumor tissues was lower than that in normal tissues, and the expression of ANGPTL5 in metastatic tissues was higher than that in primary tumor tissues. Patients with high expression of ANGPTL5 in tumor tissues had worse prognosis. The expression of HSPA8 in tumor tissues was higher than that in normal tissues, and the expression of HSPA8 in metastatic tissues was lower than that in primary tumor tissues. Patients with high expression of HSPA8 in tumor tissues had a better prognosis. The expression of SBDS in tumor tissues was lower than that in normal tissues, and the expression of SBDS in metastatic tissues was lower than that in primary tumor tissues. Patients with high expression of SBDS in tumor tissues had worse prognosis. Conclusions:Immune microenvironment of metastatic colorectal cancer is quite different from that of primary tumor. The degree of immune cell infiltration is reduced and the whole is immunosuppressed. The specific immune-related DEG related to prognosis of metastatic colorectal cancer may be new therapeutic targets of metastatic colorectal cancer.

Objectvie:To investigate the infiltration patterns of immune cells in colorectal cancer, and to explore the correlation of immune cells infiltration with clinical characteristics and overall survival (OS) of patients.Methods:The RNA sequencing data of 615 patients with colorectal cancer were downloaded from The Cancer Genome Atlas (TCGA) database. The data was updated on July 19, 2019, and included 571 colorectal cancer tissues and 44 paracancerous tissues. There were 552 cases with clinical data, such as survival time, survival status, age, gender, clinical stage, grade, tumor location and so on. Using CIBERSORT deconvolution algorithm, the relative amounts of 22 immune cell types were calculated based on standardized gene expression data. According to the results of CIBERSORT algorithm, the samples with high accuracy of deconvolution result were selected ( P < 0.05), and they were used for analysis and graphing. The correlations between the infiltration patterns of immune cells and the clinical characteristics and OS of patients were analyzed. Results:After the CIBERSORT method was used to filter and remove samples with P &ge; 0.05, a total of 282 tumor tissue samples and 16 paracancerous tissue samples were screened. In 293 cases with clinical information, there were 277 tumor tissue samples and 16 paracancerous tissue samples. In 293 samples, M0 macrophages, M1 macrophages, M2 macrophages, CD8 + T cells and unactivated CD4 memory T cells accounted for a higher proportion of total immune cells; in tumor tissue samples, the expressions of M0 macrophages, M1 macrophages, activated CD4 memory T cells, and unactivated natural killer (NK) cells were higher; in paracancerous tissues, the expressions of naive B cells, M2 macrophages, activated NK cells, unactivated dendritic cells, unactivated mast cells and plasma cells were higher; with the increase of clinical stage, the expressions of follicular helper T cells, activated CD4 memory T cells, activated NK cells, M1 macrophages decreased, and the expressions of plasma cells and regulatory T cells increased, and the differences were statistically significant (all P < 0.05). M1 macrophages, unactivated mast cells, activated CD4 memory T cells, CD8 + T cells, and follicular helper T cells were highly expressed in right colon cancer, while M0 macrophages and activated mast cells were highly expressed in left colon and rectal cancer, and the differences were statistically significant (all P < 0.05). The patients were divided into high infiltration group and low infiltration group based on the median expression level of infiltrated immune cells, and the survival analysis was performed. The result of survival analysis showed that patients with high initial B cell infiltration had good OS; however, patients with high infiltration of M2 macrophages, activated mast cells, and neutrophils had poor OS. Conclusions:There are different types of immune cell infiltration patterns in the colorectal cancer samples of different stages and locations, which are closely related to tumor progression and OS of patients. They are expected to be applied to the development of therapeutic targets and prognosis prediction.

Objective:To explore the related factors of postoperative adjuvant therapy for cervical cancer stagedⅠB1-ⅡA2 [according to 2018 International Federation of Gynecology and Obstetrics (FIGO) staging standard], and to establish a nomogram model to predict the risk of postoperative adjuvant therapy for locally advanced cervical cancer.Methods:A total of 714 patients with cervical squamous cell cancer staged FIGO ⅠB1-ⅡA2 treated by surgery in Anhui Provincial Hospital were selected as the research objects from January 2009 to December 2019, and their clinicopathological data were analyzed. Multiple logistic regression analysis was used to determine the influencing factors, and a nomogram model was established to predict the risk of postoperative adjuvant treatment of cervical cancer. The predictive performance of the model was evaluated with the consistency index (C-index), and the compliance of the model was evaluated with the calibration curve.Results:Univariate analysis suggested that postoperative adjuvant therapy for cervical cancer was associated with gravidity ( χ2=11.506, P=0.001), underlying disease (hypertension or diabetes) ( χ2=7.668, P=0.006), squamous cell cancer antigen (SCC-AG) level ( χ2=19.392, P<0.001), imaging risk factors ( χ2=16.392, P<0.001), FIGO stage ( χ2=25.686, P<0.001), tumor size ( χ2=9.392, P=0.025) and surgical path ( χ2=16.590, P<0.001). Multivariate logistic regression analysis suggested that the number of pregnancy >2 times ( OR=1.951, 95% CI: 1.355-2.808, P<0.001), SCC-Ag &ge;1.5 μg/L ( OR=2.021, 95% CI: 1.444-2.829, P<0.001), FIGO stage ⅠB3-ⅡA2 [ⅠB3 ( OR=1.933, 95% CI: 1.139-3.282, P=0.015); ⅡA1 ( OR=2.723, 95% CI: 1.556-4.765, P<0.001); ⅡA2 ( OR=3.159, 95% CI: 1.502-6.646, P=0.002)], with underlying disease (hypertension or diabetes) ( OR=1.867, 95% CI: 1.051-3.318, P=0.033), imaging risk factors ( OR=1.997, 95% CI: 1.127-3.537, P=0.018), without neoadjuvant therapy [preoperative neoadjuvant therapy for 1 cycle ( OR=0.402, 95% CI: 0.207-0.783, P=0.007)] and laparoscopic surgery ( OR=2.177, 95% CI: 1.524-3.112, P<0.001) were independent influencing factors for postoperative adjuvant treatment of cervical cancer. Based on the screened variables, the nomogram model to predict the risk of postoperative adjuvant treatment for cervical cancer has good predictive performance (C-index was 0.702) and compliance. Conclusion:The number of pregnancy >2 times, SCC-Ag &ge;1.5 μg/L, FIGO stage ⅠB3-ⅡA2, with underlying disease (hypertension or diabetes), imaging risk factors, without neoadjuvant therapy, and laparoscopic surgery are independent influencing factors for postoperative adjuvant treatment of cervical cancer. A nomogram model has been constructed to predict the risk of postoperative adjuvant therapy for locally advanced cerrical cancer, and it can provide evidence for clinical treatment selection.

Objective:To analyze the invasion characteristics and prognostic factors of patients with Masaoka-Koga stage Ⅲ thymoma.Methods:The tumor invasion characteristics of 179 patients who were diagnosed with Masaoka-Koga stage Ⅲ thymoma and treated in Affiliated Cancer Hospital of Zhengzhou University from January 2000 to June 2018 were analyzed retrospectively. According to the treatment methods, all patients were divided into the radical operation group ( n=94), palliative operation group ( n=39) and simple biopsy group ( n=46). The χ2 test was used to compare the classified variables, Kaplan- Meier method was utilized to calculate the cumulative survival rate, log-rank method was used for group comparison and univariate analysis, and Cox’s regression model was used for multivariate analysis. Results:Mediastinal pleural invasion (86.0%) was the most common site, followed by pericardium (50.8%), great vessel (40.8%) and lung (36.3%). The proportion of macrovascular invasion in the radical operation group was 14.9%, significantly lower than 79.5% and 60.9% in the palliative surgery group and biopsy group (both P<0.001). Multivariate analysis showed that the nature of operation ( P<0.001), age ( P=0.011), radiotherapy ( P=0.020) were the independent factors affecting overall survival (OS), while nature of operation ( P<0.001), age ( P=0.004), radiotherapy ( P=0.020), number of invasive organs ( P=0.023) and pathological type ( P=0.016) were the independent factors affecting progress-free survival (PFS). Conclusions:For patients with Masaoka-Koga stage Ⅲ thymoma, mediastinal pleura is the most common site of invasion, pericardium, lung and great vessels are also commonly invaded. The invasion of mediastinal pleura, pericardium and lung exerts slight effect on surgical resectability, whereas great vessel involvement can significantly affect surgical resectability. OS and PFS in patients undergoing radical resection are significantly better than those in patients treated with palliative resection and biopsy. Radical resection is the most important factor affecting prognosis.

Objective:To evaluate the long-term survival and identify prognostic factors of patients diagnosed with early-stage non-small cell lung cancer (ES-NSCLC) receiving stereotactic ablation radiotherapy (SABR).Methods:Clinical data of 109 ES-NSCLC patients treated with SABR in Henan Cancer Hospital from 2011 to 2018 were retrospectively analyzed. The overall survival (OS), cancer-specific survival (CSS) and progression-free survival (PFS) were calculated by Kaplan- Meier method and log-rank test. Multivariate prognostic analysis was performed by Cox regression model. Results:The median follow-up time was 44 months (2-93 months). The median OS, CSS and PFS were 78 months, 78 months and 44 months, respectively. The 1-year OS, CSS and PFS were 95.4%, 97.2% and 84.1%, and 75.6%, 79.1% and 56.6% for the 3-year OS, CSS and PFS, and 55.6%, 60.7% and 37.3% for the 5-year OS, CSS and PFS, respectively. Univariate analysis showed that ECOG score, age, smoking history and derived-neutrophil/lymphocyte ratio (dNLR) were the influencing factors of OS ( P=0.03, 0.02, 0.04, 0.001). Age, smoking history and dNLR were the influencing factors of CSS ( P=0.02, 0.03, 0.001). Multivariate analysis demonstrated that dNLR was an independent prognostic factor for OS and CSS ( P=0.001, 0.001). Conclusions:ES-NSCLC patients treated with SABR can achieve favorable survival. The dNLR is an independent prognostic factor of OS and CSS, which can be considered in clinical application.

Objective To evaluate the reliability and clinical practicality of colony PCR in rapid detection of pathogenic fungi causing tinea capitis.Methods Totally,17 children with tinea capitis were enrolled from the Department of Dermatology of Wuxi No.2 People's Hospital between January 2016 and March 2017.Colony PCR was performed to detect pathogenic fungi.The results of colony PCR were compared with those of routine PCR and morphological identification,so as to evaluate the reliability of colony PCR in the identification of pathogenic fungi causing tinea capitis.Results After clinical specimens (broken hairs and scales) from the 17 patients were subjected to fungal culture,the mycelia were collected and successfully amplified by colony PCR.The time of clony PCR (mean,3.82 ± 0.50 days) for DNA template preparation was short than that of traditional morphological identification (14 days).Based on the results of conventional PCR,the accuracy of colony PCR for fungal identification was 100％,which was superior to that of conventional PCR (88.2％).Conclusions Colony PCR can be applied to the clinical detection of pathogenic fungi causing tinea capitis at specy level,and is a kind of rapid,economic and reliable molecular detection technique.

Objective To assess the association of three polymorphisms in Megsin (rs1055901,rs1055902 and rs2689399) and susceptibility of IgA nephropathy in Asian population.Methods We conducted a comprehensive search of electronic CNKI,VIP,WangFang Data,CBM,Pubmed,Web of Science and Google Scholar database on the association between Megsin rs1055901,rs1055902 and rs2689399 polymorphism and susceptibility of IgA nephrology in Asian population (last search update on 2 May 2016).Stata 12.0 software was used to calculate the odds ratio (OR) and 95 ％ CI (confidence interval),as well as sensitivity and publication bias analyses.Results Six publications encompassing mine case-control studies were finally included,including 2 179 cases and 1 769 controls.Finally,no significant association between Megsin rs1055901 and rs1055902 polymorphism and IgA nephrology in Asian population was identified,while a significantly decreased risk of IgA nephrology for rs2689399 polymorphism,was identified in Asian population (G and C:OR=0.754,95％CI 0.592-0.961,P=0.022;GG and CC:OR=0.506,95％CI 0.287-0.892,P=0.019;GG and GC+CC:OR=0.551,95％CI 0.316-0.961,P=0.036).Conclusion Rs2689399 G allele and GG genotype of Megsin may be the protective factors for IgA nephropathy in Asian population.

Objective:To explore the effects of superparamagnetic iron oxide-short hairpin RNA ( SPIO-ShRNA) dual functional molecular probes of different concentrations on morphology and biological beha -vior of ovarian cancer SKOV3 cells in vitro.Methods:The dual functional molecular probes at an iron concentration of 5, 15, 30, 45, 75, and 100 mg/L were transfected into SKOV3 cells.The transfection rate of the probe was observed by fluorescence microscope .The distribution and content of iron particles in SKOV3 cells were determined by Prussian blue staining , atomic adsorption spectrometer and electron microscopy .Cell viability was observed by cell counting kit-8 ( CCK-8 ) .The apoptosis was detected by flow cytometry .The expression of protein within the cells was detected by Western blot .The changes of the signal intensity were measured by magnetic resonance imaging (MRI).Results: The SPIO-ShRNA dual functional molecular probe was uptaken in aconcentration-dependence manner within a certain range (5-30 mg/L) .When the concentration of the probe was 45 mg/L, the labeling rate of the cell was close to 100%;With the increase of the concentration of probe , the cell survival rate decreased gradual-ly.The cell survival rate of each experimental group were 94.626%±1.050%, 93.373%±1.180%, 91.700%±3.122%, 75.100%±4.362%, 72.983%±3.233%, 71.010%±2.910%,5, 15, 30mg/L cell survival rate was not significantly decreased , the difference was not statistically significant (P=0.226, P=0.068, P=0.475);When the concentration of the probe was greater than or equal to 45 mg/L,the survival rate decreased obviously ( P<0.001);Group of 45 mg/L protein expression rate was 68.905%± 3.510%, When the concentration of the probe was greater than or equal to 45 mg/L, the inhibition rate of the protein expression level of epidermal growth factor receptor was obviously higher than those of 5, 15, and 30 mg/L groups, the difference was statistically significant (P<0.001, P=0.001, P=0.003, all P<0.01);the MRI displayed that the signal intensity was decreased with increasing concentrations of the probe.The signal intensity of 45 mg/L group was 165.55 ±4.92, compared with the blank control group (same volume of phosphate buffer saline ), normal group(unlabeled ovarian cancer SKOV3 cells), 5, 15, and 30 mg/L groups , the signal intensity of 45 mg/L group decreased significantly (all P<0.001).Con-clusion:The dual functional molecular probe can effectively transfect and specifically inhibit the expression of SKOV3 cell lines at the iron concentration of 45 mg/L, and can also be detected by MRI .The role of diagnosis and treatment of the dual functional molecular probe has been initially confirmed .

In this study, we investigated the pharmacokinetics parameters of SPIO-shRNA dual functional molecular probe and observed the main organ distribution by MRI in vivo. Eighteen New Zealand white rabbits were randomly divided into three groups and injected intravenously with different doses of SPIO-shRNA molecular probe, respectively. The blood samples were collected to analyze the pharmacokinetic parameters by measuring the iron content at 30 minutes before and after the injection. Twenty-four Kun Ming (KM) mice were randomly divided into 4 groups: the control group was injected intravenously with physiological saline 200 µL per mouse via the tail vein, the other 3 groups were injected intravenously with different doses of SPIO-shRNA molecular probe. MRI observation was performed in 24 hours, and the liver, spleen, kidney, brain and muscle were collected for iron quantification with Prussian blue staining to determine distribution of the SPIO-shRNA molecular probe in the main organ in vivo. Our results suggest that the molecular probe blood half-life is more than 3 hours. The data of MRI suggest the probe was distributed in liver and spleen, and the MRI signal was reduced with the increase in probe's doses (P < 0.05). The results of Prussian blue staining confirmed the results of MRI. Most of the probe could escape the phagocytosis of mononuclear phagocyte system. Our data provide the pharmacokinetic and distribution of SPIO-shRNA molecular probe in organs. Meanwhile, it suggests the choice of the time and dose of probe for MR imaging of tumor in vivo.