[Cluster classification and clinical prognostic modeling based on m6A RNA methylation regulators in liver cancer].

Objective: Cluster classification based on m6A methylation regulators and construct prognostic evaluation model. Methods: Utilizing consensus cluster to classify the liver cancer samples form TCGA based on the expression of 13 m6A methylation regulators, and verify the function and prognostic significance of the clustered subtypes. Marker genes were further screened to construct a risk prediction model for evaluating the prognosis of liver cancer patients. Results: The two clustered subtypes based on m6A methylation regulators showed significant differences in the prognosis value of liver cancer patients (P=0.048), and 38 prognostic markers related to m6A methylation in liver cancer were screened from the subgroup with poor prognosis. Two m6A regulatory genes, YTHDF1 and YTHDF2, are proved with adverse prognosis by univariate cox analysis (P<0.05, Hazard ratio>1). We used Lasso regression method to build risk assessment model and effectively predicted the prognosis status of liver cancer patients within 4 years (4-year AUC=0.685, 3-year AUC=0.669). Moreover, the assessment model was validated in another dataset of Asia liver cancer patients. Conclusion: The study provided ideas for studying m6A methylation in liver cancer, and the risk prediction model can be used to evaluate the short-term prognosis of liver cancer patients.

Intestinal epithelium-derived BATF3 promotes colitis-associated colon cancer through facilitating CXCL5-mediated neutrophils recruitment.

Inflammation is a critical player in the development and progression of colon cancer. Basic leucine zipper transcription factor ATF-like 3 (BATF3) plays an important role in infection and tumor immunity through regulating the development of conventional type 1 dendritic cells (cDC1s). However, the function of BATF3 in colitis and colitis-associated colon cancer (CAC) remains unclear. Here, BATF3 wild-type and knockout mice were used to construct an AOM/DSS-induced CAC model. In addition, DSS-induced chronic colitis, bone marrow cross-transfusion (BMT), neutrophil knockout, and other animal models were used for in-depth research. We found that BATF3 deficiency in intestinal epithelial cells rather than in cDC1s inhibited CAC, which was depended on inflammatory stimulation. Mechanistically, BATF3 directly promoted transcription of CXCL5 by forming a heterodimer with JunD, and accelerated the recruitment of neutrophils through the CXCL5-CXCR2 axis, ultimately increasing the occurrence and development of CAC. Tissue microarray and TCGA data also indicated that high expression of BATF3 was positively correlated with poor prognosis of colorectal cancer and other inflammation-related tumors. In summary, our results demonstrate that intestinal epithelial-derived BATF3 relies on inflammatory stimulation to promote CAC, and BATF3 is expected to be a novel diagnostic indicator for colitis and CAC.

Efficacy of computed tomography-guided implantation of 125I seeds in the treatment of refractory malignant tumors accompanied with cancer pain and its influence on tumor markers in the serum.

OBJECTIVE: This study intended to explore the efficacy of computed tomography (CT)-guided implantation of iodine-125 (125I) seeds in the treatment of refractory malignant tumors with cancer pain and its influence on tumor markers in the serum. PATIENTS AND METHODS: 76 patients with refractory malignant tumors accompanied by cancer pain that received treatments in LongHua Hospital Shanghai University of Traditional Chinese Medicine from September 2013 to August 2014 were selected. They were divided into control group and observation group using a random number table (38 patients in each group). Patients in the control group received simple chemotherapy, while those in the observation group undergone CT-guided implantation of 125I seeds in combination with chemotherapy. Recent efficacy and 1-3-year survival rate were compared between the two groups of patients. The degree of pain relief after treatment was also compared between the two groups of patients. Electrochemiluminescence method was used to detect the concentrations of carcinoembryonic antigen (CEA), sugar chain antigen 199 (CA 199), sugar chain antigen 125 (CA 125), neuron-specific enolase (NSE) and cytokeratin-19-fragment (CYFRA21-1) in the two groups of patients before treatment, and 3 days, 7 days and 30 days after treatment. RESULTS: Recent disease control rate of the patients in the observation group was higher than that of the patients in the control group (p<0.05). The 1-3-year survival rate after surgery in the observation group was significantly higher than that in the control group (p<0.05). The total efficiency of pain control in the observation group was significantly higher than that in the control group (p<0.05). The levels of tumor markers in the two groups of patients were significantly decreased after treatment, while the reduction in the observation group was more evident than that in the control group (p<0.05). CONCLUSIONS: Our results showed that CT-guided implantation of 125I seeds is effective for the treatment of patients with refractory malignant tumors accompanied by cancer pain. It can reduce the levels of tumor markers, improve the survival rate and prolong the survival time of the patients.

Clinical significance and expression of PUMA, MCL-1, and p53 in human renal cell carcinoma and para-carcinoma tissues.

We investigated the expression level of p53 upregulated modulator of apoptosis (PUMA), myeloid cell leukemia-I (MCL-1), and p53 in renal cell carcinoma (RCC) and para-carcinoma tissues, as well as their clinical significance. The expression levels of PUMA, MCL-1, and p53 in RCC and para-carcinoma tissues were measured using immunohistochemical and quantitative real-time PCR methods. Correlations between protein expression and pathological characteristics were analyzed. Renal clear cell carcinoma showed elevated MCL-1 and p53 protein expression (P > 0.05) and reduced PUMA expression as compared to that in para-carcinoma tissues. Spearman ranking correlation analysis showed that expression of PUMA, MCL-1, and p53 in was negatively correlated with RCC (r = -0.504, P = 0.001; r = -0.413, P = 0.008). We also observed significant correlation between MCL-1 expression and tumor differentiation (P < 0.05), where MCL-1 expression was significantly higher in well-differentiated adenocarcinoma as compared to that in medium or lowly differentiated adenocarcinoma. In addition, p53 expression was highly correlated with TNM staging (P < 0.05). Single factor analysis on COX's proportional hazard model indicated that postoperative survival rate and prognosis of renal clear cell carcinoma was highly correlated with TNM staging (P < 0.05). Quantitative real-time PCR analysis indicated higher expression of PUMA, MCL-1, and p53 in cancer tissues as compared to that in para-carcinoma tissues (P < 0.05).The expression of PUMA, MCL-1, and p53 can reflect the biological behavior of renal cell carcinoma, and can be used to indicate tumor invasion, progression, and prognosis.

[Comparison of the anterior corneal asphericity after small incision lenticule extraction and femtosecond laser in situ keratomileusis].

OBJECTIVE: To evaluate and compare the anterior corneal asphericity after small incision lenticule extraction(SMILE)and femtosecond laser in situ keratomileusis(FS-LASIK). METHODS: In this case-control study, 45 subjects who underwent SMILE operation comprised the study group, and 33 subjects with FS-LASIK operation comprised the control group. The asphericity coefficient Q-value of the right eyes in both groups was measured at diameters of 6, 7, 8 and 9 mm, respectively, before surgery and at 1 day, 1 week, 1 month and 6 months following surgery. The correlation between the variation of Q-value and the central cutting depth was analyzed. RESULTS: The Q-value of anterior corneal surface was 0.85 ± 0.31, 0.80±0.28, 0.69±0.25 and 0.51±0.23 after SMILE, and 1.13±0.56, 1.01±0.47, 0.80±0.39 and 0.51±0.31 after FS-LASIK at 1 week. In both groups, the Q-value was significantly different before and after surgery(P< 0.05); there were interaction effects between the operation method and time; the difference between the two groups at 6-mm and 7-mm diameters was statistically significant(P<0.05). The variation of the Q-value before and after operation(ΔQ)showed significant difference(P6mm=0.004, P7mm=0.014)between the two groups at 6-mm and 7-mm diameters. The cap diameter of the SMILE group was smaller than that of the FS-LASIK group, but the cutting depth was larger. There was no correlation between ΔQ and the cap/disc diameter. It showed a linear relationship(P<0.05)between ΔQ and the central cutting depth at all examined diameters in the two groups, and the relation degree in the FS-LASIK group was superior to the SMILE group. CONCLUSIONS: Both SMILE and FS-LASIK operations can change the negative Q-value of the anterior corneal surface to the positive. The impact of SMILE on the asphericity is smaller than that of FS-LASIK. (Chin J Ophthalmol, 2016, 52: 681-685).

[An analysis of changes in posterior corneal elevation and relevant factors after small incision lenticule extraction].

OBJECTIVE: To investigate changes in posterior corneal elevation after small incision lenticule extraction (SMILE) and related factors. METHODS: Retrospective case series study. Eighty-three eyes of 44 myopic patients undergoing SMILE were examined with the Pentacam preoperatively, and at 1 day, 1 month, 3 months, and 6 months postoperatively. Posterior corneal elevation at the corneal apex and 0°, 45°, 90°, 135°, 180°, 225°, 270° and 325° points of the 2 mm and 6 mm diameter (total, 17 points) was analyzed. RESULTS: The changes in posterior corneal elevation at 1 day, 1 month, 3 months, and 6 months were(-1.72±2.59), (-0.98±2.37), (-0.45±1.81) and (-0.25±2.20) µm, respectively, at the corneal apex. The results were statistically significant (t=6.07, 3.75, 6.07; P<0.05), except 6 months. The changes in posterior corneal elevation were (-1.42±2.06),(-0.69±1.86), (-0.30±1.50) and(-0.22±1.58) µm, respectively, in the 2 mm circle. The results of 1 day and 1 month were statistically significant (t=6.28, 6.28, P<0.05). The changes in posterior corneal elevation were (1.48±1.47),(0.98±1.32),(0.90±1.31) and (0.90±1.16) µm, respectively, in the 6 mm circle .The results were totally statistically significant (t=6.28, 6.28, P<0.05). There were statistically significant differences between 1 month and 1 day postoperatively in the corneal apex, 2 mm and 6 mm circles. The changes were (0.75±2.55),(0.73±1.97) and(-0.50±1.60) µm. There were statistically significant differences between 3 months and 1 month postoperatively in the corneal apex and 2 mm circle. The changes were (0.53±2.22) and (0.39±1.80) µm. No significant change was found in the 6 mm circle. Between postoperative 6 months and 3 months, there were no statistically significant differences. The spherical equivalent, intraocular pressure, ablation depth, residual bed thickness, corneal hysteresis, and corneal resistance factor had no obvious correlation with the changes in posterior corneal elevation. CONCLUSIONS: After SMILE, the surrounding cornea was slightly forward, while the central posterior cornea was slightly backwards, and returned gradually. The spherical equivalent, intraocular pressure, ablation depth, residual bed thickness, corneal hysteresis, and corneal resistance factor had no obvious correlation with the changes in posterior corneal elevation. (Chin J Ophthalmol, 2016, 52: 494-498).

Correlation between PPARg2 gene Pro12Ala polymorphism and cerebral infarction in an Inner Mongolian Han Chinese population.

The variant of PPAR-g2 has been shown to promote the increase of carotid IMT in patients suffering from cerebral infarction and the Pro12Ala polymorphism in the peroxisome proliferator-activated receptorg2 (PPARg2) gene may be associated with cerebral infarction. However, due to the different genetic background, race, and regional variations of cerebral infarction patient, the results of investigations into this subject differ. The aim of this study was to investigate this polymorphism in relation to cerebral infarction among the Inner Mongolian Han Chinese population. A total of 574 Han Chinese individuals from Inner Mongolian were selected randomly, including 302 patients with cerebral infarction and 272 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism was used to determine genotypes of the PPARg2 Pro12Ala variant and results were confirmed by direct sequencing. Genotype frequencies were found to be 90.7 and 91.9% for P/P, 8.6 and 7.7% for P/A, and 0.7 and 0.4 for A/A in the cerebral infarction and control groups, respectively. No statistically significant differences in genotype distribution were observed between the two groups (P > 0.05). Moreover, PPARg2 Pro12Ala genotype was not significantly associated with altered fasting blood glucose, blood pressure, or serum lipid profiles. After adjustment for gender, body mass index, and smoking habit, logistic regression was used to analyze the relationship between the Pro12Ala polymorphism and cerebral infarction (odds ratio = 0.888, 95% confidence interval = 0.106-7.460, P > 0.05), revealing that this variant was not the main pathogenic factor involved. Therefore, the Pro12Ala mutation of PPARg2 may not be associated with cerebral infarction in the Inner Mongolian Han Chinese population.

Identification of Genes Associated with Papillary Thyroid Carcinoma (PTC) for Diagnosis by Integrated Analysis.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma, and our understanding of its pathogenesis is incomplete. To elucidate the mechanisms underlying such progression and identify novel diagnostic markers, we aimed to discover the underlying gene associated with PTC. Integrated analysis of microarray datasets was performed to identify differentially expressed genes (DEGs) between PTCs and normal tissues. GO enrichment analysis and KEGG pathway enrichment analysis were then performed to uncover the functions of DEGs. Furthermore, the protein-protein interaction (PPI) network of DEGs was constructed. Five GEO datasets were obtained. Totally, 154 DEGs across the studies were identified, including 26 upregulated and 128 downregulated DEGs. In the PPI network, MLLT1, DLG2, and EFEMP1 were the hub proteins, in which DLG2 and EFEMP1 were involved in tumor progression. Among the top 10 up- and downregulated genes, the dysregulation genes of TPO, CDH16, and MPPED2 may be closely related to the tumorigenesis of PTC. By integrated analysis of multiple gene expression profiles, we propose that the dysregulation genes of TPO and MPPED2 will be the promising diagnostic markers for PTCs.

High methylation of the SEPT9 gene in Chinese colorectal cancer patients.

Methylation of the septin 9 gene (SEPT9) occurs in higher frequency in colorectal cancer (CRC) compared to control samples, which suggests that SEPT9 methylation is a useful biomarker for screening CRC. However, the methylation status of SEPT9 in Chinese CRC patients is scarcely reported. In the present study, SEPT9 methylation was tested in CRC tissues obtained from a Chinese population and correlations with pathological characteristics were investigated. The methylation status of SEPT9 was detected using methylation-specific polymerase chain reaction (PCR)-denaturing high-performance liquid chromatography (MSP-DHPLC) in 234 colorectal tissues (172 cases, 62 controls). Samples were sequenced to confirm the results from MSP-DHPLC. The chi-squared test was used to analyze the correlation of SEPT9 gene methylation status and pathological characteristics in CRCs. SEPT9 gene methylation was detected in 152 of 172 (88.4%) cases of verified CRC and in 4 of 62 (6.5%) healthy controls (χ(2) = 137.62, P < 0.001). There was no association between the methylation status of SEPT9 and age, gender, Duke's stage, TNM stage, differentiation, and site of cancer (P > 0.05). Our results suggest that SEPT9 gene methylation is a valuable biomarker for screening CRC in the Chinese population.

Inhibition of hepatitis B virus gene expression by small interfering RNAs targeting cccDNA and X antigen.

To test the possible inhibition of hepatitis B virus (HBV) replication and expression by small interfering RNAs (siRNAs) targeting simultaneously covalenthy closed circular DNA (dnacccDNA) and X antigen, corresponding recombinant plasmids were transfected into HepG2.2.15 cells and the levels of cccDNA, HBXAg, HBcAg, and HBeAg were assayed at various times post transfection. As expected, the single siRNAs showed marked inhibitory effects but their combination was even more efficient. These results provide a new insight into the development of a potential anti-HBV strategy of enhancing the efficacy of individual antivirals and overcoming the high mutation rate of HBV.

cDNA, genomic sequence cloning and overexpression of ribosomal protein gene L9 (rpL9) of the giant panda (Ailuropoda melanoleuca).

The ribosomal protein L9 (RPL9), a component of the large subunit of the ribosome, has an unusual structure, comprising two compact globular domains connected by an α-helix; it interacts with 23 S rRNA. To obtain information about rpL9 of Ailuropoda melanoleuca (the giant panda) we designed primers based on the known mammalian nucleotide sequence. RT-PCR and PCR strategies were employed to isolate cDNA and the rpL9 gene from A. melanoleuca; these were sequenced and analyzed. We overexpressed cDNA of the rpL9 gene in Escherichia coli BL21. The cloned cDNA fragment was 627 bp in length, containing an open reading frame of 579 bp. The deduced protein is composed of 192 amino acids, with an estimated molecular mass of 21.86 kDa and an isoelectric point of 10.36. The length of the genomic sequence is 3807 bp, including six exons and five introns. Based on alignment analysis, rpL9 has high similarity among species; we found 85% agreement of DNA and amino acid sequences with the other species that have been analyzed. Based on topology predictions, there are two N-glycosylation sites, five protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, two tyrosine kinase phosphorylation sites, three N-myristoylation sites, one amidation site, and one ribosomal protein L6 signature 2 in the L9 protein of A. melanoleuca. The rpL9 gene can be readily expressed in E. coli; it fuses with the N-terminal GST-tagged protein, giving rise to the accumulation of an expected 26.51-kDa polypeptide, which is in good agreement with the predicted molecular weight. This expression product could be used for purification and further study of its function.

p73 expression and its clinical significance in colorectal cancer.

OBJECTIVE: To explore p73 mRNA and p73 protein expression and their clinical significance in colorectal cancer. METHOD: p73 mRNA and p73 protein expression were detected using hybridization in situ and immunohistochemical method in cancerous tissue from 60 patients of colorectal cancer and in paracancerous tissue from 23 patients among the corresponding 60 patients of colorectal cancer. Quantitative analysis was performed using Smartscape image analysis system. RESULTS: Compared with paracancerous tissue, p73 mRNA and p73 protein expression in cancerous tissue was increased with a statistically significant difference (P < 0.01) and was associated with differentiation level and lymphatic metastasis. CONCLUSION: High expression of p73 mRNA and p73 protein in colorectal cancer may be involved in the progression of colorectal cancer and p73 mRNA and p73 protein may serve as a potential index to predict differentiation level and prognosis of colorectal cancer.

Bone mass and lifestyle related factors: a comparative study between Japanese and Inner Mongolian young premenopausal women.

The purpose of this study was to evaluate the ethnic difference in bone mass between Japanese and Inner Mongolian young premenopausal women and to assess the contribution of lifestyle related and anthropometric factors to bone mass. We studied 33 Japanese and 44 Inner Mongolian healthy young women, aged 20-34 years, in urban area. Speed of sound (SOS), broadband ultrasound attenuation (BUA) and stiffness index (SI) were measured at the calcaneus using quantitative ultrasound (QUS) analysis. Age at menarche, regularity of menstruation and lifestyle related factors were estimated by a self-reported questionnaire. There were no differences between the two groups in age, height, weight, BMI, regularity of menstruation, frequency of meat intake, frequency of yellow-green vegetable intake and exercise habit. Japanese women had significantly lower age at menarche and higher proportion of milk consumption habit at junior high school, senior school and present. Before adjustment, Japanese women had significantly higher SOS and SI than Inner Mongolian women. However, after adjustment for age at menarche and milk consumption habit at junior high school, both of which were significantly different between groups, no group-differences remained in either SOS or SI. These results suggest that the differences in age at menarche and milk consumption habit at junior high school, which relate to hormonal and nutritional status during puberty, may account for the differences in bone mass between Japanese and Inner Mongolian young women.

[Detection of telomerase activity level in human non-small-cell lung cancer].

OBJECTIVE: To investigate the telomerase activity in human non-small-cell lung cancer (NSCLC) and to investigate the possibility of telomerase as a tumor biological marker. METHODS: The semi-quantitative assay and expression of PCR-based telomeric repeats amplification Protocol ELISA (TRAP-PCR-ELISA) and PAGE (TRAP-PCR-PAGE) were detected in 15 NSCLC tissues and 15 tumor-adjacent tissues, 3 normal lung tissues. RESULTS: No telomerase activity in 15 tumor-adjacent tissues and 3 normal lung tissues was detected, However, of 15 NSCLC tissues, 13 were positive for telomerase activity with a 86.7% positive rate, telomerase activity was significantly higher in NSCLC than in Tumor-adjacent tissues and normal lung tissues (P < 0.001). CONCLUSION: The results suggest that telomerase is a tumor specific gene marker.

[Effects of retinoic acid on proliferation and differentiation of human lung squamous carcinoma cell line].

Human lung squamous carcinoma cell line(TUL cell) was treated with 5 x 10(-6) mol.L-1 all-trans retinoic acid(ATRA). Cell numbers were estimated by 0.4% trypon blue stain. MTT test was used for determining the percentage of cell growth inhibition(GI). DNA syntheses of cells were evaluated by 3H-thymidine incorporation. Cell ultrastructure was examined by transmission electron microscope(TEM). TUL cells were treated with or without ATRA, and transplanted in cutaneous of Balb/c nude mice. The results showed that: 1. The speed of cell growth in study group was smaller than that in control group. 2. The GI of cell lines TUL was 42.3%. 3. The percentage of 3H-thymidine incorporation of TUL cells was declined significantly. 4. Ultrastructure under TME showed that TUL cells treated with ATRA was suppressed. These results suggest that ATRA has the effects on growth-inhibition and differentiation-induction of TUL cell.

Flow-injection determination of total ammonia and total carbon dioxide in blood based on gas-diffusion separation and with a bulk acoustic wave impedance sensor.

A novel flow-injection (FIA) system, for the rapid and direct determination of both total ammonia (T[NH3]) and total carbon dioxide (T[CO2]) in clinical blood samples, has been developed. Samples were injected into a carrier stream of H2O, then emerged with a reagent stream, where the analyte was converted into a gaseous species and diffused across a PTFE gas-permeable membrane into an acceptor stream. The trapped NH3/CO2 in the acceptor was determined on line by a bulk acoustic wave (BAW) impedance sensor. At a through-put of 20 and 65 h(-1), the proposed system exhibited a linear frequency response up to 200 micromol l(-1) ammonium and 20 mmol l(-1) bicarbonate with a detection limit of 1.0 and 10 micromol l(-1), respectively. Results obtained for T(NH3) in serum and T(CO2) in plasma were in agreement with those obtained by the conventional glutamate dehydrogenase (GDH) method and gas-sensing electrode method, respectively. The effects of composition of acceptor stream, cell constant of conductivity electrode, sample volume, flow rate and potential interferents on the FIA signals were also discussed.

Determination of ammonium in Kjeldahl digests by gas-diffusion flow-injection analysis with a bulk acoustic wave-impedance sensor.

A novel flow-injection analysis (FIA) system has been developed for the rapid and direct determination of ammonium in Kjeldahl digests. The method is based on diffusion of ammonia across a PTFE gas-permeable membrane from an alkaline (NaOH/EDTA) stream into a stream of diluted boric acid. The trapped ammonium in the acceptor is determined on line by a bulk acoustic wave (BAW)-impedance sensor and the signal is proportional to the ammonium concentration present in the digests. The proposed system exhibits a favorable frequency response to 5.0 x 10(-6)-4.0 x 10(-3) mol l(-1) ammonium with a detection limit of 1.0 x 10(-6) mol l(-1), and the precision was better than 1% (RSD) for 0.025-1.0 mM ammonium at a through-put of 45-50 samples h(-1). Results obtained for nitrogen determination in amino acids and for proteins determination in blood products are in good agreement with those obtained by the conventional distillation/titration method, respectively. The effects of composition of acceptor stream, cell constant of conductivity electrode, sample volume, flow rates and potential interferents on the FIA signals were discussed in detail.

[Studies on the chemical components and anti-tumour action of the volatile oils from Pelargonium graveoleus].

Three geranium oils were separated, identified and compared by high resolution cross-linked fused silica capillary GC and GC-MSD. It was found that their chemical constituents are very similar, but the contents of some components (linalool, citronellol, cis-caryophyllene and guaiene) differ appreciably. Pharmacological experiments showed that citronellol, citronellyl formate, geraniol and citronellyl acetate exhibited marginal antitumour activities. The activity of the latter two components has not been reported in the literature.