[Clinicopathological analysis of pseudostratified ependymal tubules in ovarian mature teratoma].

Objective: To investigate the morphology and immunohistochemical (IHC) expression of pseudostratified ependymal tubules in ovarian mature teratoma (MT). Methods: Five cases of ovarian MT with pseudostratified ependymal tubules were collected from Shenzhen Hospital(Futian) of Guangzhou University of Chinese Medicine and the Eighth Affiliated Hospital of Sun Yat-sen University from March 2019 to March 2022. In addition, 15 cases of ovarian MT with monolayer ependymal epithelium from Shenzhen Hospital (Futian) of Guangzhou University of Chinese medicine and seven cases of immature teratoma (IMT) from Hainan Provincial People's Hospital from March 2019 to March 2022 were collected as control. The morphologic characteristics and immunophenotypes of pseudostratified ependymal tubules, monolayer ependymal epithelium, and primitive neural epithelial tubules were observed and compared by H&E stain and IHC expression pattern of genes related to the differentiation status of neuroepithelium, namely SALL4, Glypican3, nestin, SOX2, Foxj1, and Ki-67. Results: Mean age of the five patients of ovarian MT with pseudostratified ependymal tubules was 26 years (range from 19 to 31 years). Two tumors were located in the left ovary and three in the right. All five cases were excised, and clinical follow-up was available (mean follow-up 1.5 years; range 0.5 to 3 years). No recurrence was noted in any cases. The pseudostratified ependymal tubules of ovarian MT, which were lined with columnar or oval epithelia up to 4-6 layers, were morphologically similar to the primitive neuroepithelial tubules of IMT and different from monolayer ependymal epithelium of ovarian MT. By immunohistochemistry, SALL4 and Glypican3 were negative, Foxj1 was positive and Ki-67 index was lower in the pseudostratified ependymal tubules and the monolayer ependymal epithelium of ovarian MT. However, the primitive neuroepithelial tubules of IMT showed variably expression of SALL4 and Glypican3, were negative for Foxj1 and high Ki-67 index. All the above three groups expressed nestin and SOX2. Conclusions: The pseudostratified ependymal tubules of ovarian MT, which have morphological similarities to the primitive neuroepithelial tubules of IMT, are similar to the monolayer ependymal epithelia of the MT in immunophenotype. IHC assessment of Foxj1 and Ki-67 is helpful to differentiate the pseudostratified ependymal tubules of ovarian MT from the primitive neuroepithelial tubules of IMT.

[Study of three kinds of primary immunization schedules with poliovirus vaccine].

Objective: To compare the immunogenicity of three kinds immunization programs with poliovirus vaccine. Methods: Healthy infants aged 2 months or over were selected and divided into three groups by complete randomization method. Basic immunization with Sabin inactivated poliovirus vaccine(sIPV) and bivalent oral poliovirus vaccine(bOPV) were completed. Three kinds of basic immunization procedures were 1sIPV+2bOPV,2sIPV+1bOPV and 3sIPV, respectively.Two qualified serums that before basic immunization and 28-42 days later were collected, and measured the poliovirus neutralizing antibody with microcell neutralization method. To compare the difference by analysis of variance, rank test and χ2 test. Results: After the basic immunization, 205 subjects of the positive conversion rate of poliovirus neutralizing antibodies of types Ⅰ, Ⅱ and Ⅲwere all higher than 97.00%, and the positive rates were all higher than 98.00%, the geometric mean titer (GMT) of neutralizing antibody was significantly higher than that before basic immunization in three groups.There were significant differences in the positive rate and GMT before and after basic immunization of typeⅠ, Ⅱand Ⅲ in the three (P<0.05). The highest GMT in three groups after basic immunization were all typeⅠ, followed by type Ⅲ, and the lowest in type Ⅱ. The GMT of type Ⅱin 2sIPV+1bOPV and 3sIPV groups were both higher than that in sIPV+2bOPV group. Conclution: After three kinds of basic immunization, the poliovirus neutralizing antibodies of serum were all at high levels in three groups, which could form an effective immune barrier against poliovirus. The immunogenicity of three kinds of basic immunization programs were all well, but there were certain differences of neutralizing antibodies among three kinds basic immunization programs. The immunogenicity in 2sIPV+1bOPV and 3sIPV groups against typeⅡpoliovirus were better than that in 1sIPV+2bOPV group.

[Six cases of acute toilet cleaners].

The oral toilet cleaners poisoning can result in devastating gastrointestinal tract injuries with the risk of perforation and/or hemorrhage, and sometimes potentially fatal systemic complications and sequelas. It should be given positive treatment. In this paper, six cases of acute toilet cleaners poisoning were analyzed, and the clinical characteristics and treatment effect were summarized, so as to improve the understanding, diagnosis and treatment level of the disease.

[Expression of lncRNA MIR210HG in preeclampsia placental tissue and its functional analysis].

Objective: To investigate the differential expression of long non-coding RNA (lncRNA) in placental tissues of women with preeclampsia (PE) and the effect of MIR210HG on the biological function of HTR8/SVneo cells. Methods: A total of 39 cases of PE women (PE group) and 39 cases of normal pregnant women (CTL group) admitted to the Affiliated Hospital of Qingdao University from July 2018 to July 2019 were collected. (1) Transcriptome sequencing (RNA-seq) was used to analyze the differentially expressed lncRNAs in the placental tissues of the two groups. (2) The expression level of MIR210HG, one of the differentially expressed lncRNAs, in the placental tissues of the two groups was detected by real-time quantitative PCR. And the correlations between the expression level of MIR210HG and systolic blood pressure, diastolic blood pressure and neonatal birth weight were analyzed. (3) The constructed small interfering RNA and negative control (NC) RNA were transfected into the HTR8/SVneo cells. The cells were divided into MIR210HG knockdown (KD) group and NC group. The effects of living cell counting (CCK-8) and transwell assay on the proliferation and migration of HTR8/SVneo cells were detected. (4) RNA interacting with MIR210HG was predicted using the Encyclopedia of RNA Interactomes (ENCORI) database. Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Gene and Genomes (KEGG) and BioCarta pathway enrichment analysis were performed. Results: (1) A total of 26 significantly differentially expressed lncRNAs were found by RNA-seq, among which 21 lncRNAs were up-regulated and 5 lncRNAs were down-regulated. (2) The relative expression level of MIR210HG in the PE group was significantly higher than that in the CTL group (9.30±1.90 and 1.10±0.20, respectively; t=4.425, P<0.01). The relative expression level of MIR210HG had positive linear correlation with systolic blood pressure (r2=0.234, P<0.05) and diastolic blood pressure (r2=0.190, P<0.05), but had a negative linear correlation with newborn birth weight (r2=0.157, P<0.05). (3) Compared with the NC group, the proliferation and migration ability of HTR8/SVneo cells in the KD group were increased (all P<0.05). (4) A total of 38 RNAs that might interact with MIR210HG were predicted by ENCORI database. GO functional annotation analysis showed that MIR210HG might be involved in the functions of 27 pathways, including the regulation of production of molecular mediator of immune response, etc; KEGG pathway analysis showed that MIR210HG might be involved in the function of 8 pathways including allograft rejection, etc; Biocarta pathway analysis showed that MIR210HG may be involved in the functions of 8 pathways, including the eukaryotic initiation factor (eIF) pathway, etc. Conclusion: The expression of MIR210HG is up-regulated in the placental tissue of PE women, and MIR210HG might be a regulator of the biological behavior of trophoblast cells.

[Application of a new type of angle-adjustable osteotomy guide in closing wedge distal femoral osteotomy].

Objective: To explore the feasibility of fast and accurate osteotomy using a new angle adjustable osteotomy guide (AAOG) in closing wedge distal femoral osteotomy(CWDFO). Methods: The clinical data of 14 patients (17 knees) with valgus knee treated with CWDFO at Department of Integrated Chinese and Western Medicine Orthopedics, Honghui Hospital, Xi'an Jiaotong University from January 2018 to July 2019 were analyzed retrospectively. There were 3 males and 11 females, aging (41.4±16.4) years (range: 18 to 56 years). The body mass index was (23.5±3.5) kg/m(2) (range: 18.1 to 28.9 kg/m(2)). The guide pins were placed with the assistance of the self-designed AAOG. Before the surgery, Solidworks software was used to calculate the correction angle and the osteotomy radius accurately. The osteotomy guide was adjusted according to these two parameters. During the surgery, the adjusted osteotomy guide was placed to the surface of bone closely and the guide pins were drilled into the bone through the guide holes. The position of the guide pins was confirmed under fluoroscopy. The osteotomy was finished under guide of pins and fixed with Tomofix plate (Synthes). The times and duration of placement of the guide pins, the times of X-ray examination, the planned and actual thickness of the osteotomy wedge, the top and bottom area of the osteotomy wedge, the posterior distal femoral angle(PDFA), the correction of the weight line, and the American Knee Society Score(AKSS) and Tegner scores were collected and compared by paired t test or Kruskal-Wallis non-parametric test. Healing time after osteotomy and complications were recorded. Results: The guide pins were successfully placed once in 10 knees, adjusted once in 5 knees and twice in 2 knees. The time spent in placing all the 6 pins was 82.4 seconds (range: 51 to 125 seconds), and the times of X-ray examination was 1.5 times (range: 1 to 5 times). The top and bottom areas of the osteotomy wedge were (5.52±0.52)cm(2) and (5.36±0.49)cm(2). PDFA was (85.2±2.6)° preoperatively and (85.5±1.4)° postoperatively (t=-0.401, P>0.05). The thickness of the osteotomy was (11.3±1.9)mm according to the preoperative plan, and the actual thickness was (8.1±1.7)mm. All the patients were followed up for 6 months after surgery and AKSS and Tegner scores improved significantly (all P<0.05). The correction of the weight lines was within the ideal range. Fractures of the hinge point occurred in 3 knees. All of the osseous healing without complications. Conclusion: The new osteotomy guide helps to place the guide pins rapidly and precisely according to the preoperative planning, which should be widely used in clinical applications with promising outcomes.

Identification of downstream target genes regulated by CX43 in hepatocellular carcinoma.

The aim of study was to identify the downstream target genes of CX43 by Human Transcriptome Array. Therefore, a gene microarray was generated which consists of CX43-overexpressed hepatocellular carcinoma (HCC) cells transfected with the constructed plasmid and negative controls to identify candidate genes. Integrated bioinformatic analysis was used to clarify biological functions of the identified genes, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein-protein interaction network, and survival analysis. The candidate genes were further validated by qRT-PCR in liver cancer tissues and CX43-silenced HCC cells. We have found the mRNA and protein levels of CX43 significantly upregulated in HCC cells transfected with CX43 constructed plasmid. We identified 928 differentially expressed genes including 394 upregulated and 534 downregulated genes, enriched in the cancer related functions and pathways by GO and KEGG pathway analysis. The protein-protein interaction network revealed 9 hub genes in this study. Statistical analysis indicated that upregulation of RALA and SRC was associated with poor prognosis in liver cancer. The differential expression of 2 candidate genes were further validated in HCC cells and tissues. In conclusion, protein-coding genes RALA and SRC could be target genes of CX43 and therapeutic targets for hepatocellular carcinoma.

[Fibroblast growth factor receptor 1 propagates estrogen and fluid shear stress driven proliferation and differentiation response in MC3T3-E1 cells].

Fluid shear stress (FSS) and estrogen exposure positively regulate bone metabolism. Fibroblast growth factor receptor 1 (FGFR1) plays a vital role in FSS-induced osteogenesis. An in vitro experiment with MC3T3-E1 cells combined with microarray analysis aided us in identification of the genes differentially expressed in response to FSS and highlighted the role of FGFR1 in this process. Both estrogen exposure and FSS increase methyl thiazol tetrazolium (MTT) values and alkaline phosphatase (ALP) activity, as well as the levels of Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). The effects of estrogen exposure and FSS were cumulative. Treatment with PD166866 inhibitor of the FGFR1 reduced the MTT values, increased ALP activity, and increased the levels of Runx2 and OCN. To investigate the regulation of FGFR1 signaling in stressed cells, a number of key components of the mitogen-activated protein kinase (MAPK) cascade were quantitatively examined. Neither estrogen nor FSS change the protein expression of extracellular signal-regulated kinase (ERK), Jun amino-terminal kinases (JNK) or p38, but positively influence their phosphorylation levels. Treatment with the FGFR1 inhibitor induced an increase in ERK phosphorylation levels only. In summary, estrogen exposure and FSS have a synergistic effect in osteogenesis. FGFR1 promotes osteoblast proliferation and inhibits the differentiation of osteoblasts. In MC3T3-E1 cells, FGFR1 signaling responds to independent and combined effects of estrogen and FSS. MAPK cascades participate in osteogenesis, but only the ERK signaling pathway responds to FGFR1.

Association between the rs4753426 polymorphism in MTNR1B with fasting plasma glucose level and pancreatic ß-cell function in gestational diabetes mellitus.

We investigated the association between rs4753426 single nucleotide polymorphisms in the melatonin receptor 1B (MTNR1B) gene and the risk of developing gestational diabetes mellitus (GDM). A total of 516 gravidas (186 with GDM and 330 non-diabetic controls) were enrolled in the study. Genotype and allele frequencies of rs4753426 in the MTNR1B gene were detected by DNA sequencing. Fasting plasma glucose and fasting insulin levels were measured to calculate the homeostasis model assessment for insulin resistance (HOMA-IR) and for ß-cell function. Three genotypes (CC, CT, and TT) were found in both groups. The frequencies of CC, CT, and TT genotypes for the GDM group were 70.97, 22.58, and 6.45% vs 53.03, 39.70, and 7.27% in the control group, respectively. Significant differences were observed in genotype frequencies between groups (P < 0.05). T and C allele frequencies in the GDM group were 17.74 and 82.26%, respectively, and in the control group were 27.12 and 72.88%, respectively. Significant differences in T and C allele frequencies were found between groups (P < 0.05). In the GDM group, the C allele was associated with increased fasting plasma glucose level and reduced pancreatic ß-cell function (P < 0.05). There were no significant differences in total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein concentration, or HOMA-IR between groups (P > 0.05). The single nucleotide polymorphism rs4753426 in MTNR1B may be a susceptibility gene locus for GDM, and the C allele may contribute to the increased fasting plasma glucose level and reduced pancreatic ß-cell function.

Association of genetic polymorphisms in TERT-CLPTM1L with lung cancer in a Chinese population.

Genome-wide association studies in several ethnic groups have reported that polymorphisms of the telomerase reverse transcriptase (TERT) and cleft lip and palate transmembrane 1-like (CLPTM1L) genes, located on 5p15.33, are associated with susceptibility to lung cancer. However, whether genetic variants of TERT-CLPTM1L are associated with an increased risk of lung cancer in the Chinese Han population is unknown. This study examined associations between five single nucleotide polymorphisms (SNPs) of TERT-CLPTM1L (rs402710, rs401681, rs465498, rs4975616, and rs2736100) and lung cancer in a Chinese Han population in the Hubei Province. The five SNPs were detected using the Sequenom MassArray(®) iPLEX System in 304 lung cancer patients and 319 controls. Of the five SNPs, rs4975616 did not conform to Hardy-Weinberg equilibrium in the controls. Only rs2736100 was significantly (P = 0.034) associated with an increased risk of lung cancer. In the linkage disequilibrium analyses, a block of strong linkage disequilibrium was observed between rs401681 and rs465498 (D' = 0.986; r(2) = 0.546). No linkage disequilibrium between rs2736100 and the other three SNPs was found. In the haplotype analyses, the frequencies of the TTCT haplotype in rs402710, rs401681, rs465498, and rs2736100 differed significantly between case and control subjects (odds ratio = 0.56; 95% confidence interval, 0.36-0.88; P = 0.012). The results of this study suggested that rs2736100 on TERT-CLPTM1L indicates a poor prognosis for lung cancer in the Chinese Han population.

Association between macrophage migration inhibitory factor rs1007888 and GDM.

We investigated the association between macrophage migration inhibitory factor (MIF) rs1007888 single-nucleotide polymorphisms and the genetic susceptibility to gestational diabetes mellitus (GDM). A total of 240 GDM pregnant women (GDM group) and 330 healthy pregnant women (control group) were included in the study. Differences in the MIF rs1007888 genotype and allele frequencies and differences between fasting blood glucose, fasting insulin, homeostatic model assessment (HOMA)-insulin resistance, and HOMA-b levels of pregnant women with different genotypes were compared. MIF genotype distributions were significantly different in the GDM group compared to the control group (P < 0.05). No significant difference was observed in the allele distributions of MIF rs1007888 between the GDM group and control group (P > 0.05). GDM patients had higher fasting blood glucose, fasting insulin, and HOMA-insulin resistance levels, but lower HOMA-b levels than normal gestational women (P < 0.05). Fasting blood glucose, fasting insulin, and HOMA-insulin resistance in pregnant women with the GG genotype were significantly higher than those with GA and AA genotypes, while HOMA-b in pregnant women with the GG genotype was lower (all P < 0.05). Our findings demonstrated the associations among MIF polymorphism rs1007888, insulin resistance, and pancreatic ß cell functions in GDM patients. The GG genotype of MIF rs1007888 may be a genetic susceptibility factor in the pathogenesis of GDM.

Implications of microRNA-197 downregulated expression in esophageal cancer with poor prognosis.

The aim of this study was to investigate the significance of the microRNA miR-197 expression level in relation to clinicopathological factors and prognoses of esophageal cancer (EC). MicroRNA was extracted using the Taqman(®) MicroRNA Assay from 46 EC patients at the same tumor node metastasis (TNM) stage, but with different prognoses, who underwent surgery. Paracancerous normal tissues were used as controls. The correlation between miR-197 expression and clinicopathologic features was analyzed, and the significance of miR-197 as a prognostic factor and its relationship with survival was determined. miR-197 expression was lower in patients with poor prognosis than in those with good prognosis (P < 0.05). Kaplan-Meier analysis results showed that the miR-197 expression level is significantly correlated with survival time (P = 0.030), and that patients with higher expression of miR-197 had longer survival times. Cox multi-factor model analysis showed that patient prognosis (P = 0.001), tumor length (P = 0.010) and expression (P = 0.042), and survival time were significantly correlated, with corresponding risks of 9.183, 2.318, and 1.925, respectively. This study supports a role of miR-197 as an anti-oncogene and a biomarker for EC and its relationship with other prognostic factors and survival.

Overregulation of microRNA-212 in the poor prognosis of esophageal cancer patients.

There have been few reports evaluating the expression and function of the microRNA miR-212 in esophageal cancer. The aim of this study was to investigate the relationship between miR-212 expression and clinicopathological factors and prognoses of esophageal cancer. MicroRNA was extracted from 46 esophageal cancer patients using the Taqman MicroRNA assay. All patients were at the same tumor node metastasis stage, but with different prognoses, and had all undergone surgery. The correlation between miR-212 expression and clinicopathological features was analyzed and the significance of miR-212 as a prognostic factor as well as its relationship with survival was determined. miR-212 expression was higher in patients with poor prognoses than in those with good prognoses (P < 0.0001). Kaplan-Meier analysis results showed that the miR-212 expression level was significantly correlated with survival time (P = 0.024). Patients with higher expression of miR-212 showed longer survival times. Cox multi-factor model analysis showed that miR-212 expression was significantly correlated with survival time (P = 0.026). mir-212 is related with prognostic factors and survival time and may be a biomarker for esophageal cancer.

Screening of PAX8 mutations in Chinese patients with congenital hypothyroidism.

BACKGROUND: Congenital hypothyroidism (CH) is a neonatal endocrine disease with an incidence of 1:2000 to 1:4000 worldwide. In about 85% of patients CH is secondary to thyroid dysgenesis, but its pathogenesis remains unclear. Thyroid transcription factors, such as paired box transcription factor 8 (PAX8), play an important role in thyroid organogenesis and development. AIM: To screen PAX8 mutations in Chinese CH patients and characterize the features of PAX8 mutations in China. MATERIALS AND METHODS: Blood samples were collected from 300 CH patients in Shandong Province, China, and genomic DNA was extracted from peripheral blood leukocytes. Using PCR and direct sequencing, exon 3 and exon 4 of PAX8 were analyzed. RESULTS: Analysis of PAX8 in 300 CH patients revealed heterozygous missense mutations or variations in two unrelated patients; one was a known missense mutation G92A, resulting in an arginine to histidine substitution at codon 31, the other was a missense variation G122T, resulting in the substitution of a glycine at position 41 by a valine residue. The patient with the R31H mutation had CH with thyroid hypoplasia, while the patient with the G41V variation had CH with a eutopic and normal-sized thyroid gland. CONCLUSION: We report a heterozygous missense mutation and a variation in PAX8 in two out of 300 unrelated Chinese CH patients, showing that the PAX8 mutation rate is very low in CH patients in China.

[Establishment of a rat model of laryngeal precancerous lesions induced by 4NQO smearing].

Objective: To establish a rat model for laryngeal precancerous lesions histologically and pathologically comparable to the human counterpart. Methods: Thirty-six Wistar rats were randomly divided into experimental group and control group, with 18 rats in each group, and 1% 4-nitroquinoline-1-oxide (4NQO) solution and saline were respectively applied to the laryngeal mucosas of rats in two groups. During subsequent 20 weeks, the changes of laryngeal mucosas were regularly observed with naked eyes and endoscope and lesions were determined by histology. SPSS 22.0 software was used for statistical analysis. Results: The food intake, water intake and body weight of the rats in the experimental group were lower than those in the control group, with statistically significant differences (all P<0.05). White plaque, superficial ulcer, erosion and miliary particles were present in the larynxes of rats in the experimental group, with histological manifestations of atypical hyperplasia or carcinoma in situ, and normal epitheliums were shown in the control group. The number of Ki67 positive cells in the laryngeal mucosas of rats in the experimental group at the 4 th, 8 th, 12 th, 16 th, and 20 th weeks were 13.5±2.4, 35.6±5.8, 53.4±8.3, 78.8±11.6, 80.6±12.4, respectively, no Ki67 positive cells were found in the control group at individual time points, and the differences were statistically significant (t=9.74, 10.63, 11.14, 11.77, 11.26, respectively, all P<0.01). Conclusion: 4NQO can credibly cause rats laryngeal precancerous lesions, which morphologically and histologically mimic laryngeal carcinnogenesis. This method is practical, easy and reliable to prepare the animal model of laryngeal precancerous lesions.

Visible-light excitable europium(III) complexes with 2,7-positional substituted carbazole group-containing ligands.

Two novel carbazole-based beta-diketones with 2- or 2,7-substituted groups in the carbazole ring, 2-(4'4'4'-trifluoro-1'3'-dioxobutyl)-carbazole (2-TFDBC) and 2,7-bis(4'4'4'-trifluoro-1''3'-dioxobutyl)-carbazole (2,7-BTFDBC), and their europium(III) ternary complexes Eu(2-TFDBC)(3)phen and Eu(2)(2,7-BTFDBC)(3)(phen)(2) were synthesized via a dexterously designed routine, where phen is 1,10-phenanthroline. The Eu(III) complexes were characterized by elementary analysis, IR and UV-visible absorption spectroscopy, thermogravimetric analysis (TGA), and photoluminescence (PL) measurements. TGA shows that the decomposition temperatures of the complexes are 361.4 and 367.3 degrees C, respectively. PL measurement results indicated that suitably expanded pi-conjugation in the complex molecules makes the excitation band redshift to the visible region, and both the Eu(III) complexes exhibit intense red emission under blue-light excitation. The triplet state energy levels of 2-TFDBC and 2,7-BTFDBC in the complexes are higher than that of the lowest excited level of Eu(3+) ion, (5)D(0), so the photoluminescence mechanism of the Eu(III) complexes was proposed as a ligand-sensitized luminescence process. All of the results indicate that Eu(2-TFDBC)(3)phen and Eu(2)(2,7-BTFDBC)(3)(phen)(2) are promising candidates as visible-light excitable red phosphor for luminescence applications.

[Study on the mechanism of miR-497-induced laryngeal squamous cell carcinoma growth inhibition by targeting CDK6].

Objective: To study the effects of miR-497 and CDK6 on the growth of laryngeal squamous cell carcinoma (LSCC). Methods: The expressions of CDK6 mRNA in fresh LSCC specimens, the adjacent normal mucosa of LSCC, and cell lines of LSCC were detected with quantitative real time polymerase chain reaction, pcDNA3.1(+) CDK6 plasmids were respectively transfected into the LSCC cells, and MTT assay and clone formation assay were performed to evaluate the growth of LSCC cells. Flow cytometry was employed for cell cycle analysis. SPSS17.0 software was used to analyze the data. Results: CDK6 was highly expressed in LSCC(t=14.01, P=0.009) and the overall survival rate of the patients with high CDK6 expression was less than that with low CDK6 expression, with a significant difference (HR=3.236, P<0.001). Double luciferase reporter gene analysis showed that fluorescence activity in wild type CDK6 group was significantly different from that in control group (P<0.01), while there was no significant difference in the fluorescence activity between mutant CDK6 group and control group (P>0.05). A(490) values were respectively 0.42±0.14 (Mean±SD) in siRNA Hep-2 group, 0.51±0.13 in siRNA TU-212 group; 0.98±0.16 in control Hep-2 group and 1.17±0.20 in control TU-212 group. Colonies were 55±4 in siRNA Hep-2 group, 51±3 in siRNA TU-212 group, 108±6 in control Hep-2 group and 105±7 in control TU-212 group, namely, cell growth and clone formation ability in CDK6 siRNA group were significantly lower than those in the control group. Cells cycle was blocked in G0/G1 phase (G0/G1: 65.20%±10.12% in siRNA Hep-2 group; 63.42%±8.97% in siRNA TU-212 group; 45.31%±7.55% in control Hep-2 group; and 42.37%±7.28% in control TU-212 group), and cells decreased obviously in S phase (S: 25.39%±5.51% in siRNA Hep-2 group; 27.21%±5.43% in siRNA TU-212 group; 42.87%±6.85% in control Hep-2 group; and 44.76%±7.02% in control TU-212 group). Compared with miR-497 group, cell growth and clone formation ability in miR-497/CDK6 group were partly restored (all P<0.05). Conclusions: CDK6 expression in LSCC is upregulated, functioning as an oncogene. High expression of CDK6 is a predictor for poor prognosis. miR-497, functioning as a tumor suppressor gene, inhibits the growth of LSCC by targeting CDK6.

Low-expression of lncRNA-ANCR promotes tibial fracture healing via targeting RUNX2.

OBJECTIVE: To explore the role of long non-coding ribonucleic acid-anti-differentiation non-coding ribonucleic acid (lncRNA-ANCR) in tibial fracture healing in rabbits by regulating the runt-related transcription factor 2 (RUNX2) expression. MATERIALS AND METHODS: A total of 60 healthy adult rabbits were evenly divided into Control group (n=20), Fracture group (n=20), and Lnc group (n=20). Then, RUNX2 transfection, Real Time Polymerase Chain Reaction (PCR) assay and relevant instruments were carried out and used to determine the differences in dry weight, bone mineral density, bone mechanical strength, and RUNX2 expression in tibiae among three groups of rabbits. RESULTS: Comparison of the bone mineral density in rabbit tibiae among the three groups showed that the bone mineral density was significantly lower in Fracture group than that in Control group (p<0.05), and it was slightly higher in Lnc group than in Fracture group (p<0.05). The dry weight of the full-length tibiae in Fracture group was significantly decreased compared with that in Control group (p<0.05), and Lnc group had an increased dry weight of tibiae in comparison with Fracture group (p<0.05). The maximum load, flexural strength, elastic stress, elastic strain, elastic modulus, maximum stress, and maximum strain in Fracture group were lower than those in both Control group and Lnc group (p<0.05). Compared with those in Fracture group, the amount of new collagen was overtly increased in Lnc group, and that of mature collagen was decreased (p<0.05). The relative expression level of RUNX2 in tibial bone tissues was evidently lower in Fracture group than that in Control group (p<0.05), and it was markedly higher in Lnc group than that in Fracture group (p<0.05). CONCLUSIONS: Down-regulating lncRNA-ANCR activates and triggers the expression of RUNX2 that facilitates the growth and metabolism of bone tissues to play an important role in the repair of bone tissues and promote the healing of the tibial fracture.

[The effect of miR-497 on laryngeal squamous cell carcinoma invasion through modulating PlexinA4].

Objective: To study the roles of miR-497 and PlexinA4 in the progression of laryngeal squamous cell carcinoma. Methods: The expressions of miR-497 and PlexinA4 in fresh tumor specimens and adjacent normal mucosa tissues as well as in cell lines of laryngeal squamous cell carcinoma (LSCC) were detected with qRT-PCR and immunohistochemistry. The association of miR-497 and PlexinA4 expressions with clinicopathologic factors and their prognostic values in LSCC were evaluated PlexinA4 siRNA and pcDNA3.1 (+ )/PlexinA4 plasmid were transfected into the LSCC and measured by Transwell to evaluate their effect on the invasion of LSCC. Results: miR-497 was low expression in LSCC, which related to pathological differentiation, while PlexinA4 mRNA was high expression in LSCC. Kaplan-Meier method showed that the prognosis of patients with high miR-497 expression was better than that of patients with low miR-497 expression (χ(2)=10.342, P=0.001); . Cox regression analysis showed that miR-497 was an independent prognostic factor for LSCC. The double luciferase reporter gene showed that the variation of the fluorescence activity of wild type PlexinA4 was significantly different from that of the control group (P<0.01). In Hep-2 and TU212 cell line, the number of cells with PlexinA4 siRNA passing through the compartments was 70.00±10.85 and 85.00±6.45, significantly higher than control (F values were 30.251 and 23.936, both P<0.05), the number of cells with pcDNA3.1 (+ ) /PlexinA4 was 170.56±11.95 and 142.00±10.43, also significantly less than control (F values were 35.104 and 29.643, both P<0.05). Conclusion: The expression of miR-497 in LSCC is decreased, indicating poor prognosis, which is as an independent risk factor for prognosis of LSCC. miR-497 may modulate LSCC invasion through PlexinA4.

A natural recombinant PRRSV between HP-PRRSV JXA1-like and NADC30-like strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically significant pathogen that has adversely affected China's swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China, and commercial vaccines provide limited protection for infected animals. The extensive recombination phenomenon among NADC30-like PRRSVs is identified as a unique molecular characteristic of the virus. However, our understanding of how recombination influences NADC30-like PRRSVs is largely inadequate. In this study, we analysed the genetic characteristics of a recombinant NADC30-like PRRSV (SC-d) and examined its pathogenicity compared with a non-recombinant NADC30-like PRRSV (SD-A19) and a highly pathogenic PRRSV (HuN4). SC-d has three discontinuous deletions in nsp2, consistent with NADC30 isolated from the United States in 2008. Furthermore, we identified four recombination breakpoints in the SC-d genome, which separated the SC-d genome into four regions (regions A, B, C and D). Regions A and C are closely related to the JXA1-like strain, one of the earliest Chinese HP-PRRSV strains, and regions B and D are closely related to the NADC30 strain. Moreover, SC-d inoculated piglets exhibited a persistent fever, moderate weight loss, mild thymus atrophy and obvious microscopic lung lesions. In summary, the recombinant NADC30-like PRRSV SC-d strain displayed a higher pathogenicity than the non-recombinant NADC30-like PRRSV SD-A19 strain; however, the pathogenicity of the NADC30-like PRRSV SC-d was lower compared with the HP-PRRSV HuN4 strain in piglets. Our findings demonstrate that recombination is responsible for the enormous genetic diversity and pathogenicity variance of the NADC30-like PRRSV in China. This study provides a theoretical basis for developing a more reasonable PRRSV control and prevention strategy.

Effects of dietary threonine levels on growth performance, serum biochemical indexes, antioxidant capacities, and gut morphology in broiler chickens.

A dose-response experiment was conducted to investigate the impacts of dietary threonine (Thr) levels on growth performance, serum biochemical indices, antioxidant capacities, and gut morphology of broiler chickens. Four hundred and thirty-two 1-d-old commercial broilers were allocated to 4 treatments consisting of 6 replicates of 18 birds. The experimental treatments received the same Thr-deficient basal diet and were labeled as follows: 85%, 100%, 125%, and 150% of NRC (1994) recommendations. The results demonstrated that on 21 d and 42 d, average daily weight gain (ADG, 22 to 42 d, 0 to 42 d) increased quadratically or cubically as the inclusion of Thr increased, while feed conversion ratio (FCR, 0 to 21 d, 0 to 42 d) decreased quadratically or cubically as dietary Thr increase from 85% to 150%. Excess dietary Thr levels triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. The concentrations of total protein (TP) and globulin (GLO) increased quadratically with increasing Thr level, and the highest concentrations of TP and GLO were obtained at the 125% Thr level. Moreover, the plasma uric acid (UA) concentration decreased linearly or quadratically with the increase in dietary Thr level. Likewise, the serum glutathione peroxidase (GSH-Px) and total superoxide dismutases (T-SOD) activities increased quadratically as dietary Thr increased, and the highest activity of GSH-Px was obtained at the 125% Thr level, while the highest T-SOD level occurred in the 100% Thr group. Gut morphology of birds showed significant response to different graded concentrations of Thr level. Villus height (VH), crypt depth (CD), and VH:CD ratio (VH/CD) were increased linearly or quadratically by Thr supplementation. Therefore, the present study suggests that the NRC (1994) recommendations Thr level that was optimum for growth performance, and 125% of the NRC (1994) recommendations Thr level had better effects on biochemical indices, antioxidant function, and gut morphology of broilers.