[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.

[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.

[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.

Tea polyphenols inactivate Cronobacter sakazakii isolated from powdered infant formula.

This study evaluated the antimicrobial activity of tea polyphenols (TP) against 4 Cronobacter sakazakii strains with different sequence types (ST) isolated from powdered infant formula (PIF). The results showed that in normal saline, 5mg/mL of TP (pH 3.44) could eliminate approximately 7.0 log cfu/mL of C. sakazakii within 1 h; in rehydrated PIF, after acidification with HCl (pH 3.55), TP showed a stronger antibacterial activity compared with the controls (malic acid, ascorbic acid, and citric acid). Further, some differences were obvious in tolerance to TP between C. sakazakii strains with different ST. The tolerance of C. sakazakii CE1 (ST4) to TP was found to be greater than that of the other 3 C. sakazakii strains (ST1, ST8, and ST64). The results of recovered test and transmission electron microscope analysis revealed that the action of TP against C. sakazakii was an irreversible bactericidal process caused by leakage of cytoplasm. Taken together, these results indicated that TP had an effective bactericidal effect against C. sakazakii, and provided a new idea for preventing and inactivating C. sakazakii in PIF.