Abstract The AMC-HN-8 cell line and the primary human laryngeal epithelial cell lines were utilized in this work to explore the molecular mechanism of miR-548-3p regulating the gene DAG1 to induce the occurrence and malignant transformation of laryngeal carcinoma. Non-coding RNA miR-548- 3p overexpression plasmid, interference plasmid and blank plasmid were constructed, and the plasmids were transfected into AMC-HN-8 cells, respectively. Meanwhile, a non-transfected plasmid group and a human laryngeal epithelial primary cell group were set up. Five groups of cells were named as NC (Normal control), Model, Ov-miR-548-3p, Sh-miR-548-3p and Blank-plasmid group. The luciferase reporter experiment was used to analyze the regulation characteristics of hsa-miR-548-3p on dystrophin-associated glycoprotein 1 (DAG1). Immunofluorescence was used to analyze the relative expression characteristics of the protein DAG1. The cell cloning experiment was used to analyze the proliferation characteristics of AMC-HN-8. The scratch healing test was used to analyze the migration ability of AMC-HN-8. The transwell test was used to analyze the invasion ability of AMC-HN-8. The RT-PCR was used to analyze the expression level of miR-548-3p. Western blot experiments were used to analyze the expression of protein DAG1, laminin α2 (LAMA2) and utrophin (UTRN). The luciferase report experiment and immunofluorescence test found that the expression of DAG1 and miR-548-3p are positively correlated. Cell cloning, scratching and migration experiments identified that the activity of laryngeal cancer cells was positively correlated with the expression of DAG1. The results of Western blot analysis further strengthened the above conclusions. Through carrying out research on the cellular levels, our work has demonstrated that miR-548-3p regulated the content of protein DAG1, and then further induced malignant transformation of laryngeal carcinoma.

Resumen En este trabajo se utilizaron la línea celular AMC-HN-8 y la línea celular epitelial laríngea humana primaria, para explorar el mecanismo molecular regulador del miR-548-3p sobre el gen DAG1 para inducir la aparición y la transformación maligna del carcinoma laríngeo. Se construyeron el plásmido de sobreexpresión de miR-548-3p de ARN no codificante, el plásmido de interferencia y el plásmido en blanco, y los plásmidos se transfectaron en células AMCHN-8 respectivamente. Mientras tanto, se establecieron un grupo de plásmidos no transfectados y un grupo de células primarias epiteliales laríngeas humanas. Se nombraron cinco grupos de células como NC (control normal), modelo, OvmiR-548-3p, Sh-miR-548-3p y grupo de plásmido en blanco. El experimento indicador de luciferasa se utilizó para analizar las características de regulación de hsa-miR-548-3p en la glicoproteína 1 asociada a distrofina (DAG1). Se utilizó inmunofluorescencia para analizar las características de expresión relativa de la proteína DAG1. El experimento de clonación celular se utilizó para analizar las características de proliferación de AMC-HN-8. Se utilizó la prueba de cicatrización por rascado para analizar la capacidad de migración de AMC-HN-8. La prueba de transwell se utilizó para analizar la capacidad de invasión de AMCHN-8. Se utilizó RT-PCR para analizar el nivel de expresión de miR-548-3p. Se usó un experimento de transferencia Western (Western blot) para analizar las expresiones de la proteína DAG1, laminina α2 (LAMA2) y utrofina (UTRN). El experimento de reporte de luciferasa y la prueba de inmunofluorescencia encontraron que la expresión de DAG1 y miR-548-3p están positivamente correlacionadas. Los experimentos de clonación celular, rascado y migración, identificaron que la actividad de las células cancerosas de laringe se correlacionó positivamente con la expresión de DAG1. Los resultados del análisis de transferencia Western fortalecieron aún más las conclusiones anteriores. A través de la investigación a nivel celular, nuestro proyecto ha demostrado que miR-548-3p regula el contenido de la proteína DAG1 y luego induce la transformación maligna del carcinoma de laringe.

ABSTRACT Quantifying the effects of coal mining subsidence on soil quality is critical for developing sustainable strategies in the local agriculture. The objective of this study was to assess the effects of slope position because of coal mining on soil quality using principal component analysis. Soil samples were collected from five positions in the subsided farmland: Top slope position (1), upper slope position (2), middle slope position (3), lower slope position (4), and bottom position (5). Samples from an adjacent non-subsided farmland (CK) were used as a reference. For each soil sample, 21 different physical, chemical and biological attributes were investigated. The principal components analysis (PCA) identified the bulk density (BD), salinity, organic matter (OM) content, urease enzymatic activity (UA), actinomycete quantities (AQ), polyphenol oxidase enzymatic activity (POA) and phosphatase enzymatic activity (PA) as the most sensitive indicators in a mínimum data set (MDS) to assess the soil quality. The soil quality index (SQI) was highest for positions 5 (1.220), 4 (1.203), CK (1.101), 1 (1.093), 3 (1.080), and 2 (1.044). Positions 5 and 4 had higher SQI values but lower crop yields than CK, which suggests that higher SQI does not represent higher production, and other soil quality indicators, which were not investigated in this study, had stronger effects on the crop productivity. Overall, the slope position because of coal mining subsidence strongly affected the soil quality and crop yields.

RESUMO Racional: É importante, mas difícil de se tratar fístula anal complexa devido à alta taxa de recorrência e de incontinência pós-operatória. A ligadura do trajeto da fístula interesfincteriana (LIFT) - um novo procedimento cirúrgico com a vantagem de evitar a incontinência anal - tem taxa de sucesso variável entre 57-94,4%. Objetivo: Avaliar os resultados em longo prazo do procedimento cirúrgico LIFT modificado - ligadura do trato interesfincteriano com fístula - para tratar fístula complexa anal. Métodos: Análise retrospectiva de 62 casos de fístula complexa no ânus tratados com abordagem modificada de LIFT (incisão curva na pele do canal anal; sutura em bolsa realizada em torno da fístula; as fístulas residuais removidas em um túnel) e teve tempo de acompanhamento de mais de um ano. A condição geral pré-operatória dos pacientes, a eficácia pós-operatória e a função anal foram comparadas. Resultados: A mediana de idade dos participantes foi de 34 anos, e 43 (69,4%) dos casos eram de homens. Quarenta e um (66,1%) casos eram de fístula transesfincteriana alta, quatro (6,5%) de fístula intra-esfincteriana alta e 17 (27,4%) de fístula anal anterior em mulheres. A mediana da duração do acompanhamento foi de 24,5 meses (12-51). A taxa de sucesso no final do acompanhamento foi de 83,9% (52/62). A pressão anorretal e a Incontinência Fecal da Cleveland Clinic Florida (CCF-FI) avaliadas três meses antes e após a operação não encontraram alterações aparentes. Conclusões: Comparado com o LIFT, o LIFT modificado reduz notavelmente a falha pós-operatória e a taxa de recorrência de fístula complexa com resultados aceitáveis em longo prazo.

ABSTRACT Background: It is important but difficult to treat complex fistula-in-ano due to the high recurrent rate and following incontinence. Ligation of the intersphincteric fistula tract (LIFT), a novel surgical procedure with the advantage of avoiding anal incontinence, has a variable success rate of 57-94.4 %. Aim: To evaluate the long-term outcomes of modified LIFT operative procedure - ligation of intersphincteric fistula tract - to treat complex fistula-in-ano. Methods: Retrospective analysis of 62 cases of complex fistula-in-ano. The group was treated with the modified approach of LIFT (curved incision was made in the anal canal skin; purse-string suture was performed around the fistula; the residual fistulas were removed in a tunnel-based way) and had a follow-up time of more than one year. Patient´s preoperative general condition, postoperative efficacy and their anal function were compared. Results: The median age of the participants was 34, and 43 (69.4%) cases were male. Forty-one (66.1%) cases were of high transsphincteric fistula, four (6.5%) cases of high intrasphincter fistula, and 17 (27.4%) cases of anterior anal fistula in female. The median follow-up duration was 24.5 (range, 12-51) months. The success rate in the end of follow-up was 83.9% (52/62). The anorectal pressure and Cleveland Clinic Florida Fecal Incontinence (CCF-FI) evaluated three months before and after the operation did not find apparent changes. Conclusions: Compared with LIFT, the modified LIFT remarkably reduces postoperative failure and the recurrence rate of complex fistula with acceptable long-term outcomes.