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Objective:To investigate effects of long non-coding growth stasis specific protein 6 antisense RNA1 (lncRNA DLX6-AS1) on the proliferation, migration and invasion of a cutaneous squamous cell carcinoma cell line A431, and to explore the underlying mechanisms.Methods:A dual-luciferase reporter system was used to verify the targeting relationship between lncRNA DLX6-AS1 and miR-16-5p, as well as between miR-16-5p and nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) mRNA. Cultured A431 cells were divided into several groups: si-DLX6-AS1 group and DLX6-AS1-NC group transfected with lncRNA DLX6-AS1 inhibitor and its negative control respectively; anti-miR-16-5p group and anti-miR-NC group transfected with miR-16-5p inhibitor and its negative control respectively; si-NUCKS1 group and NUCKS1-NC group transfected with NUCKS1 inhibitor and its negative control respectively; si-DLX6-AS1+ anti-miR-16-5p group transfected with lncRNA DLX6-AS1 inhibitor followed by miR-16-5p inhibitor, and si-DLX6-AS1+ anti-miR-NC group transfected with lncRNA DLX6-AS1 inhibitor followed by anti-miR-NC; si-DLX6-AS1+ anti-miR-16-5p+ si-NUCKS1 group transfected with lncRNA DLX6-AS1 inhibitor, miR-16-5p inhibitor and NUCKS1 inhibitor, and si-DLX6-AS1+ anti-miR-16-5p+ NUCKS1-NC group transfected with lncRNA DLX6-AS1 inhibitor, miR-16-5p inhibitor and NUCKS1-NC. After the above treatment, real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure the mRNA expression of lncRNA DLX6-AS1, miR-16-5p and NUCKS1 in A431 cells, Western blot analysis to determine the protein expression of NUCKS1, Cyclin D1 antibody, matrix metalloproteinase (MMP) 2 and MMP9, cell counting kit-8 (CCK8) assay to detect cell survival rate, and Transwell assay to evaluate cell migratory and invasive abilities. Two-independent-sample t test was used for comparisons between two groups. Results:Dual-luciferase reporter assay showed targeted binding of lncRNA DLX6-AS1 to miR-16-5p, as well as of miR-16-5p to NUCKS1. Compared with the DLX6-AS1-NC group, the si-DLX6-AS1 group showed significantly increased miR-16-5p expression in A431 cells (3.01 ± 0.31 vs. 1.02 ± 0.10, t = 18.33, P < 0.001) , but significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 (all P < 0.05) , and significantly decreased cell survival rate and numbers of migratory and invasive cells (all P < 0.05) . Compared with the NUCKS1-NC group, the si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells (all P < 0.05) , and significantly decreased cell survival rate and numbers of migratory and invasive cells (all P < 0.05) . After inhibition of lncRNA DLX6-AS1 expression, the si-DLX6-AS1+ anti-miR-16-5p group showed significantly decreased miR-16-5p expression in A431 cells (0.34 ± 0.04) compared with the si-DLX6-AS1+ anti-miR-NC group (1.00 ± 0.12, t = 15.65, P < 0.05) , but significantly increased protein expression of Cyclin D1, MMP2 and MMP9, cell survival rate and numbers of migratory and invasive cells compared with the si-DLX6-AS1+ anti-miR-NC group (all P < 0.05) . After inhibition of lncRNA DLX6-AS1 expression and knockdown of miR-16-5p, the si-DLX6-AS1+ anti-miR-16-5p+ si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells, as well as cell survival rate and numbers of migratory and invasive cells, compared with the si-DLX6-AS1+ anti-miR-16-5p+ NUCKS1-NC group (all P < 0.05) . Conclusion:lncRNA DLX6-AS1 can regulate the proliferation, migration and invasion of A431 cells by targeting miR-16-5p/NUCKS1, suggesting that lncRNA DLX6-AS1 may be a potential molecular target for the treatment of cutaneous squamous cell carcinoma.
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Objective:To evaluate the effects of long non-coding RNA (lncRNA) LEF1-AS1 on proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, and to explore their mechanisms.Methods:Cutaneous squamous cell carcinoma SCC13 cells were divided into si-LEF1-AS1 group transfected with lncRNA LEF1-AS1 interference oligonucleotides (si-LEF1-AS1) , si-NC group transfected with lncRNA LEF1-AS1 nonsense oligonucleotides (si-NC) , miR-612 group transfected with miR-612-overexpressing oligonucleotides, miR-NC group transfected with miR-612 nonsense oligonucleotides (miR-NC) , si-LEF1-AS1+anti-miR-612 group transfected with si-LEF1-AS1 and oligonucleotides against miR-612, and si-LEF1-AS1+anti-miR-NC group transfected with si-LEF1-AS1 and miR-612 nonsense oligonucleotides. Quantitative reverse transcription (qRT) -PCR was performed to determine the relative expression of miR-612 in SCC13 cells, cell counting kit-8 (CCK8) assay to evaluate cellular proliferative activity, flow cytometry to detect cell apoptosis, Transwell assay to assess migratory and invasive abilities of SCC13 cells, and Western blot analysis to determine protein expression of cyclin-dependent kinase 1 (cyclinD1) , cyclinD1 inhibitor p21, Bcl-2 family protein (Bcl-2) , Bcl-2 related X protein (Bax) , matrix metalloproteinase 2 (MMP-2) and MMP-9. The online bioinformatics database LncBase predicted v.2 was employed to predict the complementary sequence between lncRNA LEF1-AS1 and miR-612, and luciferase reporter gene plasmids were constructed by using the complementary/non-complementary sequence, which were co-transfected with miR-612-overexpressing oligonucleotides (miR-612 overexpression group) or miR-NC (overexpression control group) into SCC13 cells in order to verify the binding ability of lncRNA LEF1-AS1 to miR-612. Statistical analysis was carried out by using t test for comparison between two groups, one-way analysis of variance for comparison among multiple groups, and least significant difference (LSD) - t test for multiple comparisons. Results:Compared with the miR-NC group, miR-612 group showed significantly decreased cellular proliferative ability, number of migratory cells and invasive cells (all P < 0.05) , but a significantly increased apoptosis rate ( P < 0.05) . The relative expression of miR-612 ( F = 150.78, P < 0.001) , cellular proliferative activity at 24, 48, 72 hours (all P < 0.05) , apoptosis rate and number of migratory and invasive cells (all P < 0.05) significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1+anti-miR-612 group and si-LEF1-AS1+anti-miR-NC group. Compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased expression of miR-612 and apoptosis rates, but significantly decreased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells (all P < 0.05) ; compared with the si-LEF1-AS1+anti-miR-NC group, the si-LEF1-AS1+anti-miR-612 group showed significantly decreased expression of miR-612 and apoptosis rates, but significantly increased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells (all P < 0.05) . Western blot analysis showed that the relative protein expression of cyclinD1, p21, Bcl-2, Bax, MMP-2 and MMP-9 significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1+anti-miR-612 group and si-LEF1-AS1+anti-miR-NC group (all P < 0.001) ; compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax (all P < 0.05) ; compared with the si-LEF1-AS1+anti-miR-NC group, the si-LEF1-AS1+anti-miR-612 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax (all P < 0.05) . After co-transfection with complementary sequences, the fluorescence activity was significantly lower in the miR-612 overexpression group than in the overexpression control group ( t = 21.19, P < 0.001) ; after co-transfection with non-complementary sequences, no significant difference was observed in the fluorescence activity between the miR-612 overexpression group and overexpression control group ( t = 0.28, P = 0.78) . Conclusion:lncRNA LEF1-AS1 regulates the proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, likely by targeting miR-612.
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Objective To discuss the application and effect of predictive care in stage divided treatment and salvage of children's persistent status of epilepsy (SE).Methods Sixty-four children with SE were admitted to the First Peoples Hospital of Wenling in Zhejiang Province from December 2014 to December 2016 as the research objects, a controlled study was adopted, in which the stage divided therapeutic method was compared with the conventional method for treatment of SE, and the children were divided into an observation group and a control group, 32 cases in each group. Foresight treatment method was used in the observation group: the first stage of treatment (early treatment or pre-hospital treatment) was intravenous injection of the first-line drugs (benzodiazepines); the second stage of treatment (progressive stage treatment or initial treatment) mainly used epileptic drugs to prevent recurrence; the third stage treatment (referred to after the first-line drug in combination with one second line antiepileptic drug treatment, the disease was still unable to be controlled) adopted the mild hypothermia therapy to reduce the SE attack amplitude and decrease recurrence; in terms of aspects of predictive nursing measures, the respiratory tract nursing, basic nursing, the prediction of complications and their nursing, etc. were implemented. The routine nursing without care about the stages was carried out in the control group. The SE control time, skin lesions, false aspiration, lung infection, tongue bite, cerebral edema, etc. the incidenceof complications were observed in two groups of children.Results The SE control time was significantly shorter in observation group than that in the control group (minutes: 18.13±3.15 vs. 25.19±2.69,P < 0.05), and the incidence of complications was obviously lower in observation group than that in the control group [6.25% (2/32) vs. 28.13% (9/32),P < 0.05]. Conclusion Using different therapy at different stages of children SE combined with predictive care can effectively shorten the SE control time, decrease the incidence of complications and elevate the therapeutic and nursing effects.
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OBJECTIVE:To investigate clinical efficacy and safety of Rosuvastatin calcium tablets combined with Qishen yiqi dripping pills in the treatment of chronic heart failure (CHF) and it effects on inflammatory level,oxidative stress injury and cardiac function of patients.METHODS:Ninety CHF patients in our hospital during Aug.2014-Apr.2016 were divided into observation group and control group according to random number table,with 45 cases in each group.Control group received routine anti-heart failure treatment as cardiac,diuretic,dilating vessel,and Qishen yiqi dripping pills orally 0.5 g,half an hour after meal,tid;observation group was additionally given Rosuvastatin calcium tablet orally 20 mg,at bedtime,qd,on the basis of control group.Cardiac function,serum inflammatory factor,BNP and oxidative stress levels were compared between 2 groups before and after treatment.Clinical efficacies and the occurrence of ADR were observed in 2 groups.RESULTS:Before treatment,there was no statistical significance in LVEF,LVESD,LVEDD,TNF-α,IL-6,BNP,SOD,MPO and MMP-9 levels between 2 groups (P＞0.05).After treatment,LVEF of 2 groups were increased significantly,while LVEDD,LVESD,TNF-α and IL-6,BNP levels were decreased significantly;the above indexes of observation group was significantly better than those of control group,with statistical significance (P＜0.05).MPO and MMP-9 of observation group were significantly decreased,while SOD level was significantly increased and better than that of control group,with statistical significance (P＜0.05),but there was no statistical significance in SOD and MPO,MMP-9 levels of control group before and after treatment (P＞0.05).The clinical response rate of observation group was 97.8％,which was significantly higher than 82.2％ of control group,with statistical significance (P＜0.05).There was no statistical significance in the incidence of ADR between 2 groups (P＞0.05).CONCLUSIONS:Rosuvastatin calcium tablets combined with Qishen yiqi dripping pills show significant therapeutic efficacy for CHF,can effectively reduce inflammatory level,relieve oxidant stress injury,delay the process of ventricular remodeling,and improve cardiac function with good safety.
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Objective To investigate the influence of parental compliance on the therapeutic effect of children with epilepsy.Methods Two hundred and sixty children with epilepsy and their parents admitted to the First People's Hospital of Wenling from December 2013 to June 2016 were enrolled,and the classical Morisky medication adherence questionnaire (MMAS-8) was applied to evaluate the compliance of parents for treatment of their children with epilepsy;after the patient taking drug for 3 days,fasting venous blood was collected in the morning,the concentration of the blood drug was tested and the influence of parent compliance on the blood drug concentration of the child with epilepsy was evaluated.Results In 260 patients,122 cases took karma form,and 138 cases took valproate orally.There were parents with good medication compliance in 130 cases (50％),medium medication compliance 80 cases (30.76％) and poor adherence to the doctor order in 50 cases (19.23％).In cases using medication irregularly,there were 26 cases sometimes without taking any drug (10.0％),17 patients' medication being interrupted (6.54％) and 10 cases having excessive medication (3.85％);no relationships were found between parental compliance and each of the following items,family role,occupation and age (all P ＞ 0.05);and the compliance was related to gender,indicating that women's good compliance level was higher than that of males';the education level was positively proportional to the compliance,and the compliance of parents with senior high school or above degree was higher than those with primary school and junior secondary school levels (83 cases vs.9 cases,38 cases,both P ＜ 0.01).Under situation of parents with poor compliance,their children had blood drug concentration higher or lower than proper range of drug level (high in 22 cases,low in 41 cases,higher than the result in good compliance 0 cases and 17 cases respectively),thus seriously affected the safety and efficacy of the treatment;the patients' frequency of irregular medication in parents' good compliance group was significantly lower than that in parents' poor compliance group [3.08％ (4/130) vs.72.0％ (36/50),P ＜ 0.05].Conclusion To improve the therapeutic effect of epileptic children,their parental good cooperation is necessary.