Objective:To investigate the expression and clinical significance of long noncoding RNA (lncRNA) AP006284.1 in bladder cancer tissues, and to analyze the regulatory effect and downstream mechanism of AP006284.1 knockdown on the proliferation and migration of bladder cancer cells. Methods:The expression of AP006284.1 in bladder cancer tissues, and the relationship between AP006284.1 expression and tumor stage and disease-free survival of bladder cancer patients were analyzed in the gene expression omnibus (GEO) database. AP006284.1 gene expression in bladder cancer cell lines, including MGH-U3, T24, UMUC-3, J82 and 5637, and bladder epithelial immortalized SV-HUC-1 cell were detected by real-time reverse transcription-PCR (RT-qPCR) assay. J82 cells were divided into the control group and the transfection group, and transfected with control plasmid and AP006284.1 knockdown plasmid, respectively. The effect of AP006284.1 knockdown on the proliferation of J82 cells was detected by RT-qPCR and cell counting kit-8 (CCK-8) assay. The effect of AP006284.1 knockdown on the migration of J82 cells was determined by the scratch healing assay. The target gene of AP006284.1 was predicted by LncRNA2Target and LncRNome databases. The target fragment of wild-type AP006284.1 ( AP006284.1-Wt) or mutant AP006284.1 ( AP006284.1-Mut) was constructed into pGL3 plasmid by dual luciferase gene reporter assay. J82 cells were co-transfected with miR-205-3p or miR-negtive control (miR-NC) to validate the targeting relationship between AP006284.1 and miR-205-3p. The correlation between miR-205-3p and AP006284.1 expression in bladder cancer tissues was further analyzed by the GEO database. The effect of AP006284.1 knockdown on the expression of miR-205-3p gene in J82 cells was detected by RT-qPCR assay. The effect of AP006284.1 knockdown on the expression of phosphorylated Ras protein (p-Ras), phosphorylated Raf protein (p-Raf), phosphorylated mitogen-activated protein kinase kinase (p-MEK), and phosphorylated extracellular signal-regulated kinase (p-ERK) of the ERK pathway was detected by Western blotting in J82 cells. Results:GEO database analysis showed that the relative expression of AP006284.1 in bladder cancer tissues ( n=304) was significantly higher than that in normal tissues ( n=28, P<0.01). The relative expression of AP006284.1 was positively correlated with the tumor stage of the bladder cancer patients ( P<0.01). Compared with bladder cancer patients with low expression of AP006284.1, patients with high expression had a lower disease-free survival ( P<0.01). Compared with the SV-HUC-1 cell (1.02±0.34), the expression level of AP006284.1 gene was upregulated in MGH-U3 cell (5.77±0.37), T24 cell (3.02±0.40), UMUC-3 cell (3.62±0.59), J82 cell (7.19±0.24) and 5637 cell (5.59±0.30) (all P<0.01). The expression level of the AP006284.1 gene was the highest in J82 cells, therefore, the J82 cells were selected for the study. The expression level of AP006284.1 gene in the control group (7.20±0.26) was 6.92 times higher than that in the transfection group (1.04±0.28, t=16.16, P<0.01). Compared with the control group (0.74±0.11, 1.35±0.09, 1.63±0.14, 1.74±0.11), the absorbance ( A) values of J82 cells in the transfection group (0.49±0.06, 0.95±0.14, 1.09±0.08, 1.13±0.11) were reduced than those in the control group at the 24, 36, 48 and 60 h after AP006284.1 knockdown (all P<0.05). The migration distance of J82 cells in the control group was significantly longer than that in the transfection group. The migration rate of the control group [(65.03±6.20)%], which was 2.58 times higher than that of the transfection group [(25.22±3.45)%, t=5.61, P<0.01]. The target site of miR-205-3p containing AP006284.1 was predicted by LncRNA2Target and LncRNome databases. Compared with miR-NC group (1.00±0.11), the relative activity of dual luciferase of AP006284.1-Wt gene was significantly downregulated in the miR-205-3p group (0.31±0.07, t=5.47, P<0.01). Compared with the miR-NC group (0.97±0.14), the relative activity of dual luciferase of AP006284.1-Mut vector (0.98±0.07) was not significantly change ( t=0.09, P>0.05). The GEO database analysis showed that the expression of AP006284.1 in bladder cancer tissues was negatively correlated with the expression of miR-205-3p ( P<0.01). The expression level of miR-205-3p gene in the transfection group (5.42±0.24) was 5.21 times higher than that in the control group (1.04±0.40, t=9.40, P<0.01). Compared with the control group (3.22±0.17, 5.56±0.19, 4.38±0.17, 5.74±0.36), the expressions of p-Ras (2.33±0.12), p-Raf (1.61±0.20), p-MEK (1.57±0.25), and p-ERK (2.40±0.28) of the ERK pathway were decreased in the transfected J82 cells (all P<0.01). Conclusions:AP006284.1 is highly expressed in bladder cancer tissues. Knockdown of AP006284.1 can inhibit the proliferation and migration of bladder cancer cells by regulating the miR-205-3p and ERK pathway proteins.

ObjectiveTo study the clinical efficacy of dapagliflozin combined with Shexiang Baoxinwan （SXBXW） in the treatment of acute heart failure with reduced ejection fraction （HFrEF） and syndrome of Qi deficiency and blood stasis. MethodA total of 176 patients hospitalized due to acute HFrEF （syndrome of Qi deficiency and blood stasis） were selected and randomized into control group， SXBXW group， dapagliflozin group， and SXBXW + dapagliflozin group （the latter three groups were called the intervention groups）. The New York Heart Association （NYHA） class， 6-minute walk test （6MWT） score， Kansas City Cardiomyopathy Questionnaire （KCCQ） score， traditional Chinese medicine （TCM） syndrome score， N-terminal pro-brain natriuretic peptide （NT-proBNP）， soluble suppression of tumorigenicity 2 （sST2）， interleukin-6 （IL-6）， and hypersensitive C-reactive protein （hs-CRP） of the patients were evaluated and measured at the time of admission， 1 week after treatment， and 2 weeks of treatment. Furthermore， the hospital stay， in-hospital mortality， and 30-day re-admission rate were recorded. Statistical analysis was performed to evaluate the efficacy of each group. ResultAfter 1 week of treatment， the SXBXW group exhibited superior NYHA class， KCCQ score， TCM syndrome score and curative effect， IL-6， and hs-CRP to the control group （P<0.05， P<0.01）. After 2 weeks of treatment， the SXBXW group showed superior TCM syndrome score， TCM curative effect， and hs-CRP （P<0.05， P<0.01） to the control group. The dapagliflozin group was superior to the control group in terms of TCM syndrome score， NT-proBNP， and sST2 （P<0.05， P<0.01） after 1 week of treatment and in terms of NYHA class， KCCQ score， NT-proBNP， sST2， and hospital stay （P<0.05， P<0.01） after 2 weeks of treatment. The SXBXW + dapagliflozin group exhibited better efficacy than the control group in terms of NYHA class， 6MWT score， KCCQ score， TCM syndrome score and curative effect， NT-proBNP， sST2， IL-6， and hs-CRP （P=0.014） after 1 week of treatment and in terms of NYHA class， KCCQ score， TCM syndrome score and curative effect， NT-proBNP， sST2， IL-6， hs-CRP， and hospital stay （P<0.01） after 2 weeks of treatment. ConclusionSXBXW and dapagliflozin have good therapeutic effect on acute HFrEF and syndrome of Qi deficiency and blood stasis， and their combination demonstrated better therapeutic effect， with good safety and tolerability.