Long noncoding RNA CCAT2 functions as a competitive endogenous RNA to regulate FOXC1 expression by sponging miR-23b-5p in lung adenocarcinoma

Long noncoding RNA (lncRNA) may regulate the process of tumor formation. Although lncRNA CCAT2 has been identified as a key point in many diseases, its pathophysiological mechanism in lung adenocarcinoma remains unknown. We measured the expression level of CCAT2 in lung adenocarcinoma cells and normal lung epithelial cell line BEAS-2B by quantitative real-time polymerase chain reaction (qRT-PCR). As well, cell migration and proliferation were detected by transwell detection and CCK8 assay. At the same time, the new target point of CCAT2 was confirmed with bioinformatics analysis and dual-luciferase reporter assay. In addition, potential mechanisms were studied by Western blot analysis and RNA immunoprecipitation (RIP) analysis. The expression of CCAT2 was upregulated obviously in lung adenocarcinoma cells. Cell function analysis showed that upregulation of CCAT2 significantly promoted cell proliferation and migration, and reduction of CCAT2 inhibited cell migration and proliferation. In addition, CCAT2 positively regulated the expression of FOXC1 by competitive binding with miR-23b-5p. These findings indicated that CCAT2 may act as a competitive endogenous RNA (ceRNA) to regulate FOXC1 expression by competitively binding miR-23b-5p in lung adenocarcinoma.     

The diagnostic and prognostic value of pseudogene SIGLEC17P in lung adenocarcinoma and a preliminary functional study

Among malignant tumors, lung adenocarcinoma (LUAD) is the leading cause of death worldwide. This study explored the diagnostic, prognostic value, and preliminary functional verification of sialic acid binding Ig like lectin 17, pseudogene (SIGLEC17P) in LUAD. Prognostic lncRNAs for LUAD were identified by The Cancer Genome Atlas and quantitative real-time PCR (qRT-PCR) was used to detect the expression of SIGLEC17P in LUAD and paracarcinoma tissues. Subsequently, lentiviral vectors were used to overexpress SIGLEC17P in A549 and H1299 cells. The effects of SIGLEC17P overexpression on the proliferation, migration, and invasiveness of LUAD cells (A549 and H1299) were evaluated by Cell Counting Kit-8, wound healing, and transwell migration assays, respectively. Bioinformatics analyses were performed to reveal the potential pathways in which SIGLEC17P is involved in LUAD. qRT-PCR results revealed low SIGLEC17P expression in LUAD tissues and a significant association with the N stage, T stage, and tumor node metastasis stage. Furthermore, the receiver operating characteristic curve demonstrated a reliable diagnostic value. The proliferation, migration, and invasion of LUAD cells were inhibited by overexpression of SIGLEC17P. Bioinformatics analyses suggested that SIGLEC17P might exert antioncogenic effects in LUAD through the mir-20-3p/ADH1B or mir-4476-5p/DPYSL axis. In summary, our results revealed that SIGLEC17P acts as a prognostic biomarker, independent prognostic factor, and potential therapeutic target for patients with LUAD.     

余甘子提取物调控LINC01772/miR-153对肺癌细胞A549生物学行为的影响

摘要 目的：探讨余甘子提取物对肺癌细胞A549增殖、迁移和侵袭的影响及机制。方法：体外培养A549细胞,分为对照组、不同剂量（低、中、高剂量）余甘子提取物组、si-NC组、si-LINC01772组、高剂量余甘子提取物+pcDNA组和高剂量余甘子提取物+pcDNA-LINC01772组,细胞计数试剂盒（CCK-8）法和克隆形成实验检测细胞增殖,划痕实验检测细胞迁移,嵌入式细胞共培养法（Transwell）检测细胞侵袭,免疫印迹法（Western Blot）检测细胞中上皮型钙黏蛋白（E-cadherin）和神经型钙黏蛋白（N-cadherin）蛋白表达水平,实时荧光定量PCR（RT-qPCR）检测LINC01772和miR-153表达水平。双荧光素酶报告基因实验验证LINC01772和miR-153调控关系。结果：与对照组相比,不同剂量余甘子提取物组A549细胞中LINC01772表达降低,且光密度值（OD值）、克隆形成数、迁移以及侵袭细胞数减少（P<0.05）,而miR-153含量与E-cadherin蛋白表达升高（P<0.05）,且呈剂量依赖性（P<0.05）。LINC01772在A549细胞中负调控miR-153表达。与si-NC组相比,si-LINC01772组A549细胞增殖,侵袭及迁移能力受到抑制（P<0.05）。与高剂量余甘子提取物+pcDNA组相比,高剂量余甘子提取物+pcDNA-LINC01772组A549细胞增殖,侵袭及迁移能力增强（P<0.05）。结论：余甘子提取物可能通过调控LINC01772/miR-153轴抑制肺癌细胞A549增殖、迁移和侵袭,其可能通过下调LINC01772进而上调miR-153表达发挥作用,具有开发为治疗肺癌药物的潜在价值。     

Pseudogene HSPB1P1 contributes to renal cell carcinoma proliferation and metastasis by targeting miR-296-5p to regulate HMGA1 expression

With the aid of next-generation sequencing technology, pseudogenes have been widely recognized as functional regulators in the development and progression of certain diseases, especially cancer. Our present study aimed to investigate the functions and molecular mechanisms of HSPB1-associated protein 1 pseudogene 1 (HSPB1P1) in renal cell carcinoma (RCC). HSPB1P1 expression at the mRNA levels was determined by quantitative real-time polymerase chain reaction, and its clinical significance was assessed. Cell viability was detected by Cell Counting Kit-8 assay. Cell migration and invasion were detected by transwell assays. The location of HSPB1P1 in RCC cells was detected by subcellular distribution analysis. The direct relationship between HSPB1P1 and miR-296-5p/HMGA1 axis was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Our results identify the elevated expression of HSPB1P1 in RCC tissues and cell lines, which predicted advanced progression and poor prognosis in patients with RCC. Knockdown of HSPB1P1 suppressed cell proliferation, migration, and invasion, and reversed epithelial–mesenchymal transition process in RCC. HSPB1P1 was mostly enriched in the cytoplasm and functioned as a miRNA sponge for miR-296-5p and then regulated high-mobility group A1 expression. In conclusion, our study indicated that HSPB1P1 contributed to RCC progression by targeting the miR-296-5p/HMGA1 axis, and should be considered as a promising biomarker and therapeutic target for clinical applications.     

基于PTEN-PI3K-AKT信号通路探讨竹节参总皂苷抑制人肺腺癌A549细胞增殖和转移的机制

目的:竹节参是人参属植物,和人参成分相似,前期研究其对肺癌具有一定的抑制作用,但作用机制不清,因此,本项目拟研究竹节参皂苷对人肺癌细胞系A549增殖、迁移和侵袭能力以及PTEN-PI3K-AKT信号通路的影响。方法:CCK8法测定不同浓度和不同作用时间的竹节参皂苷对A549存活率的影响,划痕实验测定细胞迁移能力,Transwell小室测定细胞的侵袭能力,ELISA试剂盒测定培养基上清中MMP-2和MMP-9水平的变化。Western blot测定PTEN、P-PI3K和P-Akt表达的变化。结果:竹节参皂苷对A549细胞增殖具有明显的抑制作用,呈浓度和时间依赖关系,与对照组比较具有统计学差异。同时,竹节参皂苷可以浓度依赖性的抑制细胞侵袭和转移,以及MMP-2和MMP-9细胞因子的分泌。Western blot结果表明竹节参皂苷可促进PTEN蛋白表达,抑制P-PI3K和P-Akt蛋白表达,采用PTEN的特异性抑制剂SF1670证实竹节参皂苷通过抑制PTEN发挥作用。结论:竹节参皂苷可抑制肺癌A549细胞增殖、迁移和侵袭,以及分泌蛋白MMP-2和MMP-9表达,其作用机制可能是通过调控PTEN抑制PI3K和Akt磷酸化,从而发挥抗癌作用。     

Expression of periostin in the serum of NSCLC and its function on proliferation and migration of human lung adenocarcinoma cell line (A549) in vitro

Periostin is over expressed in many epithelial malignant cancers, including lung cancer, breast cancer, ovarian cancer and colon cancer. It is related with the progression and migration of breast and ovarian cancer cells in vitro. The aim of this study was to investigate the serum level of periostin in non-small cell lung cancer (NSCLC) and its relationship with established biological and prognostic factors by enzyme-linked-immunosorbent serologic assay. We also observe the function of periostin on the proliferation and migration of human lung adenocarcinoma cell line (A549) and discuss the mechanism. The mean value for serum periostin (POSTN) was elevated in NSCLC patients (242.84 ± 5.33 pg/ml) compared to the normal healthy volunteers (215.66 ± 11.67 pg/ml) (p = 0.030). The serum level of periostin of NSCLC patients had no connection with gender, age, pathological type, TNM stage, lymph node status, tumor size and invasiveness. We constructed a plasmid named pEGFP-N1/POSTN expressing full-length human periostin. Transfecting the plasmid to A549 cells and periostin was efficiently expressed in transfected A549 cells. Our data showed that periostin could promote the proliferation and migration of A549 cells by inducing vimentin and N-cadherin expression and downregulating E-cadherin expression. These results strongly suggest that periostin is a novel molecular which play an important role during the progression and development of NSCLC.     

TRB3 interacts with ERK and JNK and contributes to the proliferation,apoptosis, and migration of lung adenocarcinoma cells

Tribbles homolog 3 (TRB3) has been accounted for regulation of a few cell processes through interaction with other significant proteins. The molecular mechanisms underlying TRB3 in tumorigenesis in lung adenocarcinoma have not been entirely elucidated. The present study is aimed at determining the function and fundamental mechanisms of TRB3 in lung adenocarcinoma progression. TRB3 was highly expressed in A549 and H1299 cells and lung adenocarcinoma tissues compared with human bronchial epithelial cells (HBEpC) and adjacent normal lung tissues. Hypoxia significantly upregulated the expression of TRB3 protein in A549 and H1299 cells in a time-dependent way. Gene expression profiling interactive analysis data analysis indicated that patients with lung adenocarcinoma with excessive expression of TRB3 mRNA had fundamentally shorter survival time. TRB3 knockdown in A549 cells can inhibit cell proliferation and migration, and promote cell apoptosis. TRB3 knockdown reduced the expression of p-ERK and p-JNK, but did not affect the expression of p-P38 MAPK. TRB3 overexpression enhances the malignant transformation abilities of HBEpC such as cell proliferation, migration and colony formation, which could be reversed by U0126 and SP600125. TRB3 overexpression promotes the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but was not affected by U0126 and SP600125. The results of coimmunoprecipitation experiments indicated that TRB3 binds directly to ERK and JNK. This study suggests that TRB3 has a potentially carcinogenic role in lung adenocarcinoma by binding to ERK and JNK and promoting the phosphorylation of ERK and JNK. TRB3 can be a possible therapeutic focus for lung adenocarcinoma.     

Effects of polyphyllin I on growth inhibition of human non-small lung cancer cells and in xenograft

Polyphyllin I (PPI), a small molecular monomer extracted from Rhizoma of Paris polyphyllin, shows strong anticancer effects in previous study. Human lung adenocarcinoma A549 cells, human lung squamous cell carcinoma SK-MES-1 cells, and human lung large cell carcinoma H460 cells were cultured and then treated with PPI. Cell proliferation and apoptosis were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, flow cytometry, western blot analysis, and DNA ladder. Athymic nude mice bearing tumors were injected with PPI, and tumor growth was recorded. Our results showed that PPI significantly inhibited the proliferation of three non-small cell lung cancer (NSCLC) cell lines, with the inhibitory concentrations (IC50) of 1.24, 2.40, and 2.33 μg/ml for A549, H460, and SK-MES-1 cells, respectively. After being treated with 2.5 μg/ml of PPI for 24 h, the apoptotic rate of A549 cells was 39.68%, which was remarkably higher than that of the control. Tumor growth was significantly inhibited in the PPI-treated group compared with the group treated with cisplatin (DDP) or PBS in the nude mice. PPI exhibits antitumor ability in NSCLC cells in vitro and in vivo, which might be related to the apoptosis induced by PPI.     

IL-8对肺腺癌A549细胞迁移的影响及其机制探讨

探讨IL-8对肺腺癌A549细胞迁移的影响及其可能机制。用MTT法选择了合适的IL-8使用浓度。分别用划痕试验及Transwell试验证明了IL-8可以促进肺腺癌A549细胞的迁移。Westernblot结果表明:(1)IL-8可以促进MMP-2蛋白的表达,而对MMP-9的表达无明显影响;(2)IL-8可促进JNK/SAPK磷酸化蛋白的表达;(3)抑制剂(SP600125)可以阻断IL-8对MMP-2蛋白表达的影响。划痕试验从反面验证了低表达的MMP-2可以抑制A549细胞的迁移。表明IL-8可通过JNK/SAPK信号通路调控MMP-2蛋白的表达,进一步促进肺腺癌A549细胞的迁移。     

miR-670-5p对肺癌细胞增殖、迁移、侵袭的影响

目的: 探讨miR-670-5p对肺癌细胞增殖、迁移和侵袭的影响,分析其调控WW结构域氧化还原酶基因(WWOX)的机制。方法: 收集2016年1月至2017年10月收治的28例肺癌组织和对应癌旁组织,实时荧光定量PCR(RT-qPCR)检测肺癌组织、癌旁组织中miR-670-5p的表达水平。将肺癌细胞A549分为anti-miR-NC组(转染anti-miR-NC)、anti-miR-670-5p组(转染anti-miR-670-5p)、anti-miR-670-5p+si-NC组(转染anti-miR-670-5p与si-NC)、anti-miR-670-5p+si-WWOX组(转染anti-miR-670-5p与si-WWOX)。转染48 h后,RT-qPCR或蛋白质印记(Western blot)检测转染效果。细胞计数试剂盒(CCK-8)检测细胞活力;Transwell实验检测细胞迁移和侵袭能力;Western blot检测P21、上皮细胞钙粘蛋白(E-cadherin)和基质金属蛋白酶2(MMP-2)蛋白的表达水平。双荧光素酶报告基因实验和Western blot验证miR-670-5p和WWOX的靶向关系。结果: 肺癌组织中miR-670-5p的表达水平较癌旁组织显著升高(P＜0.05)。抑制miR-670-5p可抑制MMP-2蛋白表达(P＜0.05),促进P21和E-cadherin表达(P＜0.05),抑制A549细胞增殖、迁移和侵袭(P＜0.05)。WWOX是miR-670-5p的靶基因,miR-670-5p负调控WWOX表达。抑制WWOX可部分逆转anti-miR-670-5p对A549细胞增殖、迁移和侵袭的影响(P＜0.05)。结论: miR-670-5p通过靶向WWOX能够促进肺癌细胞增殖、迁移、侵袭。     

EGCG对LPS刺激人肺腺癌A549细胞凋亡及CUGBP1蛋白表达的影响

目的:研究表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate,EGCG)对炎性刺激的人肺腺癌A549细胞增殖和凋亡的影响及与CUGBP1表达的关系。方法:MTT法检测EGCG和LPS刺激A549细胞增殖活性的影响;流式细胞仪检测细胞凋亡;免疫细胞化学检测EGCG对LPS刺激人肺腺癌A549细胞内CUGBP1蛋白的表达。结果:与对照组相比,LPS体外显著促进A549细胞增殖,其胞核胞质内CUGBP1表达明显增强(P0.01)。加入EGCG可拮抗LPS促A549细胞增殖的作用,促进其凋亡,明显抑制LPS刺激的A549细胞内CUGBP1的表达(P0.01)。CUGBP1蛋白定量分析可知EGCG和LPS共同孵育A549细胞4h、24h时,细胞中的CUGBP1蛋白表达量较单纯LPS作用时降低。但EGCG和LPS共同孵育A549细胞24h,A549细胞中胞核CUGBP1蛋白表达量(1210.565±3.46)较4h时胞核CUGBP1蛋白表达量(67.344±3.68)高,差异有统计学意义(t=927.164,P0.001)。结论:EGCG可能通过干扰CUGBP1基因的表达抑制炎症刺激人肺腺癌细胞A549的增殖,促进其凋亡。     

柚皮素通过Notch1/Hes1通路抑制肺癌干细胞增殖、迁移和分化

为探讨柚皮素对肺癌干细胞增殖、迁移和分化的分子机制,本研究应用免疫磁珠法分选肺癌干细胞(A549-CSCs),并通过流式细胞术进行表面分子的鉴定;通过CCK8法检测不同浓度的柚皮素(25μg/m L,50μg/mL, 100μg/mL)对肺癌干细胞(A549-CSCs)活力的影响,Transwell检测柚皮素对A549-CSCs细胞迁移能力的影响,Q-PCR检测柚皮素对肺癌干细胞分化相关因子Sox2和Oct4 m RNA表达的影响,Western blotting法检测柚皮素对细胞内Notch1和Hes1蛋白表达的影响。流式细胞术检测结果显示,A549-CSCs细胞表面分子CD133呈阳性表达,符合肺癌干细胞特征。CCK8结果显示,与对照组(control)比较,25μg/m L、50μg/mL、100μg/mL柚皮素处理A549-CSCs 24 h,细胞活力显著降低(p<0.05);Transwell检测结果显示,与对照组比较,不同浓度柚皮素处理组A549-CSCs迁移能力显著降低(p<0.05);定量PCR (real-time polymerase chain reaction, Q-PCR)结果显示,与对照组比较,柚皮素处理组细胞Sox2和Oct4 m RNA表达水平显著降低(p<0.05);蛋白质印迹法(Western blotting)结果显示,与对照组相比柚皮素处理组细胞Notch1和Hes1蛋白表达水平均降低。本研究发现柚皮素可能通过抑制Notch1/Hes1通路抑制肺癌干细胞增殖、迁移和分化。这为柚皮素治疗肺癌提供临床依据。     

褪黑素对肺腺癌A549 细胞增殖及核转录因子Bp65 核移位的影响

目的:探讨褪黑素(MLT)对体外培养的肺腺癌A549细胞增殖的影响及作用机制。方法:体外培养人肺腺癌A549细胞,通过不同浓度的褪黑素(0.1、1.0、2.5、5.0mmol/L)干预24、48、72h,通过噻唑蓝(MTT)法测定细胞增殖,DNA末端原位标记染色法(Tunel)检测细胞凋亡情况,蛋白印迹(Western-blot)法检测褪黑素对A549细胞核内核转录因子Bp65(NF-Bp65)蛋白水平的影响。结果:褪黑素能够抑制人肺腺癌A549细胞增殖,呈剂量、时间依赖性性;高浓度褪黑素作用后凋亡细胞比例明显升高,同时细胞核内NF-Bp65蛋白量明显减少。结论:褪黑素能够呈剂量、时间依赖性抑制人肺腺癌A549细胞的增殖,抑制核因子Bp65的核移位诱导细胞凋亡是可能作用路径之一。     

Haishengsu, a Protein from Shellfish Tegillarca L. granosa, Inhibits the Growth and the Activity of Matrix Metalloproteinases-2 and -9 in Human Lung Carcinoma

Haishengsu (HSS) is a seashell protein extracted from Tegillarca L. granosa, a type of Malaysian shellfish. Previous in vitro studies showed that HSS might possess biological anticancer activity. In this combined in vitro and in vivo study, we investigated the inhibitory effects of HSS on tumor growth, invasion, and metastasis using human lung carcinoma cell lines A549 and NCI-H292, both intensely positive for matrix metalloproteinases-2 (MMP-2) and MMP-9. HSS significantly inhibited the proliferation of A549 and NCI-H292 as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The transwell chamber assay showed that HSS effectively blocked the invasion and migration of the carcinoma cells through the reconstituted extracellular matrix (Matrigel). Gelatin zymography analysis revealed that the secretion and activity of MMP-2 and MMP-9 in the supernatants of the cultured cells A549 and NCI-H292 were decreased after treatment with HSS. The levels of MMP-2 and MMP-9 in these cancer cells were further examined by Western blot assay in which a significant decrease of MMP-2 and MMP-9 was observed in A549 and NCI-H292 cells after 24 h of exposure to HSS. The anticancer activity of HSS was verified in a mouse model in which HSS delayed the growth of A549 xenografts after 3 weeks of oral administration. Inhibition of MMP-2 and MMP-9 expression was also demonstrated in the A549 xenografts as determined by Western blot analysis. These results suggest that HSS is a novel seashell protein that cannot only inhibit tumor growth but also prevent tumor invasion and metastasis through suppressing the activity of MMP-2 and MMP-9.     

维甲酸对A549细胞增殖和凋亡及Skp2、p27^kip1蛋白表达的影响

目的探讨维甲酸对A549细胞增殖和凋亡及相关基因表达的影响。方法MTT法观察ATRA对A549细胞增殖的抑制作用;流式细胞仪、AO/EB荧光双染法检测细胞凋亡;免疫细胞化学检测ATRA处理前后A549细胞Skp2、p27^kip1蛋白表达的情况。结果ATRA处理后①MTT法结果显示ATRA对A549细胞具有增殖抑制作用,在一定范围内呈时间-剂量依赖性。②AO/EB荧光双染色法观察到ATRA 25μmol/L作用A549细胞48h后,即可发现典型的凋亡形态学改变。③流式细胞仪结果出现凋亡峰,与对照组细胞相比,实验组细胞周期延长,主要表现为G0/G1期细胞比例增加,同时S期细胞比例减少。④免疫细胞化学结果显示,ATRA 25μmol/L处理细胞48h后,维甲酸处理组Skp2有明显下调,p27^kip1则明显上调。结论ATRA具有抑制肺腺癌A549细胞增殖,诱导细胞凋亡的作用,其机制可能与下调Skp2,上调p27^kip1蛋白的表达水平有关。     

Over-expression of LSD1 promotes proliferation, migration and invasion in non-small cell lung cancer

Background

Lysine specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics, but the pathological roles of its dysfunction in lung cancer remain to be elucidated. The aim of this study was to evaluate the prognostic significance of LSD1 expression in patients with non-small cell lung cancer (NSCLC) and to define its exact role in lung cancer proliferation, migration and invasion.

Methods

The protein levels of LSD1 in surgically resected samples from NSCLC patients were detected by immunohistochemistry or Western blotting. The mRNA levels of LSD1 were detected by qRT-PCR. The correlation of LSD1 expression with clinical characteristics and prognosis was determined by statistical analysis. Cell proliferation rate was assessed by MTS assay and immunofluorescence. Cell migration and invasion were detected by scratch test, matrigel assay and transwell invasion assay.

Results

LSD1 expression was higher in lung cancer tissue more than in normal lung tissue. Our results showed that over-expression of LSD1 protein were associated with shorter overall survival of NSCLC patients. LSD1 was localized mainly to the cancer cell nucleus. Interruption of LSD1 using siRNA or a chemical inhibitor, pargyline, suppressed proliferation, migration and invasion of A549, H460 and 293T cells. Meanwhile, over-expression of LSD1 enhanced cell growth. Finally, LSD1 was shown to regulate epithelial-to-mesenchymal transition in lung cancer cells.

Conclusions

Over-expression of LSD1 was associated with poor prognosis in NSCLC, and promoted tumor cell proliferation, migration and invasion. These results suggest that LSD1 is a tumor-promoting factor with promising therapeutic potential for NSCLC.     

Claudin-2 knockdown decreases matrix metalloproteinase-9 activity and cell migration via suppression of nuclear Sp1 in A549 cells

AimsClaudin expression is altered in lung cancer, but the pathophysiological role of claudin is not well understood. We examined the effect of claudin-2 expression on cell migration using human adenocarcinoma A549 cells.Main methodsThe mRNA level was measured by real time polymerase chain reaction. To knockdown claudin-2 expression, we made the cells expressing doxycycline-inducible claudin-2 shRNA vector. The protein level was examined by Western blotting. Cell migration was measured by wound-healing assay. The enzymatic activity of MMP-9 was assessed by gelatin zymography.Key findingsIn A549 cells, claudin-2 expression was higher than in normal lung tissue. Claudin-2 knockdown did not affect the expression of other junctional proteins including claudin-1, occludin and E-cadherin. Claudin-2 knockdown decreased cell migration concomitant with a decrease in the mRNA level and enzymatic activity of MMP-9. The expression level of Sp1 in the nuclei was decreased by claudin-2 knockdown. In contrast, the expression levels of c-Fos, c-Jun and NF-kB p65 in the nuclei were not changed by claudin-2 knockdown. The knockdown of Sp1 expression by siRNA decreased cell migration, and the mRNA expression, enzymatic activity, and promoter activity of MMP-9.SignificanceClaudin-2 may increase the mRNA level and enzymatic activity of MMP-9 mediated by the elevation of nuclear distribution of Sp1, resulting in the up-regulation of A549 cell migration.     

靶向AXL克服肺腺癌EGFR-TKIs获得性耐药

目的:探索AXL在肺腺癌细胞(Lung adenocarcinoma cell, LAC)EGFR-TKIs获得性耐药中的作用,为肺癌临床治疗和新型药物的研发提供实验依据。方法:构建EGFR-TKIs获得性耐药的肺腺癌模型并通过CCK-8法检测耐药株对肺腺癌靶向治疗药物吉非替尼(Gefitinib)、厄洛替尼(Erlotinib)和奥希替尼(Osimertinib)的敏感性。基于基因组学分析筛选出潜在的克服耐药的靶点AXL,通过Western blot和qRT-PCR技术检测AXL的表达情况,并同时检测上皮-间质转化(Epithelial-mesenchymal transition,EMT)分子标志物。R428是AXL的小分子抑制剂,通过CCK-8法、Transwell以及划痕实验等探究靶向AXL对肺腺癌亲本及耐药株增殖和迁移能力的影响。结果:AXL在构建的耐药株中显著高表达,其蛋白表达水平上调15-20倍(P0.001),m RNA水平上调2-5倍(P0.01);EGFR-TKIs耐药株发生上皮间质转化(EMT);靶向AXL选择性抑制耐药株的增殖能力并且恢复了耐药株对EGFR-TKIs的敏感性(P0.001);靶向AXL显著抑制耐药株增强的迁移能力,与亲本株相比最高抑制率可达80%左右(P0.001)。结论:用遗传学和药理学手段靶向AXL可以显著逆转肺腺癌对EGFR-TKIs耐药,逆转耐药株所增强的迁移等肿瘤生物学特征,对克服EGFR-TKIs获得性耐药有着重要的临床治疗价值以及转化医学前景。     

DUSP12 ameliorates myocardial ischemia–reperfusion injury through HSPB8-induced mitophagy

This study aimed to explore the role of dual specificity phosphatase 12 (DUSP12) in regulating myocardial ischemia–reperfusion (I/R) injury and the underlying mechanism. The expression of DUSP12 in myocardial tissues and heat-shock protein beta-8 (HSPB8) and mitophagy-related proteins in myocardial tissues and H9c2 cells were detected by western blot analysis. The serum creatine kinase isoenzymes (CK-MB) and lactate dehydrogenase (LDH), levels of reactive oxygen species and malondialdehyde, superoxide dismutase activity in myocardial tissues and H9c2 cells, and caspase-3 activity in H9c2 cells were analyzed by corresponding assay kits. The infarct area in the rat's heart was observed by triphenyl tetrazolium chloride staining. The apoptosis of myocardial cells in myocardial tissues and H9c2 cells was detected by terminal-deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay. The interaction between DUSP12 and HSPB8 was clarified by the coimmunoprecipitation assay. The transfection efficacy of si-HSPB8#1 and si-HSPB8#2 in H9c2 cells was confirmed by real-time quantitative-polymerase chain reaction and western blot analysis. As a result, DUSP12 expression was downregulated in I/R rats, which was promoted by lentivirus-expressing DUSP12. DUSP12 overexpression reduced the serum creatine kinase isoenzymes (CK-MB) and LDH, decreased the infarct area in the rat's heart, and suppressed the apoptosis and oxidative stress in myocardial tissues. DUSP12 overexpression also upregulated the expression of HSPB8 to promote mitophagy. The coimmunoprecipitation assay indicated that DUSP12 could be combined with HSPB8. In addition, DUSP12 overexpression could inhibit hypoxia/reoxygenation-elicited apoptosis as well as oxidative stress in H9c2 cells by upregulating HSPB8 expression to promote mitophagy, which was countervailed by HSPB8 deficiency. In conclusion, DUSP12 overexpression decreased the apoptosis and oxidative stress in myocardial I/R injury through HSPB8-induced mitophagy.