BI-1蛋白及其相关调控因子在非小细胞肺癌细胞系中的表达研究

目的 研究凋亡抑制因子BI-1与及其相关调控因子Bcl-2 、Bcl-XL在非小细胞肺癌细胞系中的表达差异，分析BI-1与Bcl-2、Bcl-XL的相互作用关系；方法 以9种非小细胞肺癌细胞系（NSCLC）为实验材料，采用RT-PCR和western blot 技术在cDNA和蛋白水平检测三种基因的表达；结果 发现BI-1在NSCLC中高表达，Bcl-2表达较低（在A549、PAa、GLC-82和SPC-1-A几乎检测不到），Bcl-XL表达较稳定，且明显高于Bcl-2；结论 在NSCLC细胞中，BI-1主要与Bcl-XL形成蛋白复合体作用于凋亡通路。     

Effects of long non-coding RNA (lncRNA) cancer susceptibility candidate 2c (CASC2c) on proliferation,metastasis and drug resistance of non-small cell lung cancer (NSCLC) cells through ERK1/2 and β-catenin signaling pathways

ObjectivesThis study was aimed to investigate the effects of long non-coding RNA (lncRNA) cancer susceptibility candidate 2c (CASC2c) on the proliferation, metastasis and drug resistance of non-small cell lung cancer (NSCLC) cells.MethodsThe expression of CASC2c in NSCLC tissues and cell lines was detected by real-time fluorescence quantitative PCR (RT-qPCR). MTT and Transwell assay were used to determine the proliferation and migration of NSCLC cells in the experimental group and the control group respectively. The drug sensitivity test was used to confirm whether increasing the CASC2c expression level could reverse the resistance of NSCLC cells to the chemotherapy drug cisplatin. The effects of CASC2c on the expression levels of p-ERK1/2 and β-catenin were detected by western blot.ResultsThe results of RT-qPCR showed that CASC2c was under-expressed in NSCLC tissues and cells compared with normal adjacent lung tissues cells (p < 0.05). In addition, the CASC2c expression was remarkably correlated with TNM staging, tumor cell differentiation, lymph node metastasis, smoking and other pathological indicators of patients with NSCLC (p < 0.05). MTT and Transwell assay showed that the high-expression of CASC2c significantly reduced the proliferation and migration of NSCLC cells compared to that of the control group (p < 0.05). Western blot assay showed that the high-expressed CASC2c can decrease the expression of phosphorylated-ERK1/2 (p-ERK1/2) and β-catenin.Conclusions: CASC2c was low expressed in NSCLC tissues and cells. What's more, it inhibited the proliferation and migration of NSCLC cells by inhibiting the expression of p-ERK1/2 and β-catenin and reversed NSCLC cells’ resistance to the chemotherapy drug cisplatin. Therefore, CASC2c may serve as a new biomarker and therapeutic target in the diagnosis and treatment of NSCLC.     

Over-expression of Orai1 mediates cell proliferation and associates with poor prognosis in human non-small cell lung carcinoma

Orai1 and STIM1 mediate calcium release-activated calcium current (CRAC) which is the best characterized store-operated calcium current involving in a wide range of cell progresses, such as cell proliferation, metastasis, apoptosis. Orai1 has been studied as a carcinogenic biomarker in some cancers such as esophageal cancer. However, its function and clinical significance in non-small cell lung cancer (NSCLC) have not been well studied. The present study was aimed at discussing the relationship between Orai1 and lung cancer malignant behavior with its clinical significance. We used quantitative real-time-PCR and Western blot to detect the expression of Orai1 in NSCLC cell lines and fresh cancer tissues. Immunohistochemistry were performed to test the location and expression of Orai1 in paraffin sections. We found that Orai1 was markedly overexpressed in both NSCLC cell lines and fresh cancer tissues. Immunohistochemistry data also revealed that overexpression of Orai1 was present in 42.4% of NSCLC tissues, compared with the corresponding adjacent nontumorous tissues. Furthermore, NSCLC patients with high Orai1 expression survived shorter than those with low Orai1 expression. In addition, when knockdown Orai1 by RNAi technic, we found the PI3k/AKT/ERK pathway was inhibited which may indicated that Orai1 could influence cell proliferation. Taken together, our study demonstrated that Orai1 was remarkably overexpressed in NSCLC and could be served as a potential prognostic marker for patients with this deadly disease.     

miR-106a promotes growth and metastasis of non-small cell lung cancer by targeting PTEN

MicroRNAs are a class of small non-coding RNAs that play essential roles in cancer development and progression. Recent studies suggested that abnormal expression of miRNAs occurs frequently in non-small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues. In this study, we investigated the expression and the biological roles of miR-106a in non-small cell lung cancer. Our results showed that miR-106a was up-regulated in NSCLC tissues and cell lines. Inhibition of miR-106a in NSCLC cells substantially inhibited cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a direct target of miR-106a, and over-expression of miR-106a suppressed PTEN by direct binding to its 3’-untranslated region (3’-UTR). Furthermore, the presence of miR-106a was inversely correlated with PTEN in NSCLC tissues. Overall, this study suggested that miR-106a inhibited the growth and metastasis of NSCLC cells by decreasing PTEN expression. These data provide novel insights with potential therapeutic applications for the treatment of NSCLC.     

Expression and functional analysis of Bax inhibitor-1 in human breast cancer cells

Recently, deregulated expression of the anti-apoptotic protein Bax inhibitor-1 (BI-1) has been shown in several human cancers. In this report, we show that BI-1 is expressed at various levels in six different human breast cancer cell lines. In order to investigate the function of BI-1 in oestrogen-dependent MCF-7, T-47D and oestrogen-independent MDA-MB-231 breast cancer cells, the RNA interference technique was used to knock down BI-1 expression specifically. Suppression of BI-1 expression caused a significant increase in spontaneous apoptosis in MDA-MB-231 cells, whereas MCF-7 and T-47D cells remained almost unaffected. Furthermore, BI-1 expression analysis using a cancer profiling array showed up-regulation of BI-1 expression in cancer samples of breast, uterus and ovary, whereas down-regulated BI-1 expression was identified in stomach, colon, kidney, lung and rectal cancer. In addition, immunohistochemical studies using a BI-1-specific antibody on human breast cancer specimens also revealed that BI-1 is expressed in the majority of cases. Moreover, to analyse whether BI-1 expression is oestrogen receptor-dependent, tumour cells were treated with oestradiol, ICI and tamoxifen: this showed no significant changes in BI-1 expression. Taken together, our results demonstrate that BI-1 expression is differentially deregulated in different cancers and that BI-1 plays an important role in preventing certain breast cancer cells from undergoing apoptosis. Thus, the development of novel therapeutic strategies based on targeting BI-1 gene expression in breast cancer could be restricted to selected individual cancer types.     

miR-185 acts as a tumor suppressor by targeting AKT1 in non-small cell lung cancer cells

Increasing evidence has shown that microRNAs play critical roles in the initiation and progression of non-small cell lung cancer (NSCLC). miR-185 is deregulated in various cancers, whereas its functional mechanism in NSCLC is still unclear. Here, we confirmed that the expression of miR-185 was significantly down-regulated in NSCLC tissues and cell lines. miR-185 over-expression caused significant suppression of in vitro cell proliferation, migration and invasion, and in vivo tumor growth. We subsequently identified that AKT1 was a target gene of miR-185. Re-expression of AKT1 could partially rescue the inhibitory effects of miR-185 on the capacity of NSCLC cell proliferation and motility. Collectively, we conclude that miR-185 has a critical function by blocking AKT1 in NSCLC cells, and it may be a novel therapeutic agent for miRNA based NSCLC therapy.     

Inhibition of SALL4 suppresses carcinogenesis of colorectal cancer via regulating Gli1 expression

Background: SALL4 is a novel oncogene mediating tumorigenesis in multiple carcinomas. However, its actual role and mechanisms participating in the development of colorectal cancer remains unclear. Methods: Immunohistochemical staining and Western blot were conducted to detect the expression of SALL4 and other molecules. siRNA of SALL4 was transfected to silence SALL4 expression in Caco-2 cell line. Flow cytometry was used for cell cycle and apoptosis analysis. Wound healing and transwell assay were used for invasion test. CCK-8 test was employed for cell proliferation and drug sensitivity assessment. Results: By inhibition of SALL4 expression, the proliferation, invasiveness and drug resistance were dramatically reduced while apoptosis rate was up-regulated. Gli1 was found to decrease its expression in SALL4 silencing cells. Moreover, the inhibition on tumorigenesis of Caco-2 by SALL4 silencing was antagonized by Gli1 up-regulation, suggesting Gli1 as a downstream target of SALL4 in cancer development. Conclusion: SALL4 inhibition limited oncogenesis on colorectal cancer by reducing Gli1 expression.     

X射线诱导NSCLC组织Axin表达并通过p53或JNK途径促进细胞凋亡

目的研究X-射线对非小细胞肺癌(non-smallcelllungcancer,NSCLC)组织中Axin表达的作用,以及X射线上调Axin表达的机制。方法应用Westernblot、RT-PCR方法检测15例经过X射线照射后NSCLC组织中Axin在蛋白水平及RNA水平的表达变化情况以及caspase-3活化情况。转染Axin及AxinΔp53ΔHIPK2至A549、BE1细胞中,并施加p53或JNK抑制剂,细胞经X线照射后用流式细胞仪(FCM)检测细胞凋亡,研究Axin上调细胞凋亡的机制。结果经X射线照射后,在15例NSCLC肺癌组织中,有8例Axin在RNA水平和蛋白水平上表达上调,与Axin未上调组相比,细胞凋亡显著增加(P<0.05)。转染Axin能使A549、BE1细胞凋亡明显增加,AxinΔp53ΔHIPK2不能上调A549细胞凋亡,p53抑制剂和JNK抑制剂分别抑制A549和BE1细胞凋亡。结论 X射线诱导部分NSCLC组织Axin表达增加并促进细胞凋亡,Axin通过p53或JNK途径促进X射线诱导的细胞凋亡。检测NSCLC组织中是否存在p53基因突变并不能作为判定是否对X射线敏感的指标。     

Enhanced expression of stem cell markers and drug resistance in sphere-forming non-small cell lung cancer cells

There is growing evidence suggesting that cancer stem cells (CSCs) are playing critical roles in tumor progression, metastasis and drug resistance. However, the role of CSCs in non-small cell lung cancer (NSCLC) remains elusive. In this study, we enriched for stem-like cells from tumor spheres derived from NSCLC cell line A549 cultured in serum-free medium. Our results showed that sphere-derived cells expressed various stem cell markers such as CD44, CD133, Sox2 and Oct4. Compared with the corresponding cells in monolayer cultures, sphere-derived cells showed marked morphologic changes and increased expression of the stem cell markers CD133. Furthermore, we found that sphere-derived cells exhibited increased proliferation, cell-cycle progression as well as drug-resistant properties as compared to A549 adherent cells. Consistently, expression of several drug resistance proteins, including lung resistance-related protein (LRP), glutathion-S-transferase-π (GST-π) and multidrug resistance proteins-1 (MRP1) were all significantly enhanced in sphere-derived cells. These results indicate the enrichment of CSCs in sphere cultures and support their role in regulating drug resistance in NSCLC.     

非小细胞肺癌中凋亡抑制基因Bcl-2的转录表达研究

目的 探讨Ｂｃｌ－２这度转录表达在人非小细胞肺癌发生、发展中的作用。方法 采用Ｎｏｒｔｈｅｒｎ印迹杂交技术和非放射性地高辛标记检测系统,检测了１３７份不同部位、不同性质的人肺组织中的Ｂｃｌ－２基因的ｍＲＮＡ表达。结果 肺组织中Ｂｃｌ－２ ｍＲＮＡ的表达有从良性病变组织、远离癌灶的肺组织、癌旁组织到癌灶组织逐渐增高的趋势;其中,肺癌癌灶组织中Ｂｃｌ－２ ｍＲＮＡ的表达较肺良性病变组织和远离癌灶的肺组     

An Orthotopic Model of Lung Cancer to Analyze Primary and Metastatic NSCLC Growth in Integrin α1-Null Mice

The role of matrix metalloproteinase (MMP)9 in lung cancer progression is controversial. MMP9 promotes local tumor progression and distant metastasis in mouse models by enhancing extracellular matrix degradation, releasing VEGF from extracellular matrix and promoting vascular pericyte recruitment. Furthermore, increased plasma MMP9 expression levels in human subjects with metastatic non-small cell lung cancer (NSCLC) inversely correlates with survival. In contrast, MMP9 can benefit the host by generating inhibitors of endothelial cell proliferation such as angiostatin and NC1 domains of collagen IV. To better understand the role of host MMP9 on the primary growth and metastatic potential of NSCLC, we performed an orthotopic model of NSLC in integrin α1-null mice (a genetic model for increased MMP9). In these mice we observed decreased number, size and vascularization of primary NSCLC tumors when compared to wild type controls. In addition, decreased number and size of NSCLC-derived metastases were evident in the α1-null mice. Furthermore, pharmacological inhibition of MMPs in the α1-null mice at the time of tumor cell injection resulted in an increase in the number of both primary and metastatic lung cancer as compared to untreated mice, suggesting that primary growth and metastases of NSCLC are worsened by the early inhibition of MMPs. In conclusion, although MMP9 may potentially promote tumor growth and metastasis, production of MMP-dependent anti-angiogenic factors seems to override these effects and protects the host from NSCL growth and progression.     

MiR-142-3p functions as a potential tumor suppressor directly targeting HMGB1 in non-small-cell lung carcinoma

Background: microRNAs (miRNAs) play a significant role in cancer development and progression by regulating the expression of oncogenes or tumor suppressor genes. Previous study using microarrays demonstrated that miR-142-3p was downregulated in patients with Non-small-cell lung carcinoma (NSCLC). However, the functional role of miR-142-3p in NSCLC is still unclear. Material and method: Real-time quantitative PCR was applied to evaluate the expression level of miRNA-142-3p in NSCLC and normal samples. The cell proliferation of NSCLC cells was analyzed by MTT and colony formation assay after miR-142-3p transfection. Luciferase activities assay, cotransfection and Western blot were used to reveal that the predicted target genes of miR-125b were direct and specific. Results: In this study, we demonstrate miR-142-3p was downregulated in NSCLC tissues and cell lines. We demonstrated that the overexpression of miR-142-3p inhibits NSCLC cell proliferation and induced cell apoptosis. Furthermore, we demonstrate HMGB1 was a directly target of miR-142-3p in NSCLC cells, and confirmed the target specificity between miR-142-3p and the HMGB1 3’-untranslated region by luciferase reporter assay. Conclusions: These results suggest that miR-142-3p may be a tumor suppressor through the downregulation of HMGB1 in NSCLC. miR-142-3p may be a tumor suppressor and a potential therapeutic agent for patients with NSCLC.     

miR-425 regulates cell proliferation,migration and apoptosis by targeting AMPH-1 in non-small-cell lung cancer

BackgroundMicroRNAs (miRNAs) have been proved to act as vital roles on non-small-cell lung cancer (NSCLC), and miR-425 has been proven to serve an important function in several tumors. However, the functional role of miR-425 on NSCLC is still unclear.MethodsThe mRNA and protein expression of miR-425 and AMPH-1 were determined by qRT-PCR and western blot analysis, respectively. NSCLC cells (SK-MES-1 and A549) proliferation and migration were measured by CCK-8 and transwell assay, respectively. Cell apoptosis was assessed by flow cytometry and western blotting, In addition, luciferase reporter assay was carried out to confirm the direct targeting of AMPH-1 by miR-425. Xenograft experiments were performed to observe the tumorigenesis of miR-425 in vivo.ResultsThe results showed that miR-425 was overexpressed and AMPH-1 expression was downregulated in SK-MES-1 and A549 cells. Silencing miR-425 inhibited proliferation, migration and promoted apoptosis of NSCLC cells. Moreover, we proved that miR-425 could target AMPH-1. The expression of AMPH-1was upregulated in A549 with miR-425 inhibitor. Moreover, miR-425 knockdown were less tumorigenic than the control in vivo.ConclusionsTaken together, miR-425 could promote the proliferation, invasion and suppress apoptosis by targeting AMPH-1 in NSCLC cells. miR-425/AMPH-1 axis may represent a potential therapeutic strategy or novel prognostic biomarkers to NSCLC.     

Expression of miR-32 in human non-small cell lung cancer and its correlation with tumor progression and patient survival

Introduction: miR-32 has recently been found to be implicated in many critical processes in various types of human cancer. However, its clinical significance in human non-small cell lung cancer (NSCLC) has not yet been elucidated. In the present study, we investigated the expression of miR-32 in NSCLC and analyzed its association with clinical features and prognosis of NSCLC patients. Methods: Quantitative real-time PCR (qRT-PCR) was used to measure expression level of miR-32 in lung cancer cell lines, normal bronchial epithelial cells, 90 pairs of tumor samples and adjacent non-tumor tissues. To determine its prognostic value, overall survival was evaluated using the Kaplan-Meier method. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis. Results: The expression of miR-32 was significantly decreased in lung cancer cell lines and NSCLC tissues compared with normal bronchial epithelial cells and adjacent non-tumor tissues (P < 0.05). This reduction of miR-32 was associated with tumor stage and lymph node metastasis (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with low miR-32 expression had shorter overall survival time than those with high miR-32 expression (P < 0.05). Univariate analysis revealed statistically significant correlations between overall survival and miR-32 level, tumor stage and lymph node metastasis (P < 0.05). Furthermore, miR-32 levels, tumor stage and lymph node metastasis were independently associated with overall survival (P < 0.05). Conclusions: Our results provided the first evidence that down-regulation of miR-32 was correlated with NSCLC progression, and miR-32 might be a potential molecular biomarker for predicting the prognosis of patients.     

circ0000231在非小细胞肺癌中的表达及功能研究

目的:探讨环状RNA_0000231(circ_0000231)在非小细胞肺癌(NSCLC)中的表达及功能。方法:应用RT-qPCR检测circ_0000231在NSCLC组织和细胞系中的表达,将circ_0000231的小干扰RNA(si-circ_0000231)和阴性对照siRNA(NC)分别转染NSCLC细胞,采用CCK-8法、集落形成实验和流式细胞术检测2组细胞的增殖和凋亡情况,并用RT-qPCR和Western blot检测细胞周期蛋白D1(CCND1)和抗凋亡蛋白Bcl-2的表达。结果:分别与癌旁组织和人正常支气管上皮BEAS-2B细胞相比,circ_0000231在NSCLC组织和细胞系中的表达均显著上调(P<0.05)。与NC组相比,si-circ_0000231组的NSCLC细胞活力和集落形成数显著降低,凋亡率显著增加(P<0.05)。此外,RT-qPCR和Western blot检测结果显示转染si-circ_0000231能够抑制NSCLC细胞CCND1和Bcl-2的表达(P<0.01)。结论:circ_0000231在NSCLC组织和细胞中的表达显著升高,敲减circ_0000231的表达能显著抑制NSCLC细胞的增殖。     

Aberrant expression of Per1, Per2 and Per3 and their prognostic relevance in non-small cell lung cancer

The objective of the current study was to investigate the expression pattern and clinicopathological significance of Period1 (Per1), Period2 (Per2) and Period3 (Per3) in patients with non-small cell lung cancer (NSCLC). In 130 archived NSCLC tissues, the positive rate of Per1 (86/130, 66.2%), Per2 (77/130, 59.2%) and Per3 (82/130, 63.1%) were reduced in human lung cancer samples compared with adjacent normal lung tissues (Per1, 119/130, 91.5%; Per2, 115/130, 88.5%; Per3, 121/130, 93.1%), as measured by immunohistochemical staining. Loss of Per1 was correlated with poor differentiation (P < 0.001), tumor status (P=0.04), high p-TNM stage (P < 0.001) and lymph node metastasis (P=0.045). The similar tendencies were also found in the correlation of the expression of Per2 and Per3 with clinicopathological factors. In addition, a significant correlation was found between Per1 and Per2 (P < 0.001) in 130 cases of NSCLC. Similarly, a significant correlation was found between Per2 and Per3 (P=0.045). Patients with lower expression of Per1, Per2 and Per3 had a shorter survival time than those with higher expression. These results indicate that loss of Per may promote tumor progression in NSCLC, and may serve as a novel prognostic biomarker of NSCLC.