p21基因的克隆及其对肺癌细胞生长的抑制

用反转录ＰＣＲ从正常人胚胎肺细胞中获得了ｐ２１基因ｃＤＮＡ,将其插入真核表达载体ｐＭＳＣＶｎｅｏ,构建成重组质粒,ｐＭＳ２１,并将其转染至肺癌细胞株Ａ５４９．通过集落形成观察到ｐ２１对肺癌细胞具有明显的抑制作用,经ＲＮＡ狭缝杂交、Ｗｅｓｔ－ｅｒｎｂｌｏｔ分析和免疫细胞化学实验证实这是ｐ２１表达的结果．荷瘤裸鼠实验也进一步证实了ｐ２１对肺癌细胞具有明显的抑制作用．为ｐ２１的深入研究提供了基础．     

p21基因的克隆及其对肺癌细胞生长的抑制

用反转录ＰＣＲ从正常人胚胎肺细胞中获得了ｐ２１基因ｃＤＮＡ,将其插入真核表达载体ｐＭＳＣＶｎｅｏ,构建成重组质粒,ｐＭＳ２１,并将其转染至肺癌细胞株Ａ５４９。通过集落形成观察到ｐ２１对肺癌细胞具有明显的抑制作用,经ＲＮＡ狭缝杂交、Ｗｅｓｔｅｒｎ ｂｌｏｔ分析和免疫细胞化学实验证实这是ｐ２１表达的结果。荷瘤裸鼠实验也进一步证实了ｐ２１对肺癌细胞具有明显的抑制作用。为ｐ２１的深入研究提供了基础。     

miR-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为研究

目的:探讨mi R-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为。方法:qRT-PCR法检测并比较肺癌组织、癌旁正常组织、肺癌细胞、正常肺上皮细胞中的mi R-199a-3p m RNA相对表达量。比较远处转移肺癌组织、未转移肺癌组织中mi R-199a-3p m RNA相对表达量。qRT-PCR法、Western Blot法检测并比较肺癌组织、癌旁正常组织中的CBX7 m RNA及蛋白的表达水平。荧光素酶活性法检测mi R-199a-3p与靶基因CBX7的结合。比较mi R-199a-3p模拟物转染组与阴性对照组的肺癌细胞中的CBX7 m RNA相对表达量及CBX7蛋白表达水平。CCK8实验检测mi R-199a-3p对肺癌细胞增殖的促进作用。Tranwell实验检测mi R-199a-3p对肺癌细胞侵袭与迁移能力的影响。结果:肺癌组织中mi R-199a-3p明显高于癌旁正常组织,发生远处转移的肺癌组织中mi R-199a-3p m RNA的表达量明显高于未发生转移的肺癌组织,差异有统计学意义(P<0.001)。肺癌组织中CBX7m RNA、CBX7蛋白表达水平均明显低于癌旁正常组织,差异有统计学意义(P<0.001)。荧光素酶活性法证实mi R-199a-3p可与靶基因CBX7结合抑制CBX7的表达。肺癌细胞中mi R-199a-3p m RNA的相对表达量明显高于正常肺上皮细胞,CBX7 m RNA相对表达量明显低于正常肺上皮细胞(P<0.05)。对于肺癌细胞,mi R-199a-3p模拟物转染组的CBX7 m RNA相对表达量及CBX7蛋白表达水平均明显低于阴性对照组(P<0.001)。CCK8实验证实mi R-199a-3p能够促进肺癌细胞的增殖,Tranwell实验证实mi R-199a-3p对肺癌细胞侵袭与迁移具有积极的促进作用。结论:mi R-199a-3p在肺癌的发生发展过程中发挥重要作用,能够通过抑制CBX7基因的表达,促进肺癌细胞的增殖、侵袭和转移。     

高低转移肺腺癌细胞系Anip973和AGZY83-a中P21过表达的研究

为探讨肿瘤抑制基因对肺腺癌细胞生长的抑制作用,利用FuGene转染方法将 P21基因的表达质粒转入一对分别具高、低转移能力的肺腺癌细胞系An ip973和AGZY83-a中。对p21蛋白过表达的细胞系进行了细胞生长曲线,克隆形成率,原位末端标记分析和流式细胞仪分析。p21蛋白过表达的一对细胞系细胞生长曲线斜率降低,克隆形成能力下降并出现明显的G1期阻滞,但未检测到凋亡信号。结果表明p21基因的过表达通过G1期阻滞抑制这一对肺腺癌细胞的生长,P21基因可以作为肺腺癌基因治疗的候选基因。 Abstract:In order to investigate the suppression effect of tumor suppressor genes in lung adenocarcinoma,we transfected P21 expression vector into a pair of lung adenocarcinoma cell lines with different metastasis potential:Anip973(high metastasis potential)and AGZY83-a(low metastasis potential).The suppression effects of p21 were evaluated by cell growth curve,cloning efficiency assay,flow cytometric analysis and Tunel technique.We found that increased expression of p21 in both cell lines was associated with significant lengthening of G1 phase,decreased proliferation potential and decreased cloning efficiency.No apoptosis was found in the cell lines with overexpressed P21 gene.The results showed that increased expression of P21 gene suppressed the lung adenocarcinoma cells by G1 arrest and P21 gene proved a candidate gene in lung adenocarcinoma gene therapy.     

超级干扰素抑制A549增殖诱导其衰老

该研究利用MTT和结晶紫的方法证明sIFNα对肺癌细胞A549增殖有很强的抑制作用,而同样剂量的普通干扰素IFNα2b抑制作用要小很多。与对照组相比,sIFNα处理后,细胞形态发生改变,细胞体积变大,形态呈扁平状;Hoechst33258染色发现,细胞核形态无变化并且未检测到凋亡;SA-β-gal染色发现大多数细胞呈现阳性,同时,衰老相关蛋白p53和p21表达量明显上调。而IFNα2b处理组细胞形态基本没有变化,SA-β-gal染色也只有少部分的细胞呈现弱阳性。由此说明,sIFNα比IFNα2b能更好地抑制癌细胞增殖,其机制可能为诱导癌细胞发生衰老。     

SMP30对肝癌细胞行为学影响及其与糖代谢关系研究

糖代谢是癌症的一个典型特征,研究显示SMP30(senescence marker protein 30)是一种肝癌相关抗原,与脂代谢关系密切,为了探讨SMP30与糖代谢的变化是否相关,我们构建了稳定转染SMP30的人SK-HEP1肝癌细胞株。通过迁移实验、侵袭实验和CCK8实验对比检测了过表达SMP30组与对照组肝癌细胞的迁移、侵袭、增殖的情况;同时还检测了细胞的葡萄糖吸收、乳酸排出的情况。结果显示,与对照组相比,SMP30对SK-HEP1肝癌细胞株的迁移、侵袭有明显的抑制作用(p0.01);对细胞的增殖作用不明显(p0.05)。过表达SMP30对肝癌细胞的葡萄糖吸收和乳酸排出作用不显著(p0.05)。SMP30对肝癌细胞SK-HEP1的迁移、侵袭有一定的抑制作用,但这种作用与糖代谢关系不明显。     

红色诺卡氏菌细胞壁骨架对肺癌细胞的抗癌作用及机制

前人研究发现红色诺卡氏菌细胞壁骨架(N-CWS)具有较好的抗癌活性,然而其对肺癌的抗癌作用机制尚不清楚。本研究旨在揭示N-CWS对肺癌细胞的抗癌作用及机制。通过平板克隆形成实验测定N-CWS对人肺腺癌细胞系A549菌落形成的影响,发现N-CWS可按照浓度依赖性方式降低A549细胞的菌落形成能力(p0.05)。通过5-乙炔基-2'-脱氧尿苷(EdU)法测定N-CWS对肺癌细胞增殖能力的影响,发现N-CWS可按照浓度依赖性方式降低EdU阳性细胞比例(p0.05)。通过Transwell小室和伤口愈合实验评估N-CWS对肺癌细胞侵袭和迁移能力的影响,发现N-CWS以A549浓度依赖性方式降低细胞的侵袭和迁移数量及伤口愈合百分比(p0.05)。通过qRT-PCR和Western blotting检测N-CWS对A549细胞E-cadherin、IL-6和MMP-9表达的影响,发现N-CWS以浓度依赖性方式上调了A549细胞的E-cadherin mRNA和蛋白表达(p0.05),但以浓度依赖性方式下调了IL-6和MMP-9的m RNA和蛋白表达(p0.05)。本研究证实红色诺卡氏菌细胞壁骨架可按照浓度依赖性方式降低肺癌细胞的增殖、侵袭和迁移能力,其抗癌机制与上调E-cadherin和下调IL-6和MMP-9有关。     

槲皮素对结肠癌细胞生长的影响

通过MTT试验观察槲皮素对结肠癌细胞系RKO生长的影响;通过流式细胞仪技术观察槲皮素对细胞周期和细胞凋亡的影响;采用RT-PCR和western印迹技术,确定槲皮素对p21、p27和p53表达的影响.MTT法显示槲皮素加药组生长抑制作用明显,且具有剂量依赖性;流式细胞仪分析结果显示,经5μmol/L槲皮素作用后细胞周期明显阻滞在G0/G1期;5、10、20μmol/L3个剂量组的细胞凋亡率分别为23.4%、24.2%、47.9%,而对照组为13.2%;p21和p27的mRNA及蛋白质表达水平上调,促凋亡蛋白p53表达水平上调.因此,槲皮素对RKO生长有明显的抑制作用,槲皮素可能通过上调p21和p27表达使RKO细胞周期阻滞于G0/G1期,可能通过上调促凋亡蛋白p53表达诱导RKO细胞发生凋亡.     

鸡马立克病病毒Meq蛋白抑制p53的转录激活作用

目的　构建马立克病病毒野生型Meq基因（Meq-wt）和p53结合区域缺失的突变型Meq基因（Meq-mut）表达质粒,研究Meq蛋白对p53的转录激活功能的影响,为进一步了解其生物学功能奠定基础。方法 采用PCR方法克隆马立克氏病毒Meq-wt基因,并采用突变PCR方法缺失Meq与p53结合区域,分别构建了Meq-wt和Meq-mut基因表达质粒。转染鸡胚成纤维细胞（CEF）后,用western印迹法检测Meq蛋白的表达,利用荧光素酶报告基因系统证实Meq蛋白对p53转录激活的抑制作用。同时借助免疫荧光技术,采用荧光显微镜观察Meq蛋白与p53蛋白在细胞中的定位。结果 DNA序列测序表明克隆的Meq-wt、Meq-mut基因插入位点和核苷酸序列完全正确。荧光素酶报告基因系统证实Meq蛋白对p53转录激活的具有明显的抑制作用。间接免疫荧光试验证实野生型Meq蛋白主要在胞浆中表达,而缺失与p53结合区域的突变型Meq在胞浆/胞核中均有表达。野生型Meq蛋白与内源性p53蛋白在胞核内能够很好地重合。 结论 Meq蛋白对p53的转录激活具有抑制作用,可能是通过直接与p53相互结合,来抑制对p53转录活性功能。     

HMBA对人肝癌SMMC-7721细胞周期相关基因表达的影响

本文研究HMBA对人肝癌SMMC-7721细胞周期G_0/G_1期阻滞相关基因表达的影响。免疫细胞化学和核酸原位杂交检测结果显示,HMBA可明显上调p21~(WAFl/CIPl)、p16蛋白表达并增强p21~(WAFl/CIPl)基因转录,同时对CDK4、Cyclin D1蛋白表达以及c-myc基因转录均具有明显的下调作用。结果表明,HMBA可通过增强p21~(WAFl/CIPl)、p16基因表达而抑制Cyclin D1-CDK4活性,最终导致细胞进入S期所需的c-myc等基因转录活性下降,从而将细胞周期阻滞于G_0/G_1期,诱导人肝癌细胞分化。     

Transcriptional activation of the MUC2 gene by p53

MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline-regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types.     

Multifaceted p21 in carcinogenesis,stemness of tumor and tumor therapy

Cancer cells possess metabolic properties that are different from those of benign cells. p21, encoded by CDKN1A gene, also named p21^Cip1/WAF1, was first identified as a cyclin-dependent kinase regulator that suppresses cell cycle G1/S phase and retinoblastoma protein phosphorylation. CDKN1A (p21) acts as the downstream target gene of TP53 (p53), and its expression is induced by wild-type p53 and it is not associated with mutant p53. p21 has been characterized as a vital regulator that involves multiple cell functions, including G1/S cell cycle progression, cell growth, DNA damage, and cell stemness. In 1994, p21 was found as a tumor suppressor in brain, lung and colon cancer by targeting p53 and was associated with tumorigenesis and metastasis. Notably, p21 plays a significant role in tumor development through p53-dependent and p53-independent pathways. In addition, expression of p21 is closely related to the resting state or terminal differentiation of cells. p21 is also associated with cancer stem cells and acts as a biomarker for such cells. In cancer therapy, given the importance of p21 in regulating the G1/S and G2 check points, it is not surprising that p21 is implicated in response to many cancer treatments and p21 promotes the effect of oncolytic virotherapy.     

Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas.

BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.     

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。     

稳定表达外源性p16基因肺癌A549细胞株的建立及鉴定

为构建稳定表达外源性抑癌基因p16的肺癌A549细胞株,用脂质体介导的基因转染方法,借助真核质粒表达载体（pcDNA3)。将抑癌基因p16转移入此基因缺失的人肺癌细胞株A549细胞中,经G418筛选,获得稳定表达的细胞克隆,用逆转录聚合酶链反应（RT-PCR）及免疫组织化学鉴定p16基因的表达,同时对克隆细胞分泌蛋白进行活性检测。结果显示转染p16基因的A549细胞中可以检测到p16mRNA及蛋白的表达,说明建立的p16真核表达载体能在肺肿瘤细胞中分泌表达蛋白,表达P16抑癌蛋白的A549细胞株的建立有助于研究抑癌基因p16在肺癌发生中的作用。     

MicroRNA-7 Compromises p53 Protein-dependent Apoptosis by Controlling the Expression of the Chromatin Remodeling Factor SMARCD1

We previously demonstrated that the epidermal growth factor receptor (EGFR) up-regulated miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. In this study, we identified SMARCD1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. We used luciferase reporters carrying wild type or mutated 3′UTR of SMARCD1 and found that miR-7 blocked SMARCD1 expression by binding to two seed regions in the 3′UTR of SMARCD1 and down-regulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 down-regulated p53-dependent apoptosis-related gene BAX (BCL2-associated X protein) and p21 expression by interfering with the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. We found that although SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was down-regulated in patients with non-small cell lung cancer and lung adenocarcinoma cell lines, and SMARCD1 and miR-7 expression levels were negatively correlated in clinical samples. Our investigation into the involvement of the EGFR-regulated microRNA pathway in the SWI/SNF chromatin remodeling complex suggests that EGFR-mediated miR-7 suppresses the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells.     

MiR-517a-3p accelerates lung cancer cell proliferation and invasion through inhibiting FOXJ3 expression

Aims

Aberrant expression of microRNAs (miRNAs) results in alterations of various biological processes (e.g., cell cycle, cell differentiation, and apoptosis) and cell transformation. Altered miRNAs expression was associated with lung carcinogenesis and tumor progression. This study aimed to investigate the function and underlying molecular events of miR-517a-3p on regulation of lung cancer cell proliferation and invasion.

Main methods

Transfected miR-517a-3p mimics or inhibitors into 95D and 95C cells respectively, the effects of miR-517a-3p on lung cancer cell proliferation, migration, and invasion were detected. Bioinformatics software forecasted potential target genes of miR-517a-3p and dual luciferase reporter gene system and western blot verified whether miR-517a-3p regulates FOXJ3 expression directly.

Key findings

MiR-517a-3p was differentially expressed in lung cancer 95D and 95C cell lines that have different metastatic potential. Manipulation of miR-517a-3p expression changed lung cancer cell proliferation, migration and invasion capacity. MiR-517a-3p directly regulated FOXJ3 expression by binding to FOXJ3 promoter.

Significance

This study demonstrated that miR-517a-3p promoted lung cancer cell proliferation and invasion by targeting of FOXJ3 expression.