lncRNA LEF1-AS1通过靶向miR-612调控皮肤鳞状细胞癌细胞株增殖、凋亡、迁移侵袭的体外实验研究

【摘要】 目的 探讨长链非编码RNA（lncRNA）LEF1-AS1对皮肤鳞状细胞癌细胞增殖、凋亡、迁移、侵袭的影响及其机制。方法 通过干扰皮肤鳞状细胞癌SCC13细胞LEF1-AS1的表达和过表达、抑制miR-612的表达，将SCC13细胞分为转染LEF1-AS1干扰序列、无义序列的干扰LEF1-AS1组、干扰对照组，转染miR-612过表达序列、无义序列的miR-612过表达组、过表达对照组，以及转染LEF1-AS1干扰序列与miR-612抑制序列的干扰抑制组，和转染LEF1-AS1干扰序列与miR-612抑制无义序列的干扰抑制对照组。采用qRT-PCR法检测各组细胞miR-612的相对表达，CCK8法检测细胞增殖，流式细胞仪分析细胞的凋亡情况，Transwell试验检测细胞的迁移、侵袭能力，Western印迹检测细胞周期蛋白依赖激酶1（cyclinD1）、细胞周期蛋白依靠性激酶抑制剂（p21）、Bcl-2家族蛋白（Bcl-2）、Bcl-2相关X蛋白（Bax）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）的表达。用LncBase Predicted v.2 在线预测网站预测lncRNA LEF1-AS1与miR-612之间的互补序列，将互补/非互补序列用于构建荧光素酶报告基因质粒，分别与miR-612过表达及过表达对照基因共转染SCC13细胞，验证lncRNA LEF1-AS1与miR-612的结合能力。两组间比较采用t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。结果 miR-612过表达组细胞增殖能力、迁移及侵袭细胞数均低于过表达对照组（均P < 0.05），凋亡率高于过表达对照组（P < 0.05）。干扰LEF1-AS1组、干扰对照组、干扰抑制组、干扰抑制对照组miR-612的相对表达差异有统计学意义（F = 150.78，P < 0.001），24、48、72 h时的增殖能力差异有统计学意义（均P < 0.05），凋亡率、迁移及侵袭细胞数差异均有统计学意义（均P < 0.05）。干扰LEF1-AS1组细胞中miR-612表达、凋亡率高于干扰对照组，48、72 h细胞增殖能力、迁移细胞数和侵袭细胞数低于干扰对照组（均P < 0.05），而干扰抑制组细胞中miR-612表达、细胞凋亡率低于干扰抑制对照组，48、72 h细胞增殖能力、迁移细胞数和侵袭细胞数高于干扰抑制对照组（均P < 0.05）。Western印迹显示，4组细胞cyclinD1、p21、Bcl-2、Bax、MMP-2、MMP-9蛋白相对水平差异均有统计学意义（均P < 0.001），干扰LEF1-AS1组cyclinD1、Bcl-2、MMP-2、MMP-9蛋白相对水平均高于干扰对照组，p21、 Bax蛋白相对水平低于干扰对照组（均P < 0.05）；而干扰抑制组cyclinD1、Bcl-2、MMP-2、MMP-9蛋白相对水平均高于干扰抑制对照组，p21、 Bax蛋白相对水平低于干扰抑制对照组（均P < 0.05）。共转染互补序列后，miR-612过表达组细胞荧光活性低于过表达对照组（t = 21.19，P < 0.001）；而共转染非互补序列后，miR-612组细胞荧光活性与过表达对照组差异无统计学意义（t = 0.28，P = 0.78）。结论 lncRNA LEF1-AS1调控皮肤鳞状细胞癌细胞的增殖、凋亡、迁移和侵袭，机制与靶向miR-612相关。     

LncRNA SNHG8靶向调控miR-152对子宫内膜癌细胞增殖、凋亡的影响机制

目的探讨LncRNA SNHG8靶向调控miR-152对子宫内膜癌细胞增殖、凋亡的影响及机制。方法以子宫内膜上皮细胞(EEC)为对照,qRT-PCR检测人子宫内膜癌细胞株AN3CA、HEC-1A、HEC-1B和Ishikawa中SNHG8 mRNA表达。参照Lipofectamine 2000试剂盒说明,将si-SNHG8、si-NC、anti-control及si-SNHG8+anti-miR-152转染AN3CA细胞,MTT法检测转染24h～96h细胞活力,流式细胞术检测转染48h的细胞凋亡率,Western blotting检测AKT、p-AKT、PCNA和cleaved-caspase3蛋白表达。结果 SNHG8在4个子宫内膜癌细胞的mRNA表达水平均明显高于在EEC细胞表达,差异具有统计学意义(P0.05)。选择表达最高的AN3CA细胞为研究对象。与si-NC组比较,si-SNHG8组细胞活力明显降低,凋亡率升高,p-AKT和PCNA表达明显降低,cleaved-caspase3表达明显升高,差异具有统计学意义(P0.05),与si-SNHG8+anti-control组比较,si-SNHG8+anti-miR-152组细胞活力明显升高,凋亡率降低,p-AKT和PCNA表达明显升高,cleaved-caspase3表达明显降低,差异具有统计学意义(P0.05)。结论 LncRNA可SNHG8靶向调控miR-152抑制子宫内膜癌AN3CA细胞活力,诱导细胞凋亡,机制可能与调节AKT、PCNA和cleaved-caspase3表达有关。     

槲皮素对瘢痕疙瘩成纤维细胞生长的影响

目的探讨槲皮素对人体瘢痕疙瘩成纤维细胞在生长增殖、细胞凋亡方面的作用,为临床瘢痕疙瘩的治疗提供实验基础。方法MTT法测定瘢痕疙瘩成纤维细胞干预前后的生长曲线;光学显微镜观察干预后细胞的形态学变化;流式细胞仪分析成纤维细胞在干预前后的细胞周期变化;原位细胞凋亡检测成纤维细胞干预后的细胞凋亡率;免疫细胞化学和蛋白印迹实验检测成纤维细胞干预前后p53、bcl-2、bax蛋白表达水平的变化。结果MTT法显示槲皮素对体外培养的瘢痕疙瘩成纤维细胞有抑制作用,抑制效应呈剂量时间依赖关系;槲皮素作用后48h瘢痕疙瘩成纤维细胞发生G2/M期阻滞,并且出现细胞凋亡,光镜可见细胞形态学上出现细胞皱缩,染色质浓集;流式细胞仪结果显示有凋亡峰出现;原位细胞凋亡检测显示凋亡率为16.5%±2.7%。槲皮素作用后细胞p53蛋白和bax蛋白表达升高,bcl-2蛋白表达降低。结论槲皮素通过抑制细胞增殖和促进细胞凋亡来抑制瘢痕疙瘩成纤维细胞的生长。     

miR-195靶向MYB激活PI3K-Akt通路调控皮肤鳞癌细胞增殖与恶性转移

 目的:探讨微小RNA-195（miR-195）对皮肤鳞癌细胞增殖与恶性转移的影响及其机制。方法:通过实时荧光定量PCR（RT-qPCR）检测miR-195和MYB基因在皮肤鳞癌细胞（A431细胞）和人正常皮肤细胞（HaCaT细胞）中的表达。CCK-8实验检测过表达及下调miR-195对A431细胞增殖能力的影响;Western blot检测过表达及下调miR-195对A431细胞凋亡相关蛋白Bcl-2相关蛋白X（Bax）、B淋巴细胞瘤-2（Bcl-2）表达的影响;Western blot分析过表达及下调miR-195对A431细胞上皮细胞-间充质转化（EMT）的影响。TargetScan和双荧光素酶报告基因实验分析miR-195和MYB之间的关系,分析下调MYB对A431细胞增殖和EMT的影响。Western blot检测过表达MYB后对PI3K-Akt通路的影响。LY294002作用细胞后,检测过表达MYB对A431细胞增殖和EMT的影响。结果：与HaCaT细胞相比,A431细胞中miR-195表达明显下调（P<0.01）,MYB表达明显上调（P<0.01）。过表达miR-195抑制A431细胞增殖与EMT,促进细胞凋亡,下调miR-195则起到相反作用;miR-195和MYB具有靶向和负调控关系;下调MYB的表达抑制A431细胞增殖与EMT;过表达MYB激活细胞内PI3K Akt通路,且通过该通路促进A431细胞增殖与EMT。结论：miR-195通过靶向MYB激活PI3K-Akt通路调控A431细胞的增殖和转移。     

miR-519d-3p通过调控Toll样受体4/核因子-κB信号通路对前列腺癌细胞的影响机制研究

目的 探讨通过微小RNA-519d-3p(miR-519d-3p)调控Toll样受体4/核因子-κB(TLR4/NF-κB)信号通路对前列腺癌细胞的机制研究。方法 于2021年6月购买前列腺癌细胞株PC3,培养至对数时期,随机分为对照组、下调miR-519d-3p组、上调miR-519d-3p组。采用聚合酶链反应检测miR-519d-3p表达情况,对比干预24h、48h、72h细胞增殖、细胞迁移、侵袭率;检测细胞TLR4/NF-κB信号通路蛋白表达状况。结果 与对照组相比,下调miR-519d-3p组细胞增殖率、细胞侵袭数、迁移数、miR-519d-3p表达量、B淋巴细胞瘤-2基因/Bcl-2相关X基因(Bcl-2/Bax)、TLR4/β-actin、NF-κB/β-actin蛋白表达量上升,细胞凋亡率下降(P<0.05);与下调miR-519d-3p组相比,上调miR-519d-3p组细胞增殖率、细胞侵袭数、迁移数、miR-519d-3p表达量、Bcl-2/Bax、TLR4/β-actin、NF-κB/β-actin蛋白表达量降低,细胞凋亡率升高(P<0.05)。结论 上...     

MicroRNA-21对瘢痕疙瘩成纤维细胞mTOR信号通路的调控作用研究

目的:探讨MicroRNA(miR)-21对瘢痕疙瘩成纤维细胞哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的调控作用及机制。方法:应用miR-21抑制物(inhibitor)及阴性对照(NC)序列转染人瘢痕疙瘩成纤维细胞后,反转录(RT)-PCR检测miR-21及10号染色体张力缺失蛋白磷酸酶(PTEN)mRNA表达水平;western blot检测m TOR信号通路关键分子PTEN、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶BPKB(p-Akt,又称Akt)及p-mTOR蛋白的表达。生物信息学方法预测miR-21与PTEN mRNA 3′-UTR结合位点,并用双萤光素酶报告基因验证miR-21对PTEN m RNA的靶向作用;四甲基偶氮唑蓝(MTT)法检测瘢痕疙瘩成纤维细胞增殖。结果:miR-21能够靶向调控PTEN m RNA表达,抑制miR-21表达可导致PTEN的m RNA和蛋白表达增加,进而抑制p-Akt及p-mTOR蛋白的表达,从而抑制瘢痕疙瘩成纤维细胞增殖。结论:miR-21可通过靶基因PTEN调控瘢痕疙瘩成纤维细胞mTOR信号通路关键分子p-Akt和p-mTOR的表达。     

miR-142-3p/Sema3A轴调节角质形成细胞增殖凋亡及银屑病皮肤炎症反应

目的旨在探讨miR-142-3p在M5刺激人表皮角质形成细胞HaCaT中的作用及潜在机制。方法MTT法检测细胞增殖;ELISA凋亡检测试剂盒评估细胞凋亡。ELISA试剂盒测定炎症介质TNF-α、IL-22和IL-17A的分泌量。RT-PCR和Western blot分别测定目的基因mRNA和蛋白表达。荧光素酶报告实验评估荧光素酶活性。结果M5刺激的HaCaT细胞中miR-142-3p表达升高。下调miR-142-3p显著抑制M5诱导HaCaT细胞的增殖并促进凋亡,抗凋亡蛋白Bcl-2减少,伴随促凋亡蛋白Bax增加。此外,下调miR-142-3p通过降低多种炎症因子的表达改善M5诱导的炎症反应。重要的是,miR-142-3p负调控靶基因Sema3A的表达。从机制上讲,沉默Sema3A有效逆转miR-142-3p下调对HaCaT细胞的抗增殖、促凋亡及抗炎作用。结论下调miR-142-3p通过调控靶基因Sema3A保护HaCaT细胞免受M5诱导的过度增殖和炎症损伤,揭示miR-142-3p/Sema3A轴可能是防止角质形成细胞损伤的新靶点。     

HPA-siRNA促进皮肤鳞癌A431细胞凋亡并抑制其增殖、侵袭和迁移的机制

目的探讨乙酰肝素酶(HPA)小干扰RNA(siRNA)对皮肤鳞状细胞癌A431细胞增殖、凋亡、侵袭和迁移的影响及其机制。方法将体外培养的A431细胞分为空白对照组(未转染)、阴性对照组(转染NC-siRNA)和HPA沉默组(转染HPA-siRNA),RT-PCR检测各组细胞中HPA mRNA的表达,MTT法、流式细胞仪和Transwell分别检测细胞增殖、凋亡、侵袭和迁移;Western blot检测细胞中HPA蛋白和PI3K/AKT信号通路相关蛋白PCNA、MMP-2、Bcl-2、p-AKT和AKT的表达。结果与空白对照组相比,HPA沉默组细胞在2～3 d生长过程中的A值明显下降,侵袭和迁移细胞数均明显减少,HPA mRNA和HPA、PCNA、MMP-2、Bcl-2、p-AKT蛋白的表达水平均明显降低,细胞凋亡率明显升高(P0.05);而空白对照组与阴性对照组间差异无统计学意义(P0.05)。结论 HPA siRNA可抑制A431细胞增殖、侵袭和迁移并诱导细胞凋亡,其作用机制可能与抑制PI3K/AKT信号通路的活化有关。     

沉默c-Met对宫颈癌细胞增殖、细胞周期及细胞凋亡的影响

目的探讨沉默c-Met对宫颈癌细胞Hela生物学特性的影响。方法利用小RNA干扰技术在宫颈癌细胞Hela中沉默c-Met,将实验分为Sic-Met(实验组)和SiCon(阴性对照组),平板克隆实验检测Hela细胞生长情况,流式细胞术检测Hela细胞的周期和凋亡情况。Western blot检测抑凋亡基因Bcl-2和促凋亡基因Bax的蛋白表达情况。结果平板克隆实验显示,沉默c-Met 7天后,Hela细胞形成的克隆团少于SiCon (P0.05)。流式结果显示,Hela细胞中Sic-Met组的凋亡细胞数量明显多于SiCon组(P0.05)。细胞周期分布结果显示,沉默c-Met对宫颈癌Hela细胞周期变化没有影响(P0.05)。Western blot结果显示,沉默c-Met后Bcl-2的蛋白表达量下降而Bax的蛋白表达量升高。结论沉默c-Met抑制了宫颈癌Hela细胞的生长增殖,并且通过增加Bax、降低Bcl-2的蛋白表达量来促进细胞凋亡,但沉默c-Met对宫颈癌Hela细胞周期变化没有影响。     

Mansonone F对前列腺癌细胞增殖、凋亡、侵袭和迁移的影响及其机制研究

目的研究Mansonone F在前列腺癌细胞增殖、凋亡、侵袭和迁移中的作用及其机制。方法噻唑蓝(MTT)检测0μmol/L、2μmol/L、4μmol/L、8μmol/L Mansonone F作用于前列腺癌DU145细胞48h后的细胞增殖。流式细胞术检测细胞凋亡,蛋白质印迹法(Western blot)检测B细胞淋巴瘤/白血病-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸化蛋白激酶B(p-Akt)、蛋白激酶B(Akt)蛋白表达,Transwell小室法检测细胞侵袭和迁移。使用8μmol/L Mansonone F和激活剂IGF-1处理DU145细胞,观察激活PI3K/Akt信号通路对Mansonone F干预的DU145细胞增殖、凋亡、侵袭和迁移的影响。结果 Mansonone F明显抑制DU145细胞增殖(P0.05);8μmol/L Mansonone F显著提高细胞凋亡率、Bax蛋白表达(P0.05),降低细胞侵袭数、迁移数、Bcl-2、p-Akt蛋白水平(P0.05),对Akt蛋白表达无明显影响。激活PI3K/Akt信号通路逆转Mansonone F表现出对DU145细胞增殖、侵袭、迁移、Bcl-2蛋白表达的抑制作用,以及细胞凋亡、Bax蛋白表达的促进作用。结论 Mansonone F通过抑制PI3K/Akt信号通路,进而抑制前列腺癌细胞的增殖、侵袭和迁移并促进其凋亡。     

土贝母苷甲通过调控circ_0000376/miR-203轴影响皮肤鳞状细胞癌SCL-1细胞恶性表型

目的:探讨土贝母苷甲对皮肤鳞状细胞癌SCL-1细胞恶性表型的影响及可能机制.方法:将体外SCL-1细胞分为对照组、不同剂量(5、10、20μg/mL)土贝母苷甲组、si-NC组、si-circ_0000376组、土贝母苷甲+pcDNA组和土贝母苷甲+pcDNA-circ_0000376组,CCK-8法检测细胞增殖抑制率...     

Bcl-2 and Bax in congenital naevi

BACKGROUND: Melanocytes represent a static component of the epidermis, and the role of apoptosis in basal melanocyte function and melanocytic tumour formation has not been fully elucidated. OBJECTIVES: The aim of this study was to investigate the expression of Bcl-2 anti-apoptotic and Bax apoptotic proteins in congenital naevi in correlation with p-27 protein and Ki-67 proliferative index. METHODS: Our material comprised 30 congenital naevi (eight giant) excised from children aged from 15 days to 14 years old. The immunohistochemical streptavidin-biotin method was performed on paraffin sections for the detection of Bcl-2 (cl100/D5), Bax (cl2D2) , Ki-67 (MIB-1) and p-27 (1B4) proteins with monoclonal antibodies. RESULTS: Bcl-2 protein was detected in all cases showing a strong diffuse cytoplasmic expression in >70% of the naevocytes and was preserved in the deeper parts of the naevi. On the other hand, Bax was detected in 13 of the cases, showing a fainter cytoplasmic expression in 40-50% of the naevocytes without any particular topographic distribution. Ki-67 was detected in all cases showing a limited expression in 1-2% of the nuclei mainly in the junctional and upper dermal components. p-27 protein showed a broad diffuse nuclear expression (>70% of the nuclei) in all cases with a particular increase in the deeper parts of the naevi. Bcl-2 expression showed a parallel correlation with p-27 protein. CONCLUSIONS: Broad Bcl-2 expression in congenital naevi suggests that suppression of apoptosis may play an important role in the maintenance of naevocytes despite the low proliferative activity.     

Bax expression and growth behavior of basal cell carcinomas

BACKGROUND: To understand the typical growth behavior of basal cell carcinoma (BCC) we searched for the correlation between proliferation and apoptosis and progression of BCC. METHODS: Expression of Bcl-2, Bax, and Ki-67 was immunohistochemically investigated in both normal skin and BCC cells, as well as in the epidermis overlying BCC. RESULTS: The results showed that in normal epidermis, Bcl-2 was homogeneously expressed in the basal cell compartment, whereas Ki-67 expression was largely restricted to the parabasal layer, the layer just above the basal cell layer, and exhibited a more scattered staining pattern. Bax was occasionally expressed in the basal layer and widely in the suprabasal compartment. Strikingly, the apparently normal epidermis overlying BCC showed an increased Bd-2 staining. In BCC, cells stained homogeneously for Bcl-2, whereas Bax and Ki-67 showed scattered staining patterns. Simultaneous expression was seen for Bcl-2 and Bax in 80 +/- 7% of the tumor cells, and co-expression of Bcl-2 and Ki-67 in 20 +/- 7% of the tumor cells. The cells expressing Bcl-2 and Ki-67, but lacking expression of Bax, the progressive fraction, comprised on average 7 +/- 3% of the tumor cell population. CONCLUSION: These results suggest that this small progressive fraction of tumor cells, in combination with the relatively high percentage of cells still prone to apoptosis, can explain the indolent growth behavior of BCC.     

Differential expression of cell survival and cell cycle regulatory proteins in cutaneous squamoproliferative lesions

Previous models of cutaneous carcinogenesis have primarily focused on the regulation of keratinocyte (KC) proliferation and differentiation. However, it has become clear in many neoplastic systems that altered rates of cell death and/or inabilty to undergo growth arrest can also contribute to the development of cancer. Apoptosis-regulatory proteins include those that block apoptosis such as Bcl-2 and Bcl-x, whilst a related protein Bax promotes apoptosis. Cell cycle regulatory proteins include those associated with growth arrest, i.e. p21^waf1, p53, and those associated with proliferation, i.e. Ki-67. Paraffin embedded samples from ten different lesions of squamous cell carcinoma (SCC), Bowen’s disease (BD), keratoacanthomas (KA), and nine normal adult skin samples were stained by immunohistochemistry to detect expression of Bcl-2, Bcl-x, Bax, Ki-67, p21^waf1, p53 and apoptosis (TUNEL assay). Compared to low levels of Bcl-x and Bcl-2 immunostaining in normal skin, all the squamoproliferative lesions had strong and diffuse KC expression of Bcl-x (>80%) but minimal to absent KC Bcl-2 expression (<15%). Bax immunopositivity was limited to the basal layer in normal skin and BD. In contrast, by examining serial sections both Bcl-x and Bax appeared to be coexpressed by the majority of malignant KCs in KA and SCC (>70%). These immunostaining profiles reveal that squamoproliferative lesions, including invasive transformed KCs, preferentially express Bcl-x over Bcl-2, in addition to upregulating their Bax levels. Even though there were numerous TUNEL positive cells in these squamoproliferative lesions, no other evidence of apoptosis was seen reinforcing the necessity to use caution when relying on TUNEL staining for identification of programmed cell death in skin biopsies. Normal sun-exposed skin had low but detectable p53 and rare p21^waf1 KC expression. Significantly higher numbers of p21^waf1 and p53 immunopositive KCs were noted throughout the lesions in BD and SCC in contrast to KA where p53 and rare p21^waf1 immunopositive KCs were primarily limited to the periphery of the tumor cell islands. In general, p53 KC expression was higher in all squamoproliferative lesions and sun-exposed normal skin compared to p21^waf1 expression. Summary of the expression of cell cycle regulatory proteins for both p21^waf1 and p53 KC expression was: SCC>BD>KA, in marked contrast to Ki-67 KC expression which was: BD>KA>SCC. The relatively few malignant cells in SCC that were actively participating in the cell cycle (i.e. Ki-67 positive) suggests that these neoplasms may arise primarily by increased cell survival and resistance to apoptosis rather than by hyperproliferation. These studies emphasize the importance of examining multiple members of protein families that regulate apoptosis, proliferation, growth arrest, and differentiation. It is the overall balance between these cellular phenomena that determine whether a cell remains viable or undergoes programmed cell death and contributes to the appearance of a neoplasm. The overexpression of Bcl-x may confer a survival advantage to malignant KCs unable to growth arrest to repair damaged DNA (mutant p53) and/or undergo terminal differentiation (increased p21^waf1). Thus, mutation or aberrant expression of such proteins may participate in the multistep process of carcinogenesis that gives rise to these squamoproliferative lesions.     

Apoptosis and expression of Bax/Bcl-2 proteins in pyogenic granuloma: a comparative study with granulation tissue and capillary hemangioma

BACKGROUND: Pyogenic granuloma (PG) has been regarded as a florid expression of granulation tissue (GT) proliferation and shows distinct biological behavior compared to conventional GT. PG has not been examined from a standpoint of apoptosis. METHODS: PG (15 cases), GT (14 cases) and capillary hemangioma (CH, 8 cases) were compared using in situ 3'-tailing reaction (ISTR), a histochemical method for identifying apoptotic cells, and immunohistochemistry for Ki-67, Bcl-2 and Bax proteins, in order to clarify and involvement of apoptosis in the difference of biological characteristics between PG and GT. RESULTS: PG showed significantly lower ISTR-labeling indices than GT and CH and more frequent Bcl-2/Bax expression than GT, whereas Ki-67-labeling indices were variable from case to case and did not show any significant differences among any groups. CONCLUSIONS: It is consequently suggested that the low apoptotic rate in PG is closely related to its characteristic rapid growth and is regulated by Bcl-2 family proteins.     

HDAC6siRNA对SCL-1细胞增殖和细胞凋亡的影响及分子机制

目的研究组蛋白去乙酰化酶6(histonedeacetylase6,HDAC6)siRNA对皮肤鳞状细胞癌(简称鳞癌)SCL-1细胞增殖和凋亡的影响及分子机制。方法将SCL-1细胞分为三组:A为未处理组;B为siRNA对照组;C为HDAC6siRNA转染组。利用脂质体2000分别介导HDAC6siRNA和对照siRNA至SCL-1细胞中,利用Westernblot研究HDAC6siRNA对SCL-1细胞中HDAC6蛋白表达的影响。采用CCK-8试剂和流式细胞术分别研究HDAC6siRNA对SCL-1细胞增殖和细胞凋亡的影响,进一步利用WesternBlot技术分析细胞增殖相关蛋白P21和细胞凋亡相关蛋白Bcl-2及Bax的表达。结果 HDAC6siRNA转染组中HDAC6蛋白表达显著低于未处理组和siRNA对照组,差异具有统计学意义(P<0.05)。与未处理组和siRNA对照组相比,HDAC6siRNA转染组中SCL-1细胞的增殖明显受到抑制(P<0.05)。HDAC6siRNA转染组中SCL-1细胞的早期凋亡率明显高于未处理组和siRNA对照组,差异具有统计学意义(P<0.05)。HDAC6siRNA转染组中Bcl-2表达明显下调,而P21和Bax表达显著上升。结论 HDAC6可能在皮肤鳞癌的发生发展中发挥重要作用,下调皮肤鳞癌中HDAC6的表达有望成为有用的分子治疗靶点。     

miR-141-3p靶向调控DAPK1对多囊卵巢综合征大鼠卵巢颗粒细胞增殖与凋亡的影响

目的 探究miR-141-3p对多囊卵巢综合征(PCOS)大鼠卵巢颗粒细胞增殖与凋亡的影响及其机制.方法 选取雌性SD大鼠20只,随机分为对照组和模型组,每组10只.模型组构建大鼠PCOS模型,提取两组大鼠的卵巢颗粒细胞,实时荧光定量聚合酶链式反应(RT-qPCR)测定两组大鼠颗粒细胞中miR-141-3p表达水平.将...     

HPV6／11相关尖锐湿疣皮损中PCNA和Ki-67蛋白的表达

目的 :为了解增殖细胞核抗原 (proliferatingcellnuclearantigenPCNA)和细胞增殖相关核抗原Ki- 6 7在尖锐湿疣 (CA)皮损中的表达和意义。方法 :用链霉菌抗生物素蛋白—过氧化物酶免疫组化方法 (简称SP法 )检测经多聚酶联反应 (polymerasechainreactionPCR)证实人乳头瘤病毒 (HPV)DNA6 / 11阳性的 2 7例CA皮损和 10例正常皮肤。结果 :(1)CA皮损和正常皮肤均表达PCNA和Ki- 6 7。 (2 )CA皮损基底细胞和棘细胞表达PCNA和Ki- 6 7均显著高于正常皮肤。结论 :PCNA和Ki- 6 7只能反映CA皮损的增殖状态而不能反映其增殖性质。     

Keratotic melanocytic nevus: a clinicopathologic and immunohistochemical study

BACKGROUND: Epidermal hyperplasia in melanocytic nevi is a common but little-investigated phenomenon. METHODS: We prospectively examined all melanocytic nevi diagnosed in our department over an 8-month period, for the criteria of keratotic melanocytic nevus (KMN), namely the presence of marked epidermal hyperplasia with or without horn pseudocyst formation, hyperkeratosis, and papillomatosis. In addition to routine histologic review, we studied 12 representative cases with immunohistochemistry to examine expression of Ki-67, epidermal growth factor receptor (EGFR), Bcl-2, and Bax. RESULTS: From a total of 1,527 melanocytic nevi, 95 were KMN (prevalence 6%). The average age was 34 years, with a male:female ratio of 1:2. The predominant location was the trunk (76%), followed by head and neck (20%), and extremities (4%). Clinical diagnoses were atypical nevus (44%), nevus not otherwise specified (43%), and others including seborrheic keratosis, acrochordon, and basal cell carcinoma. Two KMN were junctional, 44 compound, and 49 intradermal. Twenty-three KMN (24%) had histologic features suggesting congenital onset, and 15 (16%) had mild to moderate dysplastic features. Two cases demonstrated induction of sebaceous glands. Significantly increased Ki-67 expression was detected in the hyperplastic epidermis, particularly in deeper areas related to keratinous cysts and hair follicles. Bcl-2 and Bax (anti- and pro-apoptosis proteins, respectively) and EGFR were expressed similarly in both normal and hyperplastic epidermis overlying the KMN. CONCLUSIONS: KMN are commonly biopsied skin lesions, mostly located on the trunk. Many such lesions are clinically considered atypical, in contrast to their benign histologic appearance. The epidermal hyperplasia on top of KMN demonstrates increased cellular proliferation, in the context of adequately regulated apoptosis and EGFR expression. The cellular proliferation seems to commence in hair follicles.