miR-320a靶向KLF5抑制结肠癌HCT116细胞增殖和侵袭的分子机制

目的 探讨过表达miR-320a对结肠癌HCT116细胞的生物学行为的影响,并初步分析miR-320a对KLF5的调控机制.方法 将miR-320a模拟物转染结肠癌HCT116细胞,建立过表达miR-320a结肠癌HCT116细胞系,采用CCK-8方法检测转染后HCT116细胞增殖能力,Transwell实验检测转染后HCT116细胞侵袭能力.通过网站Targetscan7.2预测KLF5是否为miR-320a的靶基因,双荧光素酶实验进行验证.Western blot检测转染后各组HCT116细胞中KLF5蛋白表达.结果 CCK-8实验结果显示,miR-320a过表达抑制HCT116细胞增殖能力,Transwell实验结果显示,miR-320a过表达抑制HCT116细胞的侵袭能力,Western blot结果显示,上调miR-320a可使HCT116细胞中KLF5蛋白表达下降,TargetScan7.2网站分析及双荧光素酶实验证实KLF5为miR-320a的靶基因.结论 过表达miR-320a可以抑制结肠癌HCT116细胞增殖及侵袭能力,其机制可能与下调KLF5的表达有关.     

结肠癌中miR-144、KLF12mRNA的表达及临床意义

目的研究结肠癌中miR-144与Krüppel样因子12(KLF12)的表达,并探讨二者表达的相关性及其临床意义。方法通过Oncomine公共数据库分析结直肠癌中KLF12基因mRNA的表达,通过UALCAN、GEPIA公共数据库分析结肠癌中KLF12基因mRNA的表达、通过GEPIA公共数据库分析KLF12表达与肿瘤病理分期的关系以及KLF12表达与患者生存期的关系。通过OncomiR公共数据库分析miR-144在结肠癌中的表达以及miR-144表达与患者生存期的关系。通过在线预测工具(TargetscanHuman 7.2, miRDB)预测KLF12是否为miR-144的靶基因。结果结肠癌中KLF12 mRNA表达水平下降,与结直肠癌的分期显著正相关,与无病生存率(Disease Free Survival, DFS)显著负相关。miR-144在结肠癌中表达水平升高,在生存组患者表达水平升高。在KLF12基因的3'UTR区域,有3处miR-144的结合位点。结论 miR-144可能通过影响KLF12的表达参与直肠癌的发生、发展。     

微小RNA-1273g-3p对人结直肠癌HCT116和SW480细胞增殖和迁移的影响

目的:探讨微小RNA(MicroRNA,miR)-1273g-3p对结直肠癌细胞增殖和迁移的调控作用.方法:将miR-1273g-3p模拟物分别转染人结肠癌细胞(Human colon cancer cell,HCT116)和人结肠腺癌细胞(Human colon adenocarcinoma,SW480)细胞株,通过聚合酶链反应(Polymerase chain reaction,PCR)测定miR1273g-3p的表达情况;四唑盐(Methyl thiazolyl tetrazolium,MTT)比色法检测miR-1273g-3p对细胞增殖的作用;Transwell实验检测细胞的迁移能力.结果:转染miR-1273g-3p模拟物后,结直肠癌HCT116和SW480细胞miR-1273g-3p相对表达量明显上调;MTT和Transell实验结果显示,过表达miR-1273g-3p能明显促进HCT116和SW480细胞的增殖和迁移(P<0.01).结论:miR-1273g-3p具有促进结直肠癌HCT116和SW480细胞株增殖和迁移的作用.     

miR-451a调控BAP31诱导结直肠癌细胞凋亡

目的:探讨miR-451a及靶蛋白BAP31在结直肠癌中的作用。方法:体外培养HCT116、SW620、SW480、DLD细胞,以Real-time PCR法检测结直肠癌细胞系中miR-451a和BAP31的相对表达量;通过建立miR-451a不同表达情况的结直肠癌细胞HCT116模型,应用抑制消减杂交方法建立抑制消减文库,从中筛选miR-451a的调控蛋白,并通过萤光素酶报告基因检测miR-451a的直接调控靶基因;采用MTT法、流式细胞术以及Hoechst染色法评价miR-451a调控的靶蛋白BAP31对结直肠细胞凋亡的调节作用。结果:在抑制杂交消减文库中得到了miR-451a可能调控的相关基因,其中在正向文库中得到BAP31、EEF1A1和CDC20等7个基因,在反向文库中得到DKK1和PSME1等4个基因。在HCT116、HT29、SW480、SW620和DLD结直肠癌细胞中miR-451a的相对表达量是正常结肠上皮细胞NCM460中的0.32、0.44、0.53、0.43和0.73倍,BAP31在DLD、HT29、SW620和HCT116结直肠癌细胞中的表达量是正常结肠上皮细胞NCM460中的1.85、2.84、2.37和3.71倍。双萤光素酶报告基因实验证明,miR-451a可作用于与BAP31开放阅读框上游177的位点,通过miR-451a作用使海肾荧光素酶的活性降低80.3%。在HCT116细胞和SW620细胞中过表达miR-451a后72h抑制率分别为39.50%和39.50%;沉默BAP31后72h抑制率分别为45.32%和53.56%。过表达miR-451a48h后HCT116凋亡增加13.57%,SW620细胞凋亡率增加13.2%;沉默BAP31 48h后HCT116细胞凋亡增加5.62%,SW620细胞凋亡率增加8.68%。结论:miR-451a在结直肠癌细胞中能够直接通过调控BAP31诱导细胞的凋亡从而抑制细胞的增殖。     

高尔基体磷酸化蛋白2对人结直肠癌细胞放射敏感性的影响

目的观察人结直肠癌细胞的放射敏感性及辐射对其高尔基磷酸化蛋白2表达的影响。方法体外培养人结直肠癌细胞株HCT116、SW480及正常结直肠细胞株FHC,检测GOLPH2基因转录及蛋白表达。选取指数生长期人结直肠癌细胞进行0、2、4、6、8、10 Gy照射,计数细胞克隆形成率,制备细胞存活曲线。应用RT-PCR和Western blotting检测2种结直肠癌细胞株在不同剂量照射时GOLPH2基因mRNA转录水平及GOLPH2蛋白表达情况。应用Transwell侵袭实验观察不同照射剂量对人结直肠癌细胞株HCT116、SW480迁移能力的影响。结果随着照射剂量在一定范围内增高时,人结直肠癌细胞株HCT116、SW480克隆形成率逐渐下降,两种细胞株中GOLPH2基因mRNA转录水平及GOLPH2蛋白表达水平均逐渐下降。不同X射线照射剂量对人结直肠癌细胞株HCT116、SW480迁移有抑制作用,并且在8Gy、10Gy时对人结肠癌细胞株迁移能力抑制最明显。结论 X射线对人结直肠癌细胞株HCT116和SW480的增殖及迁移能力均有抑制作用,可降低人结直肠癌细胞株HCT116和SW480中GOLPH2基因mRNA转录水平及GOLPH2蛋白表达水平。     

结直肠癌中miR-200家族启动子甲基化状态分析

目的 检测HCT116、HT29、LS174t、SW480、SW620和LoVo细胞中miR-200家族启动子甲基化状态,分析其与结直肠癌临床分期、分化程度及预后的相关性;同时进行miR-200家族甲基化程度与患者生存相关性分析.方法 重亚硫酸盐测序法(bisulfite genomic sequence PCR,BSP)检测6株不同恶性程度的结直肠癌细胞株中miR-200家族启动子甲基化程度;分析其甲基化程度与细胞恶性程度、miR-200a表达量的相关性.甲基化特异性(methylationspecific PCR,MSP)技术检测138例结直肠癌组织中miR-200家族启动子甲基化程度.结果 6株不同恶性程度的结直肠癌细胞株中miR-200家族启动子甲基化程度不同,且其甲基化程度与细胞恶性程度成反比,与细胞株中miR-200a表达量的检测结果一致.结直肠癌组织中miR-200家族启动子甲基化程度与患者年龄无关,与患者性别、肿瘤最大径、淋巴结转移及远处转移、患者术后生存时间相关.结论 miR-200家族的表达受甲基化修饰的调控,且该调控成为影响表达量的主要原因.miR-200家族甲基化程度与结直肠癌患者生存期密切相关,可作为预后指标.     

miR-376a、KLF15在胃癌中的表达及对胃癌AGS细胞增殖的影响

目的探讨过表达Cdh1蛋白对高糖诱导的视网膜Müller细胞株GFAP表达的影响。方法通过GEPIA公共数据库分析胃癌中KLF15的表达,通过OncomiR公共数据库分析miR-376a在胃癌中的表达。在AGS细胞系中分别转染miR-376a模拟物和miR-376a抑制物,qRT-PCR和Western blot法检测KLF15的表达,采用CCK-8法检测转染后AGS细胞的增殖能力。通过在线预测工具(TargetscanHuman 7.2, miRDB)预测KLF15是否为miR-376a的靶基因。结果在胃癌中,miR-376a表达升高,KLF15表达水平下降(P0.05);在AGS细胞系中,转染miR-376a模拟物后,miR-376a表达升高,KLF15表达下降(P0.05),AGS细胞的增殖显著升高;转染miR-376a抑制物后,miR-376a的表达水平下降,KLF15表达水平显著升高(P0.05),AGS细胞的增殖能力显著降低。生物信息学分析结果显示KLF15是miR-376a的靶基因之一。结论 miR-376a调节胃癌AGS细胞的增殖能力与下调KLF15的表达相关。     

上调miR-433表达抑制结直肠癌细胞系增殖

目的研究miR-433在结直肠癌中的表达,并初步探讨其功能。方法选择不同分期结直肠癌癌组织(CRC)、癌旁组织与正常结直肠组织,用实时定量PCR检测miR-433表达并比较;体外培养结直肠癌细胞系HT-29、HCT-116和SW480以及正常结直肠细胞系NCM460,用实时定量PCR检测miR-433表达。随后将miR-433模拟物转染SW480细胞,CCK-8法检测细胞增殖;划痕实验检测细胞迁移;Transwell小室法检测细胞侵袭;流式细胞测量术检测细胞周期。结果与癌旁组织相比,miR-433在结直肠癌组织中表达显著降低(P0.05)。在结肠癌患者血清中miR-433水平也显著低于健康人群(P0.05)。此外,从TNM分期Ⅰ期～Ⅳ期,结直肠癌组织内miR-433表达逐渐降低(P0.05)。与NCM460相比,HT-29、HCT-116和SW480中,miR-433表达下调(P0.05)。SW480转染miR-433模拟物后,与对照miRNA组相比,在SW480细胞增殖水平明显降低(P0.05),G_2和M期细胞比例增多。上调miR-433后,细胞侵袭和迁移明显减弱(P0.05)。结论 miR-433在结直肠癌中表达降低。在SW480细胞中上调其表达后,可抑制细胞系增殖、侵袭与迁移。     

结直肠癌中SPOCD1的表达及相关分子机制

目的 探讨含SPOC结构域蛋白1(SPOC domain-containing protein 1, SPOCD1)在结直肠癌中的表达及相关分子机制。方法 利用TCGA数据库和GEO数据库分析结直肠癌和癌旁正常组织中SPOCD1 mRNA的表达;采用免疫组化和Western blot法检测结直肠癌和癌旁正常组织或细胞系中SPOCD1蛋白表达,并分析其与临床病理特征的关系;应用GEPIA和Prognoscan数据库分析SPOCD1表达与结直肠癌患者预后的关系;基因集富集分析SPOCD1可能参与的通路;采用GEPIA数据库分析基因表达之间的相关性;利用siRNA沉默结直肠癌细胞系HCT116中SPOCD1蛋白表达,Western blot法验证下游基因。结果 TCGA和GEO数据库分析结果显示,SPOCD1 mRNA在结直肠癌组织中的表达显著增加;免疫组化结果显示,SPOCD1蛋白在结直肠癌组织中高表达(67.8%,40/59),显著高于癌旁正常组织(37.3%,22/59)(P<0.001);SPOCD1在结直肠癌细胞系(HCT116和LoVo)中的表达高于正常结直肠细胞系(NCM...     

miR-496过表达抑制结肠癌细胞生长和转移

目的:研究miR-496过表达对结肠癌细胞生长和转移的影响及其分子机制。方法:运用生物信息学软件筛选miR-496靶向相互作用蛋白;real-time PCR和Western blot法测定结肠癌细胞系HT29、HCT116、SW480以及正常结肠上皮细胞NCM460中miR-496、CTNNB1 mRNA和β-catenin蛋白的表达;运用Lipofectamine 2000将miR-496 mimics转染HT29、HCT116和SW480细胞,分别命名为HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞,转染scramble为阴性对照;运用MTT法、乳酸脱氢酶(LDH)试剂盒法、克隆形成实验和Transwell分别测定细胞活力、LDH漏出率、克隆形成能力和转移能力;萤光素酶报告基因实验测定miR-496启动子活性;Western blot法测定β-catenin、真核细胞翻译起始因子4E结合蛋白1(4E-BP1)、p-4E-BP1、低密度脂蛋白受体相关蛋白6(LRP6)、p-LRP6、MMP-7、MMP-9、MMP-13以及TIMP-2的蛋白水平。结果:miR-496与β-catenin内源性相互作用;miR-496在HT29、HCT116和SW480细胞中低表达,而在NCM460高表达;β-catenin在HT29、HCT116和SW480细胞中高表达,而在NCM460低表达;培养24 h、48 h、72 h、96 h的HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞活力、LDH漏出率、克隆形成率和转移的细胞数均显著低于对照组(P0.05);萤光素酶报告基因实验结果显示转染miR-496 mimics细胞中的miR-496启动子活性明显增加(P0.05),分别是对照组的1.75倍、2.04倍和1.61倍。Western blot实验结果显示miR-496过表达抑制β-catenin蛋白表达,p-4E-BP1和p-LRP6的蛋白水平降低;siRNA或miR-496过表达介导的β-catenin表达下调能显著抑制MMP-7和MMP-9的表达,促进TIMP-2的表达。结论:miR-496在结肠癌细胞中低表达,在正常结肠上皮细胞中高表达;miR-496过表达抑制结肠癌细胞的生长和转移,其机制是通过抑制Wnt/β-catenin通路进一步抑制MMP-7和MMP-9表达,促进TIMP-2表达,从而抑制结肠癌细胞的恶性表型。     

Pain and Effusion and Quadriceps Activation and Strength

Context:

Quadriceps dysfunction is a common consequence of knee joint injury and disease, yet its causes remain elusive.

Objective:

To determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion affect the magnitude of quadriceps dysfunction.

Design:

Crossover study.

Setting:

University research laboratory.

Patients or Other Participants:

Fourteen (8 men, 6 women; age = 23.6 ± 4.8 years, height = 170.3 ± 9.16 cm, mass = 72.9 ± 11.84 kg) healthy volunteers.

Intervention(s):

All participants were tested under 4 randomized conditions: normal knee, effused knee, painful knee, and effused and painful knee.

Main Outcome Measure(s):

Quadriceps strength (Nm/kg) and activation (central activation ratio) were assessed after each condition was induced.

Results:

Quadriceps strength and activation were highest under the normal knee condition and differed from the 3 experimental knee conditions (P < .05). No differences were noted among the 3 experimental knee conditions for either variable (P > .05).

Conclusions:

Both pain and effusion led to quadriceps dysfunction, but the interaction of the 2 stimuli did not increase the magnitude of the strength or activation deficits. Therefore, pain and effusion can be considered equally potent in eliciting quadriceps inhibition. Given that pain and effusion accompany numerous knee conditions, the prevalence of quadriceps dysfunction is likely high.Key Words: arthrogenic muscle inhibition, central activation failure, voluntary activation, muscles

Key Points

• Knee pain and effusion resulted in arthrogenic muscle inhibition and weakness of the quadriceps.
• The simultaneous presence of pain and effusion did not increase the magnitude of quadriceps dysfunction.
• To reduce arthrogenic muscle inhibition and improve muscle strength, clinicians should employ interventions that target removing both pain and effusion.

Quadriceps weakness is a common consequence of traumatic knee joint injury^1,2 and chronic degenerative knee joint conditions.^3,4 Arthrogenic muscle inhibition (AMI), a neurologic decline in muscle activation, results in quadriceps weakness and hinders rehabilitation by preventing gains in strength.⁵ The inability to reverse AMI and restore muscle function can lead to decreased physical abilities,⁶ biomechanical deficits,⁷ and possibly reinjury.⁵ Furthermore, researchers^8,9 have suggested that quadriceps weakness resulting from AMI may place patients at risk for developing osteoarthritis in the knee. In light of the substantial influence of quadriceps AMI on these clinically relevant outcomes, we need to improve our understanding of the factors that contribute to this neurologic decline in muscle activity so efforts to target and reverse it can be implemented and gains in strength can be achieved more easily.Joint injury and disease are accompanied by numerous sequelae (ie, pain, swelling, tissue damage, inflammation), so ascertaining which one ultimately leads to neurologic muscle dysfunction is difficult. Whereas a joint effusion can result in AMI,^10–12 the effects of pain are less understood despite many clinicians attributing AMI to pain. Using techniques that introduce knee pain without accompanying injury may provide insights into the role of pain in eliciting AMI.The degree of knee joint damage may play a role in the quantity of AMI that manifests. Hurley et al^13,14 demonstrated that quadriceps AMI, measured using an interpolated-twitch technique, was greater in patients with extensive traumatic knee injury (eg, fractured tibial plateau, ruptured medial collateral ligament, and medial meniscectomy) than patients with isolated joint trauma (ie, isolated anterior cruciate ligament [ACL] rupture). Similarly, patients with more knee joint symptoms (ie, greater number of symptoms and increased severity of symptoms) may present with greater magnitudes of quadriceps inhibition. Recently, investigators¹⁵ have suggested that patients with more pain display less quadriceps strength, supporting this tenet. Given that effusion and pain often present simultaneously with joint injuries and diseases, such as ACL injury and osteoarthritis, examining both the isolated and cumulative effects of these sequelae appears warranted to determine if they influence the magnitude of muscle inhibition.Experimental joint-effusion and pain models are safe and effective experimental methods that allow for the isolated examination of their effects on muscle function. The effusion model, whereby sterile saline is injected directly into the knee joint capsule,⁷ produces a clinically relevant magnitude of the joint effusion that may be present with traumatic injury. Effusion is thought to activate group II afferents responding to stretch or pressure,^16–18 which in turn may facilitate group Ib interneurons and result in quadriceps AMI.⁵ The pain model involves injecting hypertonic saline into the infrapatellar fat pad to produce anteromedial knee pain similar to that described in patients with patellofemoral pain syndrome.¹⁹ Pain is considered to initiate AMI through activation of group III and IV afferents that act as nocioceptors to signal damage or potential damage to joint structures.^16–18 The firing of these afferents then may lead to facilitation of group Ib interneurons, the flexion reflex, or the gamma loop, ultimately resulting in quadriceps inhibition.²⁰ Thus, these models allow us to create symptoms that are associated with knee injury and have the added benefit of providing a way to examine their effects in isolation.Therefore, the purpose of our study was to determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion would affect the magnitude of quadriceps dysfunction. We hypothesized that pain alone would result in quadriceps inhibition and that the magnitude of inhibition would be greater when effusion and pain were present simultaneously.     

即早基因c-fos与脑血管病及学习记忆

即早基因c-fos是广泛存在于原核细胞和真核细胞的高度保守基因.在正常情况下,c-fos基因参与细胞生长、分化、信息传递、学习和记忆等生理过程,而在病理情况下c-fos基因表达及调控变化与多种疾病的发生和发展有关.C-fos在中枢神经系统的某些部位可有基础水平的表达,但表达很低,当受到如脑缺血、脑出血、痫性发作、应激等刺激后,其在数十分钟内做出反应,在对外界刺激-转录耦联的信忠传递过程中起着核内第三信使的重要作用.     

Opportunities and challenges in the prevention and control of cancer and other chronic diseases: children's diet and nutrition and weight and physical activity

OBJECTIVE: The purpose of this article is to review the role of behavioral research in disease prevention and control, with a particular emphasis on lifestyle- and behavior-related cancer and chronic disease risk factors--specifically, relationships among diet and nutrition and weight and physical activity with adult cancer, and tracking developmental origins of these health-promoting and health-compromising behaviors from childhood into adulthood. METHOD: After reviewing the background of the field of cancer prevention and control and establishing plausibility for the role of child health behavior in adult cancer risk, studies selected from the pediatric published literature are reviewed. Articles were retrieved, selected, and summarized to illustrate that results from separate but related fields of study are combinable to yield insights into the prevention and control of cancer and other chronic diseases in adulthood through the conduct of nonintervention and intervention research with children in clinical, public health, and other contexts. RESULTS: As illustrated by the evidence presented in this review, there are numerous reasons (biological, psychological, and social), opportunities (school and community, health care, and family settings), and approaches (nonintervention and intervention) to understand and impact behavior change in children's diet and nutrition and weight and physical activity. CONCLUSIONS: Further development and evaluation of behavioral science intervention protocols conducted with children are necessary to understand the efficacy of these approaches and their public health impact on proximal and distal cancer, cancer-related, and chronic disease outcomes before diffusion. It is clear that more attention should be paid to early life and early developmental phases in cancer prevention.