靶向干扰前列腺源性ETS因子促进HT29细胞的增殖与侵袭

目的:结直肠癌是常见的消化道肿瘤之一,研究发现在结直肠组织,前列腺源性ETS因子(Prostate-derived Ets factor,PDEF)的表达可以促进分泌性祖细胞向杯状细胞分化,因此结直肠癌的发生可能与PDEF的表达有关。本研究旨在探讨靶向干扰前列腺源性ETS因子对结直肠癌细胞株HT29增殖与侵袭的影响。方法:阳离子脂质体法将PDEF干扰质粒和空白对照空质粒瞬时转染HT29细胞,通过荧光显微镜、RT-PCR、Western blot技术测定正常对照组、空白对照组和shRNA组中PDEF mRNA和蛋白的表达情况;MTT法检测HT29细胞的增殖情况;采用Transwell迁移实验观察细胞侵袭能力。结果:干扰质粒、空质粒成功转染HT29细胞,通过荧光显微镜可以观察到绿色荧光蛋白的表达;Western blot结果可见shRNA组PDEF蛋白的表达较正常对照组和空白对照组降低;MTT法检测发现干扰PDEF基因的表达能够明显促进HT29细胞的增殖(P<0.05);48 h后,shRNA组细胞侵袭能力明显强于正常对照组和空白对照组(P<0.05)。结论:在HT29细胞中干扰PDEF的表达,可以促进细胞的增殖与侵袭。     

沉默C20ORF32表达对结直肠癌LOVO细胞增殖与侵袭的影响

目的探讨沉默Crk相关底物家族成员C20ORF32基因表达后,对结直肠癌细胞增殖和侵袭的影响。方法应用蛋白印迹法(Western blot)检测4种结直肠癌细胞系(LOVO、SW620、HT-29和SW480)中C20ORF32的表达状况;用装载了C20ORF32 shRNA的慢病毒颗粒,感染高表达C20ORF32的结直肠癌细胞系LOVO后,运用免疫荧光染色和蛋白质免疫印迹的方法检测其对C20ORF32基因的沉默效果,并通过MTT法和软琼脂克隆形成实验检测对细胞增殖能力的影响,用Transwell侵袭实验检测细胞的体外侵袭能力。结果与其他3株细胞系相比,C20ORF32蛋白在LOVO细胞中明显高表达(t=15.33,P0.001)。下调C20ORF32表达后,LOVO细胞的增殖速度在24h以及48h显著降低(24hF=81.32,P0.001;48hF=13.33,P0.05),集落数目明显减少(F=74.21,P=0.0002),细胞侵袭能力明显减弱(F=144.38,P0.001)。结论 C20ORF32的高表达可能与结直肠癌细胞的快速增殖与侵袭有关,参与了结直肠癌的恶性进展过程。     

长链非编码RNA TINCR 通过miR-7调控肝癌细胞系Huh7侵袭和迁移

目的研究长链非编码RNA(lncRNA)组织分化诱导非蛋白质编码RNA(TINCR)通过miR-7调控肝癌Huh7细胞侵袭和迁移的机制。方法 Real-time PCR测定Huh7细胞中TINCR表达。Huh7细胞中转染TINCR shRNA,CCK-8法测定增殖;Transwell小室法测定侵袭和迁移;Western blot测定MMP-2和MMP-9蛋白表达。生物信息学软件预测发现TINCR和miR-7有结合位点,荧光素酶报告系统鉴定二者靶向关系。Huh7细胞中共转染miR-7 inhibitor和TINCR shRNA,利用上述方法测定细胞增殖、迁移和侵袭变化。结果肝癌细胞中TINCR表达水平明显高于正常肝细胞(P0.05)。转染TINCR shRNA后的Huh7细胞中TINCR水平降低(P0.05),细胞增殖、迁移以及侵袭能力均下降(P0.05),细胞中MMP-2和MMP-9蛋白水平也降低(P0.05)。TINCR靶向负调控miR-7表达。miR-7 inhibitor可以提高下调TINCR后的肝癌细胞增殖、迁移、侵袭能力以及MMP-2、MMP-9蛋白表达水平(P0.05)。结论下调TINCR可以通过靶向促进miR-7表达抑制肝癌细胞Huh7侵袭和迁移。     

长链非编码RNA EVADR促进人结直肠癌细胞系增殖及迁移

目的探讨lncRNA EVADR对人结直肠癌HCT116和LOVO细胞系的增殖和迁移的影响以及其作用机制。方法利用慢病毒感染方式分别构建稳定过表达lncRNA EVADR的HCT116和LOVO细胞系;用CCK-8检测细胞的增殖;Transwell小室法检测细胞迁移能力,Western blot检测E-cadherin的表达,实时荧光定量PCR检测Snail、Slug、ZEB1和ZEB2 mRNA的表达。结果成功构建了稳定过表达lncRNA EVADR的结直肠癌HCT116和LOVO细胞系。与HCT116-NC和LOVO-NC细胞组对比,HCT116-EVADR与LOVO-EVADR细胞组的增殖和迁移能力明显提高(P<0.05)。同时,上皮标志物E-cadherin表达明显降低,而Snail、Slug、ZEB1和ZEB2间质化标志物的表达升高。结论过表达lncRNA EVADR具有促进结直肠癌HCT116和LOVO细胞的增殖与迁移能力,可能通过调节结直肠癌细胞上皮间质转化(EMT)发挥作用。     

WNT信号抑制因子DKK3基因 对结直肠癌细胞系HCT116迁移与侵袭的影响

目的分析DKK3在结直肠癌组织中的表达及对患者预后的影响,同时探索DKK3对结直肠癌细胞增殖、迁移以及侵袭能力的影响,并对机制进行一定的探索。方法在线数据库分析DKK3在结直肠癌组织中的表达及其对患者预后的影响。用RNA干扰技术,下调DKK3;用MTT检测细胞增殖,同时运用细胞小室迁移实验检测细胞迁移及侵袭能力,并通过RT-qPCR技术以及Western blot技术检测迁移相关蛋白表达。结果与正常组织相比,DKK3在结直肠癌组织中的表达显著上调并会导致患者整体预后情况变差(P0.05),干涉DKK3后细胞增殖速率明显减缓(P0.05),并且细胞迁移以及侵袭能力减弱(P0.01)。MMP2、 MMP7以及MMP9与DKK3的表达呈正相关关系(P0.001)。在敲低DKK3后,MMP2、 MMP7、 MMP9以及p-ERK的表达均显著下调(P0.01)。结论 DKK3在结直肠癌细胞系HCT116中表达上调,可能通过MAPK/ERK信号通路促进细胞增殖以及迁移能力。     

外泌体circRPPH1促进结直肠癌增殖和转移北大核心CSCD

目的:探讨外泌体circRPPH1在结直肠癌(CRC)细胞中的生物学功能及潜在作用机制。方法:对GEO数据库GSE126094进行分析,筛选并验证CRC组织差异表达circRNA。SW620和SW480细胞转染circRPPH1 shRNA(sh-circRPPH1)后,MTT、流式细胞术和Transwell实验观察细胞增殖活性、细胞周期、迁移和侵袭能力变化。将转染Oe-circRPPH1质粒和sh-circRPPH1的SW620和SW480细胞(供体细胞)分别与未经处理的SW620和SW480细胞(受体细胞)共培养,qRT-PCR检测供体细胞、细胞外泌体及受体细胞circRPPH1表达。在线生物信息学数据和双荧光素酶报告基因实验预测并验证circRPPH1的靶miRNA及其下游靶基因。结果:circRPPH1在CRC肿瘤组织和癌细胞中高表达;敲除circRPPH1可抑制SW620和SW480细胞增殖、阻滞细胞周期、抑制细胞迁移和侵袭。SW620和SW480细胞可通过外泌体将circRPPH1传递给周围癌细胞。生物信息学和荧光素酶报告基因实验显示,circRPPH1在CRC细胞中起miR-330-5p海绵作用,miR-330-5p在CRC肿瘤组织和癌细胞中均呈低表达;NRAS是miR-330-5p的下游靶基因,在CRC肿瘤组织和癌细胞中均呈高表达。结论:circRPPH1在CRC细胞增殖和转移过程中发挥重要作用,并可能通过海绵miRNA发挥调控作用,提示外泌体circRPPH1在CRC中起致癌作用,可能是CRC的潜在生物标志物。     

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。     

Tiam1对结直肠癌细胞生物学特性的影响

目的探讨Tiam1(Tlymphomainvasionandmetastasis)对结直肠癌细胞株生物学特性的影响。方法用逆转录聚合酶链反应方法检测Tiam1基因在结直肠癌细胞株中的表达,筛选Tiam1不表达的细胞株;将Tiam1/C1199HAcDNA导入内源性不表达Tiam1基因的人结直肠癌HT29细胞株中,G418筛选抗性克隆,免疫组织化学和Western蛋白印迹法鉴定转染后Tiam1基因在细胞中的表达,利用四甲基偶氮唑盐(MTT)法检测Tiam1对细胞增殖的影响,体外侵袭实验检测Tiam1对结直肠癌侵袭转移的影响。结果Tiam1基因在高转移的LoVo和SW620中高表达,在低转移的HT29中不表达,在SW480和HCT116细胞表达相应较低。通过7d的连续比较,HT29/mock和HT29/Tiam1两组的细胞增殖能力差异有统计学意义(F=10.512,P=0.003)。Tiam1基因的导入使结直肠癌细胞的增殖能力明显增强,体外侵袭转移能力显著增加,HT29/Tiam1穿过细胞为(88.6±9.2)个/200倍视野,HT29/mock为(46.8±3.4)个/200倍视野,二者的差异具有统计学意义(t=9.54,P<0.01)。结论Tiam1基因具有促进结直肠癌细胞增殖的能力,Tiam1与结直肠癌的侵袭转移密切相关,Tiam1表达可作为结直肠癌侵袭转移过程中一个有价值的指标。     

miR-186上调Dicer1抑制结肠癌细胞侵袭转移的分子机制

为探讨miR-186通过Dicer1影响结肠癌细胞侵袭转移及其作用机制,RT-qPCR检测miR-186在结肠癌组织及正常癌旁组织、人结肠癌细胞及正常人肠上皮细胞HIEC中的表达;荧光显微镜观察稳定过表达/下调miR-186细胞系GFP荧光并采用RT-qPCR检测miR-186表达效率;Transwell及划痕实验检测miR-186对结肠癌细胞SW620和HT29侵袭、迁移能力的影响;免疫荧光法检测上皮间质转化(epithelial mesenchymal transition, EMT)相关分子N-cadherin表达;生物信息学预测及双荧光素酶报告基因实验验证miR-186和Dicer1的靶向关系;免疫组织化学法检测Dicer1在结肠癌组织及正常癌旁组织中的表达;Western blotting检测Dicer1在各细胞株中的表达。结果显示,肿瘤组织中miR-186的表达水平显著低于正常癌旁组织(P0.05);miR-186在肿瘤细胞中的表达水平显著低于HIEC(P0.05)。转染过表达/下调miR-186慢病毒阳性质粒及阴性对照的SW620和HT29细胞均出现大量绿色荧光;RT-qPCR显示稳定过表达细胞SW620-mimic-miR-186中miR-186水平显著升高,HT29-inhibitor-miR-186中miR-186水平显著下降,分别与SW620和SW620-mimic-NC、HT29和HT29-inhibitor-NC相比差异有统计学意义(P0.05)。过表达miR-186能显著降低SW620的侵袭迁移能力,同时抑制N-cadherin水平,而下调miR-186能显著增加HT29的侵袭迁移能力。经www.microRNA.org网站预测发现miR-186和Dicer1有互补结合序列,双荧光素酶报告实验证实两者的靶向结合关系。SW620中Dicer1表达水平显著低于HIEC(P0.05);稳定过表达细胞系SW620-mimic-miR-186中Dicer1表达水平明显升高,分别与SW620和SW620-mimic-NC相比差异有统计学意义(P0.05)。由此,miR-186通过靶向正调控Dicer1的表达降低N-cadherin水平从而抑制EMT进程,最终抑制结肠癌细胞的侵袭迁移能力。     

长链非编码RNA Linc00467在结直肠癌中的表达及影响

目的探讨长链非编码RNA Linc00467在结直肠癌中的表达和意义。方法实时荧光定量PCR检测Linc00467在结直肠癌组织和结肠癌细胞系中的表达水平。通过Transwell迁移实验、MTS实验和克隆形成实验检测Linc00467对结肠癌细胞迁移、增殖等行为的影响。统计纳入的67例患者的临床病理资料,分析Linc00467的表达水平和患者临床病理学特征之间的关系。结果 Linc00467在结直肠癌组织和细胞系中高表达。敲低Linc00467可以抑制结肠癌细胞的迁移和增殖。Linc00467的表达丰度与结直肠癌患者的临床病理特征之间无显著相关性。结论 Linc00467在结直肠癌中高表达,可以促进结肠癌的迁移和增殖。     

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.