Abstract ：

The molecular mechanism of the tyrosine kinase inhibitor (TKI) resistant lung adenocarcinoma is currently unclear, and the role of methylated adenosine at the N6 position in the resistance of cancer stem cells (CSCs) therapy is unknown. This study identified a novel and effective strategy to enhance TKIs therapy response. We first confirmed the sensitization of Metformin enforcing on Osimertinib treatment and revealed the mature miRNAs signatures of the Osimertinib resistant H1975 and HCC827 cells. Let-7b expression was stimulated when adding Metformin and then increasing the therapy sensitivity by decreasing the stem cell groups expanding. Methyltransferase-like 3 (METTL3) increased the pri-Let-7b, decreased both the pre-Let-7b and mature Let-7b, attenuating the Let-7b controlling of stem cell renewal. The addition of Metformin increased the bindings of DNA methyltransferase-3a/b (DNMT3a/b) to the METTL3 promoter. With the help of the readers of NKAP and HNRNPA2B1, the cluster mediated m6A formation on pri-Let-7b processing increased the mature Let-7b, the key player in suppressing Notch signaling and re-captivating Osimertinib treatment. We revealed that the maturation processing signaling stimulated the methylation regulation of the miRNAs, and may determine the stemness control of the therapy resistance. Our findings may open up future drug development, targeting this pathway for lung cancer patients.

Keyword ：

cancer stem-like cells miRNAs maturation n6-methyladenosine therapy resistance tyrosine kinase inhibitor

Cite：

Copy from the list or Export to your reference management。

Abstract ：

目的初步研究let-7b和SALL4在肝母细胞瘤(hepatoblastoma,HB)细胞恶性行为中的作用及二者间的相互作用机制。方法采用qRT-PCR检测let-7b在正常肝细胞(HL-7702)与HB细胞系、HB组织及癌旁组织中的表达水平,采用免疫组化法检测SALL4在HB组织及癌旁组织中的表达水平,并分析HB组织中let-7b与SALL4表达水平的相关性。通过MTT、细胞周期和凋亡等实验方法上调或抑制HB细胞中let-7b的表达水平,观察let-7b对HB细胞生物学功能的影响;通过生物信息学预测软件、双荧光素酶报告系统、qRT-PCR和western blot等实验,从体外水平验证let-7b与SALL4的靶向关系。采用Kaplan-Meier生存曲线分析不同let-7b与SALL4表达水平的肝母细胞瘤患者总体生存率的差异。结果与癌旁组织相比,let-7b在HB组织中表达量显著降低,而SALL4表达量显著升高;let-7b在肝母细胞瘤组织中的表达水平与PRETEXT分期存在相关性(P=0.002),且HB组织标本中let-7b与SALL4的表达水平存在负相关性(r=-0.716,P<0.001)。let-7b低表达组HB患者的总体生存率显著低于let-7b高表达组(P=0.017)。SALL4阳性组HB患者的总体生存率显著低于SALL4阴性和弱阳性组(P=0.034)。MTT、细胞周期和凋亡实验结果显示,let-7b可抑制肝癌Hep G2细胞增殖,促进细胞的凋亡。而抑制内源性的let-7b可促进Hep G2细胞增殖,抑制细胞的凋亡。生物信息学软件预测及构建双荧光报告载体荧光检测等实验结果显示,let-7b与SALL4存在一定的靶向关系。结论let-7b和SALL4可作为肝母细胞瘤不良预后的潜在标记物;let-7b作为抑癌基因可以靶向调控SALL4的表达,抑制HB细胞的增殖。

Keyword ：

let-7b SALL4 肝肿瘤 细胞增殖

Cite：

Copy from the list or Export to your reference management。

Abstract ：

目的初步研究let-7b和SALL4在肝母细胞瘤(hepatoblastoma,HB)细胞恶性行为中的作用及二者间的相互作用机制。方法采用qRT-PCR检测let-7b在正常肝细胞(HL-7702)与HB细胞系、HB组织及癌旁组织中的表达水平,采用免疫组化法检测SALL4在HB组织及癌旁组织中的表达水平,并分析HB组织中let-7b与SALL4表达水平的相关性。通过MTT、细胞周期和凋亡等实验方法上调或抑制HB细胞中let-7b的表达水平,观察let-7b对HB细胞生物学功能的影响;通过生物信息学预测软件、双荧光素酶报告系统、qRT-PCR和western blot等实验,从体外水平验证let...

Keyword ：

let-7b SALL4 肝肿瘤 细胞增殖

Cite：

Copy from the list or Export to your reference management。

Abstract ：

Matrine, a natural product extracted from the root of Sophora flavescens Ait, was the main chemical ingredient of compounds of Kushen injection, which has been widely used for its remarkable anticancer effects for years. The underlying mechanisms for Matrine regulations of human breast cancer stem cells (BrCSCs) are barely known. LIN28, a well-characterized suppressor of Let-7 microRNA biogenesis, playing vital roles in regulations of stem cells' renewal and tumorigenesis. Here we show that the compounds of Kushen injection derived Matrine could suppress the BrCSCs differentiation and self-renewal through downregulating the expression of Lin28A, resulting in the inactivation of Wnt pathway through a Let-7b-dependent way. In opposite to Matrine, Cisplatin treatment increases the ability of tumorsphere formation and the expression of BrCSCs markers, which was partially blocked by either Let-7b overexpression or CCND1 inhibition. Furthermore, Matrine sensitized BrCSCs to cisplatin's suppression of cancer expansion in vitro and in vivo. Our study uncovers the role of the LIN28A/Let-7 in BrCSCs renewal, and more importantly, elucidated a novel mechanism by which Matrine induces breast cancer involution.

Keyword ：

breast cancer stem cells Let-7 LIN28A matrine

Cite：

Copy from the list or Export to your reference management。

Abstract ：

Matrine is one of the major alkaloids extracted from Sophora flavescens Ait of the traditional Chinese medicine, was the main chemical ingredient of compounds of Kushen injection. The Matrine is considered as a promising therapeutic agent for curing nonsmall cell lung cancer (NSCLC), used either alone or combined with chemotherapeutic agents. In the present study, we focused on the possible roles of Matrine exerted on the self-renewal ability of stem-like cells of the NSCLC group, as well as the cytotoxicity of chemotherapeutic agents, in vitro and in vivo. Here we reported that Matrine inhibits cancer stem-like cell (CSC) properties through upregulation of Let-7b and suppression of the Wnt pathway. Overexpression of Let-7b suppressed the ability of tumorsphere formation, decreased Wnt pathway activation through inhibiting its transcriptional activity in lung CSCs. Further studies revealed that Let-7b directly targeted CCND1 and decreased its expression, whereas Matrine increased Let-7b levels and followed by inactivation of the CCND1/Wnt signaling pathway and inhibition of EMT, which was characterized by loss of epithelial markers and acquisition of a mesenchymal phenotype in lung CSCs. What is more, we found that Matrine increased Let-7b level in an endoribonuclease DICER1-dependent manner. And xenografts in nude mice evidenced that Matrine increased the sensitivity of lung CSCs to 5-FU and inhibited the accumulation of CCND1 in tumor tissues induced by 5-FU. Taken together, these data illustrate the role of Let-7b in regulating lung CSCs traits and DICER1/let-7/CCND1 axis in Matrine or in combination with 5-FU intervention of lung CSCs' expansion, helping to fulfill the anti-cancer action of Matrine.

Keyword ：

cancer stem cells division manners Matrine NSCLC therapy resistance

Cite：

Copy from the list or Export to your reference management。

Abstract ：

背景： 肝细胞肝癌（Hepatocellular carcinoma，HCC）是全球范围内常见的临床肿瘤疾病，其发病率和死亡率居高不下，严重危害人类健康。HCC的早期诊断困难，初诊时多数患者已无法进行手术治疗，且肿瘤的术后复发率非常高。基于上述问题，探索并完善肝癌的发展机制，为肝癌的治疗寻找更有效的靶点具有非常重要的临床意义。 长链非编码RNA（long noncoding RNA, lncRNA）在生物体RNA中的占比可以达到98%，其序列保守性低，具有十分复杂的生物学功能，可以从表观遗传水平、转录水平、翻译水平多方面发挥细胞功能调控作用。既往大量研究证明lncRNA在肿瘤的发生和发展过程中发挥重要作用，可以影响肿瘤细胞增殖、转移、衰老、自噬等多种生物学过程。有研究证实长链非编码RNA HOXA11-AS在肿瘤中发挥关键作用，但是研究尚不完整。HOXA11-AS在肝癌中的机制研究较少，需要进一步进行探索。 目的： 本课题旨在分析长链非编码RNA HOXA11-AS在肝细胞肝癌中的临床病理意义，探索HOXA11-AS在肝癌发展中的作用及其分子机制，为寻找新的预后分子标志物和治疗分子靶点提供理论依据。 方法： 1.采用RT-PCR实验，对我院单中心样本数据和不同肝癌细胞系进行HOXA11-AS的表达检测，结合公共数据库对HOXA11-AS表达水平进行验证； 2.采用卡方检验分析HOXA11-AS表达水平与HCC患者临床病理特征间的关系； 3.采用Kaplan-Merier法独立或联合分析HOXA11-AS、CCND1表达水平与HCC患者预后之间的关系；采用COX单因素和多因素计算分析法评估HOXA11-AS表达水平对HCC患者预后的指导意义； 4.在MHCC-97H和HepG2肝癌细胞中分别构建稳定敲低HOXA11-AS的肝癌细胞系，分别转染let-7a、let-7b、let-7c的类似物或者抑制物； 5.CCK-8细胞增殖实验、平板克隆形成实验、流式细胞实验、Transwell实验检测细胞增殖、迁移、周期、凋亡、衰老能力； 6.不同细胞系构建裸鼠皮下移植瘤模型，体内实验验证细胞实验发现的分子机制； 7.RNA免疫杂交（FISH）实验、RT-PCR、western blot、免疫组化实验验证HOXA11-AS的细胞定位和调控下游基因的作用； 8.RNA-pull down实验、RIP实验、荧光素酶报告实验验证HOXA11-AS与let-7a/b/c间的相互作用关系，明确CCND1与let-7a/b/c间的相互作用关系； 9.ChIP实验明确SUV39H2对P21转录时的甲基化修饰调控作用。 结果： 1.HOXA11-AS在TCGA肝癌样本、我院50例肝癌样本、不同肝癌细胞系中表达出现不同程度升高，与正常肝脏组织和正常肝脏细胞相比较，差异有统计学意义； 2.HOXA11-AS高表达与多种肿瘤恶性临床特征密切相关。TCGA中肝癌患者数据分析发现高HOXA11-AS提示患者预后更差，高血清AFP水平、较大肿瘤、肿瘤低分化程度、肿瘤转移、TNM分期较晚、高HOXA11-AS表达是提示患者较低术后生存率和较高术后复发率的独立危险因素； 3.HOXA11-AS在肝癌细胞的胞质和胞核中均有表达； 4.在肝癌细胞中稳定下调HOXA11-AS显著抑制了MHCC-97H和HepG2两种肝癌细胞的增殖、迁移和侵袭，造成细胞周期阻滞，显著提高细胞的凋亡比例和衰老比例；裸鼠皮下移植瘤模型中抑制HOXA11-AS的表达能够显著促进肿瘤组织衰老，抑制组织生长； 5.稳定敲低HOXA11-AS的肝癌细胞中发现CCND1表达显著降低，P21表达显著升高；肝癌细胞中过表达CCND1显著抑制肝癌细胞衰老表型的发生，同时抑制HOXA11-AS的表达，细胞发生衰老的比例有所回升； 6.生物素标记的HOXA11-AS探针与Ago2形成复合物，同时与let-7a、let-7b、let-7c结合沉淀，过表达let-7a/b/c能够显著下调携带野生型HOXA11-AS结合序列的肝癌细胞的荧光强度； 7.肝癌组织中CCND1表达水平与let-7a/b/c表达水平间存在显著负相关关系，肝癌细胞中分别过表达let-7a/b/c，CCND1的表达量在转录水平和蛋白水平均出现了显著下降。过表达let-7a/b/c能够显著下调携带野生型CCND1 3-UTR的肝癌细胞的荧光强度，另外，抑制HOXA11-AS表达引起的CCND1表达降低在分别加入let-7a/b/c的抑制物后P21表达明显升高； 8.生物素标记的HOXA11-AS探针能够下拉SUV39H2蛋白，干扰SUV39H2表达后P21mRNA水平显著升高，Anti-SUV39H2抗体、Anti-H3K9me3抗体捕获复合物中可以检测发现结合P21启动子区域序列表达； 结论： 1.长链非编码RNA HOXA11-AS在肝癌中表达异常升高，HOXA11-AS与HCC多种恶性临床病理特征关系密切，高表达HOXA11-AS是提示HCC患者不良预后的独立危险因素； 2.抑制长链非编码RNA HOXA11-AS能够促进肝癌细胞衰老，抑制肝癌细胞增殖和迁移的能力，发挥抑制肿瘤生长的作用； 3.CCND1是HOXA11-AS抑制肝癌细胞衰老表型的关键分子，HOXA11-AS在胞质种作为ceRNA竞争性结合let-7a/b/c，激活其下游靶mRNA CCND1翻译，进而抑制肝癌细胞衰老的发生； 4.HOXA11-AS在胞核中SUV39H2至P21基因启动子区域，通过H3K9三甲基化修饰抑制P21的转录水平表达。

Keyword ：

ceRNA lncRNA HOXA11-AS SUV39H2 肝细胞肝癌 衰老

Cite：

Copy from the list or Export to your reference management。

Abstract ：

Reactivation of Lin28 accelerates hair, cartilage, bone and mesenchyme regrowth after ear and digit injuries. However, the relationship of Lin28 to reparative dentin has been under investigation. The aim of the present study was to examine whether Lin28 participates in the reparative dentin process and lipopolysaccharide (LPS)stimulated human dental pulp cells (HDPCs) and to identify the underlying signaling pathway mechanisms. The study established a wound-healing model of the dentin-dental pulp complex in vivo and LPS-induced dental pulp cell inflammation in vitro. In vivo, the results of hematoxylin and eosin staining demonstrated the obvious appearance of reparative dentin and odontoblast-like cells were arranged along the reparative dentin. Immunohistochemical examination demonstrated that Lin28 expression was increased by 72 h after cavity preparation but was decreased by 21 d after cavity preparation. In vitro, HDPCs were exposed to 100 ng/ml LPS for 24 h, and the expression of Lin28 was increased. Overexpression of Lin28 was associated with the downregulated expression of let-7b, let-7g and miR-98. These findings suggest that the wound-healing model was successfully established. Lin28 was involved in the reparative process of the dentin-dental pulp complex and HDPCs exposed to LPS, and Lin28/let-7 may be the underlying mechanism.

Keyword ：

Dentin-dental pulp complex let-7 families Lin28 lipopolysaccharide wound-healing model

Cite：

Copy from the list or Export to your reference management。

Abstract ：

目的:探究miR-129在乳腺癌中对肿瘤干细胞自我更新能力的调控作用及其机制.方法:应用免疫组化检测乳腺癌肿瘤干细胞在肿瘤组织与癌旁组织中的数量及其与乳腺癌肿瘤分期的关系,验证miR-129和Numb与乳腺癌肿瘤干细胞之间的相关关系.在体外实验中应用Western Blotting及RT-PCR验证miR-129通过阻断雌激素受体alpha(ER alpha,ESR1)对Notch信号通路的调节作用及其调节的具体机制;应用裸鼠成瘤实验验证miR-129在裸鼠体内水平对乳腺癌肿瘤干细胞的影响.结果:肿瘤组织中乳腺癌肿瘤干细胞的比例高于癌旁组织并与肿瘤分期具有相关性;乳腺癌肿瘤干细胞比例与临床样本中miR-129和Numb的表达水平呈负相关;乳腺癌肿瘤干细胞中miR-129的过表达与Notch信号通路的抑制直接相关,这种作用很可能是miR-129通过调控ESR1所引起的Let-7b的表达下调进而导致Numb的释放来实现的;miR-129在裸鼠体内实验中可以抑制乳腺癌肿瘤干细胞的成瘤.结论:miR-129在乳腺癌组织中可以通过调控Notch信号通路影响乳腺癌肿瘤干细胞自我更新的能力,具体的调节机制可能是通过调控ESR1所引起的Let-7b的表达下调进而导致Numb的释放来最终抑制Notch信号通路的激活.

Keyword ：

Let-7b miR-129 Notch信号通路 Numb 雌激素受体alpha

Cite：

Copy from the list or Export to your reference management。

Abstract ：

Alzheimer's disease is very difficult to clinically diagnose. miRNAs constitute the promising next generation of Alzheimer's disease (AD) biomarkers and have the potential to diagnose neurodegenerative diseases. In this study, 94 subjects underwent extensive dementia screening. Let-7b miRNA was found to increase in association with the progression of AD, and the increase in let-7b miRNA was mainly due to CD4+ T cells in the cerebrospinal fluid (CSF). Additionally, let-7b was positively correlated with the expression of t-Tau and p-Tau. The inclusion of let-7b in A beta 40-A beta 42 or t-tau p-tau logistic regression and receiver operating characteristic predictive models can significantly improve the diagnostic performance. Overall, let-7b has an important association with the pathology of AD and can be used as an adjunct to improve the diagnostic performance of traditional AD biomarkers. (C) 2017 Elsevier Inc. All rights reserved.

Keyword ：

A beta AD biomarkers CD4+T lymphocytes Let-7b Tau

Cite：

Copy from the list or Export to your reference management。

Abstract ：

The poor therapy response and poor prognosis of esophageal cancer has made it one of the most malignant carcinoma, and the complicated multidisciplinary treatment failed to achieve a long-term disease-free survival. To diagnose esophageal cancer at an earlier stage, and to improve the effect of anticancer therapy would improve the therapeutic efficacy. After retrospective analysis of the cancer samples of patients who received esophagectomy, we found the relevance between ratio of either ALDH1 or CD133-positive cancer stem cells and 2-year recurrence. Higher ratios of cancer stem cells indicated later clinical stages, and Wnt signaling activation was more frequent in later esophageal carcinoma. Further in bench studies, we explored the suppressive roles and the mechanisms involved in Let-7 on self-renewal in ECA-109 and ECA-9706 esophageal cancer stem cells. Isolated cancer stem cells naturally divide symmetrically and are therapy resistant. Therapy of fluorouracil and docetaxel both enriched the stem cells, proving the resistant characteristics of cancer stem cells. Wnt activation stimulated more symmetric division of stem cells, resulting in self-renewal promotion, which could be blocked by Let-7 overexpression. Furthermore, enforced Let-7 sensitized the stem cells to chemotherapies in a Wnt pathway inhibition-dependent manner, contributing to Let-7 sensitization of chemotherapeutic response. Wnt activation weakened the suppressive Let-7b through the sponge functions of CCAT-1, forming the negative feedback loop of Let-7b/Wnt/CCAT1. These results identified the crucial participation of stem cells in esophageal cancer occurrence and progression as the potent indicator, and also indicate the potential powerful agent of Let-7 nano-particles in treatment of cancer.

Keyword ：

cancer stem-like cells division manner esophageal cancer Let-7b Wnt signaling

Cite：

Copy from the list or Export to your reference management。