The Lin28/Let-7 System in Early Human Embryonic Tissue and Ectopic Pregnancy

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7–9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans.     

MiR-146a promotes the asymmetric division and inhibits the self-renewal ability of breast cancer stem-like cells via indirect upregulation of Let-7

MiR-146a could stimulate tumor growth or block tumor proliferation in systemic malignancies, referring to the specific downstream targeted gene. However, its roles in breast cancer stem-like cells (BrCSCs) are barely known. To dig out its mechanistic functions, we explored the indicative roles of miR-146 in preclinical study, regardless of the hormone receptor status, and the positive correlation between miR-146 and better prognosis was proved, as its correlation to Let-7c was. To uncover the implicated mechanisms, we first identified the suppressive role of miR-146a in stem cells’ renewal, which was achieved by promoting the asymmetric division of BrCSCs. Let-7c was previously revealed with its suppressive functions in stem-like cells expansion, and miR-146 was predicated and successfully proved to bind to and degrade the 3’UTR of LIN28, a maturation blocker of Let-7 family. Results further showed that miR-146a increased the Let-7c level through degrading LIN28, and LIN28 inhibition is required for miR-146a induction of asymmetric stem cells’ division. Moreover, Let-7 controlled Wnt signaling pathway activity could be strengthened due to the miR146 inhibition of H19, later of which was often activated in stem cells group with functional existence of Wnt signaling. H19 itself in turn formed the positive feedback regulation with Let-7. Our results suggested the miR-146a/LIN28/Wnt signaling circle in restraining the symmetric cells division, which was specifically referred to the controlling of the small circle of Let-7c and H19, and together, this dual axis could help to prohibit the stem cells expansion.     

Matrine suppression of self-renewal was dependent on regulation of LIN28A/Let-7 pathway in breast cancer stem cells

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.     

miR-367 stimulates Wnt cascade activation through degrading FBXW7 in NSCLC stem cells

Lung carcinoma tops the categories of cancer related motility, and has been treated as the main threat to human health. The functions and related mechanism of FBXW7 controlled lung cancer stem cells' signatures is barely unknown, and the miR-367 regulations of FBXW7 via Wnt signaling have not been explored. Cancer stem cells of either ALDH1+ or CD133+ phenotype were found to be referred to advanced stages in patients with NSCLC (non-small cell lung carcinoma). To study the roles of miR-367, we found greater miR-367 level or FBXW7 level was reserved in NSCLC than that of paired adjacent normal tissues, and their upregulations were positively correlated with Wnt signaling activation. On the contrary, increased miR-367 was correlated with Let-7 repression. MiR-367 was related to stronger sphere forming ability in stem cells of NSCLC. We then explored the functions of the endogenous miR-367 in stem-like cells isolated from NSCLC cell lines. In HEK-293 cells, we identified FBXW7 as the direct downstream gene of miR-367, which consequently released the LIN-28 dependent inhibition of suppressive Let-7. Through informatics analysis, miR-367 was predicated to function through Wnt signaling, and decreased Let-7 played the pivotal role to maintain TCF-4/Wnt pathway activity. The reintroduction of FBXW7 abolished the oncogenic stimulation of miR-367 on TCF-4 activity, with Wnt signaling factors depression. In conclusion, our findings demonstrated the oncogenic roles of miR-367 exerting on the self-renewal ability of cancer stem-like cells through degrading the suppressive FBXW7, eventually helping to maintain Wnt signaling activation through a LIN28B/Let-7 dependent manner.     

Ovarian Cancer Cell Line Panel (OCCP): Clinical Importance of In Vitro Morphological Subtypes

Epithelial ovarian cancer is a highly heterogeneous disease and remains the most lethal gynaecological malignancy in the Western world. Therapeutic approaches need to account for inter-patient and intra-tumoural heterogeneity and detailed characterization of in vitro models representing the different histological and molecular ovarian cancer subtypes is critical to enable reliable preclinical testing. There are approximately 100 publicly available ovarian cancer cell lines but their cellular and molecular characteristics are largely undescribed. We have characterized 39 ovarian cancer cell lines under uniform conditions for growth characteristics, mRNA/microRNA expression, exon sequencing, drug response for clinically-relevant therapeutics and collated all available information on the original clinical features and site of origin. We tested for statistical associations between the cellular and molecular features of the lines and clinical features. Of the 39 ovarian cancer cell lines, 14 were assigned as high-grade serous, four serous-type, one low-grade serous and 20 non-serous type. Three morphological subtypes: Epithelial (n = 21), Round (n = 7) and Spindle (n = 12) were identified that showed distinct biological and molecular characteristics, including overexpression of cell movement and migration-associated genes in the Spindle subtype. Comparison with the original clinical data showed association of the spindle-like tumours with metastasis, advanced stage, suboptimal debulking and poor prognosis. In addition, the expression profiles of Spindle, Round and Epithelial morphologies clustered with the previously described C1-stromal, C5-mesenchymal and C4 ovarian subtype expression profiles respectively. Comprehensive profiling of 39 ovarian cancer cell lines under controlled, uniform conditions demonstrates clinically relevant cellular and genomic characteristics. This data provides a rational basis for selecting models to develop specific treatment approaches for histological and molecular subtypes of ovarian cancer.     

Lin28A促进结肠癌细胞对5-FU 化疗敏感性的实验研究

目的:探讨Lin28A对结肠癌5-氟尿嘧啶(5-FU)化疗敏感性的影响及其分子机制。方法:免疫组化方法检测人结肠癌和正常粘膜组织中Lin28A蛋白表达情况;荧光素酶法检测HCT116细胞活性;实时荧光定量PCR方法检测HCT116细胞中Let-7表达水平。结果:73.3%人结肠癌患者Lin28A蛋白表达上调;过表达Lin28A组与对照组相比,5-FU处理后细胞活性显著降低(P_(60μg)0.01,P_(80μg)0.01),Let-7表达显著降低;过表达Let-7组与对照组相比,5-FU治疗后细胞活性显著降低(P_(40μg)0.05,P_(60μg)0.01,P_(80μg)0.01)。结论:Lin28A可增强肿瘤细胞化疗敏感性,而且这一效应不依赖Let-7。     

The diagnostic and molecular characteristics of malignant mesothelioma and ovarian/peritoneal serous carcinoma

B. Davidson The diagnostic and molecular characteristics of malignant mesothelioma and ovarian/peritoneal serous carcinoma Malignant mesothelioma and ovarian/peritoneal serous carcinoma are two of the most common tumours affecting the serosal cavities. Unlike other malignant tumours diagnosed at this anatomical site, such as lung and breast carcinoma, malignant mesothelioma and serous carcinoma share a common histogenesis, may be difficult to differentiate morphologically, and co‐express many of the diagnostic markers used by cytopathologists in effusion diagnosis. Selected markers have nevertheless shown sufficient sensitivity and specificity to differentiate serous carcinoma from malignant mesothelioma effectively. Recently, our group applied high throughput technology to the identification of new markers that may aid in differentiating these two cancer types and validated several of these markers in follow‐up studies. This review will present current data regarding the diagnostic and biological aspects of malignant mesothelioma and ovarian/peritoneal serous carcinoma.     

OVCA1 inhibits the proliferation of epithelial ovarian cancer cells by decreasing cyclin D1 and increasing p16

OVCA1, a tumor suppressor gene, is deleted or lower expressed in about 80% of ovarian cancer. Over expression of OVCA1 in human ovarian cancer A2780 cells inhibits cell proliferation and arrests cells in G1 stage. However, the fact that the molecular mechanism of OVCA1 inhibits cell growth is presently elusive. Here we investigated the potential signaling pathway induced by over-expression of OVCA1. Our results show that over-expression of human OVCA1 in ovarian cancer cells A2780 leads to down-regulation of cyclin D1, and up-regulation of p16, but no effect on the expression of NF-κB. It indicates that OVCA1 could inhibit the proliferation of ovarian cancer cell A2780 by p16/cyclin D1 pathway, but not by NF-κB.     

LIN28A Modulates Splicing and Gene Expression Programs in Breast Cancer Cells

LIN28 is an evolutionarily conserved RNA-binding protein with critical functions in developmental timing and cancer. However, the molecular mechanisms underlying LIN28''s oncogenic properties are yet to be described. RNA-protein immunoprecipitation coupled with genome-wide sequencing (RIP-Seq) analysis revealed significant LIN28 binding within 843 mRNAs in breast cancer cells. Many of the LIN28-bound mRNAs are implicated in the regulation of RNA and cell metabolism. We identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28-interacting partner. Subsequently, we used a custom computational method to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 small interfering RNA (siRNA)-treated cells. The results reveal that these proteins regulate alternative splicing and steady-state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of the ENAH exon 11a isoform. The expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Intriguingly, analysis of publicly available array data from the Cancer Genome Atlas (TCGA) reveals that LIN28 expression in the HER2 subtype is significantly different from that in other breast cancer subtypes. Collectively, our data suggest that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes.     

Differential analysis of ovarian and endometrial cancers identifies a methylator phenotype

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.     

Long noncoding RNA CASC9 promotes LIN7A expression via miR-758-3p to facilitate the malignancy of ovarian cancer

The long noncoding RNA cancer susceptibility 9 (CASC9) has been reported to be a pivot modulator in growth and metastasis of breast cancer, liver cancer, esophageal squamous cell carcinoma, lung adenocarcinoma, gastric cancer, and nasopharyngeal cancer. However, its potential roles in ovarian cancer remain unclear. In this study, we aimed at its functions and molecular mechanism in ovarian cancer progression. We showed that CASC9 was highly expressed in ovarian cancer tissues and cell lines. An elevated level of CASC9 predicts an unfavorable prognosis in patients with ovarian cancer. Loss-of-function and gain-of-function assays illustrated that CASC9 promotes ovarian cancer cell proliferation, migration, and invasion in vitro, and accelerates tumor growth in vivo. We showed that CASC9 works as a competing endogenous RNA (ceRNA) for miR-758-3p which targets LIN7A. CASC9 inhibits the level of miR-758-3p, and in turn stimulates LIN7A expression in ovarian cancer. Overexpression of LIN7A reverses the suppressive roles of CASC9 depletion on ovarian cancer. In sum, our findings reveal a novel undefined regulatory signaling pathway, namely CASC9/miR-758-3p/LIN7A axis, involved in ovarian cancer progression.     

LIN28B regulates androgen receptor in human trophoblast cells through Let‐7c

LIN28B is an RNA‐binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let‐7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let‐7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first‐trimester trophoblast cell line, ACH‐3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral‐based vectors. LIN28B knockdown in ACH‐3P cells resulted in significantly decreased levels of AR and increased levels of Let‐7 miRNAs. Moreover, treatment of ACH‐3P cells with Let‐7c mimic, but not Let‐7e or Let‐7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin‐induced syncytialization and Let‐7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW‐1 and a significant decrease in AR in ACH‐3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let‐7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.     

An interview with Dr. Marcus E. Peter on his highly cited paper published in Cell Cycle

Comment on: Let-7 prevents early cancer progression by suppressing expression of the embryonic gene HMGA2. Sun-Mi Park, Scott Shell, Amir Reza Radjabi, Robert Schickel, Christine Feig, Ben Boyerinas, Daniela M. Dinulescu, Ernst Lengyel and Marcus E. Peter     

LncRNA H19-elevated LIN28B promotes lung cancer progression through sequestering miR-196b

LncRNA H19 is involved in the development of multiple cancers. Here, we firstly provide new evidence that H19 can induce LIN28B, a conserved RNA binding protein, to accelerate lung cancer growth through sponging miR-196b. Abundance in LIN28B was observed in clinical lung cancer samples. A positive link was observed between H19 and LIN28B in clinical lung cancer samples. In lung cancer cells, H19 was capable of increasing LIN28B expression. Mechanistically, miR-196b directly targeted LIN28B to inhibit LIN28B expression. H19 was capable of promoting LIN28B expression through sequestering miR-196b. Functionally, H19-increased LIN28B conferred the cell proliferation of lung cancer. Our finding indicates that H19 depresses miR-196b to elevate LIN28B, resulting in accelerating cell proliferation in lung cancer.     

Calcitonin administration improves endometrial receptivity via regulation of LIF,Muc-1 and microRNA Let-7a in mice

Calcitonin (CT) is one of the factors affecting the embryo implantation, but its effects on the implantation window have not been fully investigated. The current study investigated the effects of CT on the endometrium receptivity by morphological study and evaluation of leukemia inhibitory factor (LIF), mucin 1 (Muc-1), and microRNA (miRNA) Let-7a in the ovarian stimulation and the normal ovarian cycle. Then the mechanism of the CT effects through the mammalian target of rapamycin (mTOR) signaling pathway was studied by using PP242. A total of 64 BALB/c mice were divided into the normal ovarian cycle and ovarian stimulation groups. Each group consisted of four subgroups: control, calcitonin, PP242, and calcitonin+PP242. CT and PP242 were injected on the fourth of pregnancy into the mice and 24 hr later all the mice were killed. The uterine tissue samples were used for morphological analysis, and endometrial cells were mechanically isolated for evaluation of gene and protein expression. The results showed that ovarian stimulation induced mTOR phosphorylation as well as increased expression of the Let-7a miRNA. In addition, CT injection increased the expression of LIF and miRNA Let-7a in ovarian stimulation similar to that in normal ovarian cycles. However, injection of PP242 reduced expression of miRNA Let-7a and increased Muc-1 expression in ovarian stimulation group. In conclusion, the administration of CT improved endometrial receptivity in mice. This phenomenon occurred by upregulation of LIF, miRNA Let-7a and downregulation of Muc-1 via mTOR signaling pathway.