Fibroblast Growth Factor (FGF) Signaling during Gastrulation Negatively Modulates the Abundance of MicroRNAs That Regulate Proteins Required for Cell Migration and Embryo Patterning

FGF signaling plays a pivotal role in regulating cell movements and lineage induction during gastrulation. Here we identify 44 microRNAs that are expressed in the primitive streak region of gastrula stage chicken embryos. We show that the primary effect of FGF signaling on microRNA abundance is to negatively regulate the levels of miR-let-7b, -9, -19b, -107, -130b, and -218. LIN28B inhibits microRNA processing and is positively regulated by FGF signaling. Gain- and loss-of-function experiments show that LIN28B negatively regulates the expression of miR-19b, -130b, and let-7b, whereas negative modulation of miR-9, -107, and -218 appears to be independent of LIN28B function. Predicted mRNA targets of the FGF-regulated microRNAs are over-represented in serine/threonine and tyrosine kinase receptors, including ACVR1, ACVR2B, PDGFRA, TGFBR1, and TGFBR3. Luciferase assays show that these and other candidates are targeted by FGF-regulated microRNAs. PDGFRA, a receptor whose activity is required for cell migration through the primitive streak, is a target of miR-130b and -218 in vivo. These results identify a novel mechanism by which FGF signaling regulates gene expression by negatively modulating microRNA abundance through both LIN28B-dependent and LIN28B-independent pathways.     

MiR-4282 inhibits tumor progression through down-regulation of ZBTB2 by targeting LIN28B in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most aggressive type of head and neck cancer with an unsatisfactory 5-year survival rate. MicroRNAs are a group of small noncoding RNAs reported to serve important roles in carcinogenesis, inhibiting certain gene expression via targeting the 3′-untranslated region of messenger RNAs (mRNAs). MiR-4282 has been newly discovered to be a tumor suppressor in colorectal cancer, but it has never been studied in OSCC. The present study aimed to uncover the role of miR-4282 in OSCC. We first confirmed that miR-4282 was downregulated in OSCC and validated its prognostic significance. Through gain-of-function assays, miR-4282 was discovered to inhibit proliferation, migration, and epithelial-to-mesenchymal transition, and induce apoptosis. By mechanistic research, we predicted via bioinformatics tools and confirmed by luciferase reporter and pulldown assays that miR-4282 targeted LIN28B, an RNA-binding protein, which has been reported to regulate RNA stability in cancers. Furthermore, we confirmed the interaction between LIN28B and zinc finger and BTB domain containing 2 (ZBTB2), and validated that miR-4282 regulated mRNA stability of ZBTB2 by inhibiting LIN28B. Rescue assays proved that miR-4282 inhibited tumor progression through LIN28B/ZBTB2 axis. In vivo assays proved that miR-4282 inhibited tumor growth in OSCC. In conclusion, the present study revealed that miR-4282 inhibited tumor progression through downregulation of ZBTB2 by targeting LIN28B in OSCC cells, indicating miR-4282 as a novel biomarker for OSC.     

MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells

microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.     

鸡miR-19a、miR-19b通过靶向抑制LIN9促进前脂肪细胞增殖

miR-17-92基因簇编码包括miR-19a、miR-19b在内的至少6个miRNA,在鸡细胞的增殖、分化、凋亡及发育等多种生物学过程中发挥重要作用,但其作用机制尚不清楚。生物信息学分析显示,细胞周期调控子LIN9是 miR-17-92 基因簇成员miR-19a和miR-19b的潜在靶点。为验证这一预测,构建含野生型LIN9 3′-UTR荧光素酶报告基因载体（psi-CHECK2-LIN9-3′-UTR-WT）及突变型报告基因载体（psi-CHECK2-LIN9-3′-UTR-MUT）,开展靶标LIN9的鉴定。复合转染结合报告基因酶活性测定结果表明,过表达miR-19a和miR-19b能显著抑制含野生型LIN9 3′-UTR的报告基因表达,而过表达miR-19a和miR-19b抑制剂显著提高野生型LIN9 3′-UTR报告基因的表达。实时定量PCR（RT-qPCR）证明,miR-19a和miR-19b 抑制剂对内源性LIN9 mRNA的表达没有影响,提示miR-19a和miR-19b可能不是通过降解mRNA调控LIN9表达。转染结合CCK-8细胞增殖分析显示,在鸡前脂肪细胞过表达LIN9 明显抑制细胞增殖。与此相一致的是,细胞增殖标志分子CyclinD1、c-Myc、PCNA、Ki67 的mRNA表达量明显降低。本研究证实,LIN9是miR-17-92 基因簇成员miR-19a和miR-19b的靶标,同时证实,LIN9抑制鸡前脂肪细胞的增殖。是否miR-17-92基因簇编码的其它mi-RNA成员也以LIN9为靶标尚不得而知,我室正在研究中。     

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.     

LIN28B/IRS1 axis is targeted by miR-30a-5p and promotes tumor growth in colorectal cancer

Insulin receptor substrate 1 (IRS1) is a potential oncogene that has been implicated in several malignant tumors. However, the regulatory mechanism of IRS1 remains to be investigated. The aim of our current study is to unveil the mechanism by which IRS1 exerts functions in tumorigenesis of colorectal cancer (CRC). The expression level of IRS1 was found to be higher in CRC cells in comparison with the normal cell. To determine the role of IRS1 in regulating CRC cellular processes, loss-of-function assays were designed and carried out in two CRC cell lines. Both in vitro and in vivo functional assays indicated that silencing of IRS1 suppressed CRC cell survival. Based on bioinformatics prediction and mechanism experiments, IRS1 was identified as a downstream target of miR-30a-5p. Furthermore, RNA-binding protein lin-28 homolog B (LIN28B) was determined to be a stabilizer of IRS1 messenger RNA. More importantly, LIN28B also acted as a target of miR-30a-5p.Through rescue assays, we proved that LIN28B-stablized IRS1 mediated miR-30a-5p-mediated CRC cell growth. In conclusion, this study revealed that LIN28B and LIN28B-stablized IRS1 promoted CRC cell growth by cooperating with miR-30a-5p.     

miR-152/LIN28B axis modulates high-glucose-induced angiogenesis in human retinal endothelial cells via VEGF signaling

Diabetic retinopathy (DR) is a serious complication of diabetes contributing to blindness in patients. Inhibiting retinal neovascularization is a potent strategy for diabetic retinopathy treatment. Reportedly, the stable expression of lin-28 homolog B (LIN28B), a member of the highly conserved RNA-binding protein LIN28 family, could promote vascular endothelial growth factor (VEGF) expression; herein, we investigated the role and mechanism of LIN28B in diabetic retinopathy progression from the perspective of microRNA (miRNA) regulation. We identified miR-152 as a miRNA that may target the LIN28B 3′-untranslated region and can be significantly downregulated under high-glucose (HG) condition. The expression of miR-152 was remarkably suppressed, whereas the expression of LIN28B was significantly increased under HG condition within both human retinal endothelial cells (hRECs) and retinal microvascular endothelial cell line (hRMECs). miR-152 overexpression significantly suppressed, while LIN28B overexpression promoted the angiogenesis and the protein levels of proangiogenesis factors in both hRECs and hRMECs. More importantly, LIN28B overexpression could remarkably attenuate the effect of miR-152 overexpression. In summary, miR-152 overexpression could inhibit HG-induced angiogenesis in both hRECs and hRMECs via targeting LIN28B and suppressing VEGF signaling. Further, in vivo experiments are needed for the application of miR-152/LIN28B axis in the treatment for diabetic retinopathy.     

miR-210 promotes lung adenocarcinoma proliferation,migration, and invasion by targeting lysyl oxidase-like 4

Accumulating evidence has revealed that various microRNAs are deregulated and involved in lung cancer development and metastasis. miR-210 is implicated in several cancer progression. However, the detailed biological function and role of miR-210 in lung adenocarcinoma remains unclear. Our current study was aimed to investigate the mechanism of miR-210 in lung adenocarcinoma progression. We observed that miR-210 was significantly upregulated in lung cancer cell lines (A549 and H1650) in comparison to BEAS-2B cells. In addition, we found that miR-210 was greatly elevated in lung adenocarcinoma tissues. Then, it was shown that overexpression of miR-210 was able to promote lung cancer cell proliferation and colony formation ability while inhibitors of miR-210 exhibited a reversed phenomenon. Subsequently, A549 and H1650 cell migration and invasion capacity were obviously restrained by miR-210 inhibition whereas induced by miR-210 mimics. Lysyl oxidase-like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases has been found to be increased or decreased in different cancer types. Here, we confirmed that LOXL4 could serve as a downstream target of miR-210 and miR-210 promoted lung cancer progression via targeting LOXL4. In A549 and H1650 cells, knockdown of LOXL4 dramatically repressed lung cancer cell proliferation, migration, and invasion. In conclusion, our study implied that miR-210 might indicate a new perspective for lung cancer.     

LINC01451 drives epithelial-mesenchymal transition and progression in bladder cancer cells via LIN28/TGF-β/Smad pathway

BackgroundThe pathogenesis of bladder cancer (BLCa) is still unclear. Long non-coding RNAs (lncRNAs) participate in diverse biological processes across every branch of life, especially in cancer. Dysregulated lncRNAs in BLCa and their biological significance require further investigations.MethodsHerein, a differential expression profile of lncRNAs in BLCa was conducted by microarray data. The expression level of lncRNA LINC01451 in 70 pairs of BLCa tissue samples and different BLCa cell lines were analyzed via real-time quantitative PCR. The CRISPR-CAS9 technique was employed to establish the LINC01451 stably transfected cell lines. Loss-of-function, as well as gain-of-function assays were carried out to evaluate the effects of LINC01451 on cell proliferation, migration, and invasion. Patient-derived xenograft (PDX) mouse models were adopted in the in vivo experiments. Western blot, biotinylated RNA probe pull-down assay, fluorescence in situ hybridization, and immunohistochemistry were utilized to assess the underlying molecular mechanisms of LINC01451 in BLCa.ResultsLINC01451 was identified a novel functional lncRNA, whose expression level in BLCa tissues was significantly higher compared with the normal tissues. Furthermore, it was found that LINC01451 directly docked LIN28A and LIN28B, and promoted the proliferation, invasion, and metastasis of BLCa. Mechanistically, LINC0145 was shown to depend on LIN28A and LIN28B, facilitated epithelial-mesenchymal transition (EMT) through activating the TGF-β/Smad signaling pathway, which subsequently aggravated BLCa progression.ConclusionsWe demonstrates that LINC01451 drives EMT-induced BLCa progression by activating the LIN28/TGF-β/Smad signaling pathway. Promisingly, LINC01451 acts as a prognostic biomarker and a novel therapeutic target for BLCa.     

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.     

MicroRNA-19a targets tissue factor to inhibit colon cancer cells migration and invasion

The over-expression of tissue factor (TF) and its roles in colon cancer progression have attracted much attention. However, the mechanisms regulating TF expression have not yet been shown in detail. In this study, we over-expressed miR-19a, miR20a and miR-106b in colon cancer cells, and evaluated their impact on TF expression and cellular function. We provide evidence demonstrating that miR-19a inhibited TF expression in vitro. Luciferase reporter assay confirmed that TF was a direct target of miR-19a because the miR-19a mediated repression of luciferase activity was abolished by mutation of the putative binding site. Moreover, miR-19a suppressed colon cancer cell migration and invasion. This effect was due to the indirect down-regulation of matrix metalloproteinase 9. Finally, we investigated the relevance of TF and miR-19a expression in a total of 48 paired colon cancer samples and revealed that miR-19a was inversely correlated with TF expression in stages I and II cases. Therefore, our results suggested that miR-19a was capable of suppressing TF expression in vitro and inhibiting cell migration and invasion. Although it was not the unique mechanism responsible for the expression of TF in vivo, miR-19a was inversely correlated with TF expression in early stage colon cancer patients.     

miR-146a Inhibits Cell Growth,Cell Migration and Induces Apoptosis in Non-Small Cell Lung Cancer Cells

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P<0.05). The patients with high miR-146a expression in their tumors showed longer progression-free survival (25.6 weeks in miR-146a high patients vs. 4.8 weeks in miR-146a low patients, P<0.05). miR-146a is therefore a strong candidate prognostic biomarker in NSCLC. Thus inducing miR-146a might be a therapeutic strategy for NSCLC.     

miR-125b慢病毒表达载体的构建及其对卵巢癌SKOV3细胞增殖和迁移的影响

构建并鉴定miR-125b慢病毒过表达载体,研究miR-125b对卵巢癌细胞增殖和迁移的影响及其可能机制。将PcR扩增的rniR-125b前体序列与经过酶切后的GP—SupersilencingVector进行连接,产生miR-125b重组慢病毒表达载体。将重组慢病毒载体质粒、pGag／Pol、pRev和pVSV-G共转染293T细胞,包装产生慢病毒。使用收获的病毒颗粒感染卵巢癌SKOV3细胞,嘌呤霉素筛选稳定感染细胞株;实时荧光定量PCR（Real．timeqPCR）检测miR-125b在SKV03细胞中的表达;Westernblot检测其潜在靶基因HER-2的表达：MTT实验和Transwell侵袭实验分别观察miR-125b过表达后SKOV3细胞增殖和迁移能力的改变。该研究成功构建miR-125b陧病毒过表达载体,感染卵巢癌SKOV3细胞后,能够过表达miR-125b,并抑制SKOV3细胞的增殖及迁移,降低潜在靶基因HER-2的表达。该研究证叽miR-125b能够抑制SKOV3细胞的增殖及迁移,并可能通过降低潜在靶基因HER-2的表达而实现。     

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.