Lin28 Mediates Radiation Resistance of Breast Cancer Cells via Regulation of Caspase,H2A.X and Let-7 Signaling

Resistance to radiation therapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to breast tumorigenesis; however, the relationship between Lin28 and radioresistance remains unknown. In this study, we investigated the association of Lin28 with radiation resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines. The results showed that the expression level of Lin28 was closely associated with resistance to radiation treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to radiation than MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which have low-level Lin28 expression. Transfection with Lin28 siRNA significantly led to an increase of sensitivity to radiation. By contrast, stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to radiation treatment. Stable expression of Lin28 also significantly inhibited radiation-induced apoptosis. Moreover, further studies have shown that caspases, H2A.X and Let-7 miRNA were the molecular targets of Lin28. Stable expression of Lin28 and treatment with radiation induced H2AX expression, while inhibited p21 and γ-H2A.X. Overexpression of Let-7 enhanced the sensitivities to radiation in breast cancer cells. Taken together, these results indicate that Lin28 might be one mechanism underlying radiation resistance, and Lin28 could be a potential target for overcoming radiation resistance in breast cancer.     

Lin28 mediates paclitaxel resistance by modulating p21, Rb and Let-7a miRNA in breast cancer cells

Resistance to chemotherapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to tumor relapse after chemotherapy; however, the relationship between Lin28 and chemoresistance remained unknown. In this study, we investigated the association of Lin28 with paclitaxel resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines and tumor tissues. We found that the expression level of Lin28 was closely associated with the resistance to paclitaxel treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to paclitaxel than the MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which had low-level expression of Lin28. Knocking down of Lin28 in Lin28 high expression T47D cells increased the sensitivity to paclitaxel treatment, while stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to paclitaxel treatment, resulting in a significant increase of IC50 values of paclitaxel. Transfection with Lin28 also significantly inhibited paclitaxel-induced apoptosis. We also found that Lin28 expression was dramatically increased in tumor tissues after neoadjuvant chemotherapy or in local relapse or metastatic breast cancer tissues. Moreover, further studies showed that p21, Rb and Let-7 miRNA were the molecular targets of Lin28. Overexpression of Lin28 in breast cancer cells considerably induced p21 and Rb expression and inhibited Let-7 miRNA levels. Our results indicate that Lin28 expression might be one mechanism underlying paclitaxel resistance in breast cancer, and Lin28 could be a potential target for overcoming paclitaxel resistance in breast cancer.     

Increased Expression of Lin28B Associates with Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

Recent studies showed that incomplete cell reprogramming can transform cells into tumour-like cells. Lin28A is associated with fibroblast and sarcoma cell reprogramming, whereas its homologue Lin28B is associated with hematopoietic cell reprogramming. This study aimed to investigate the expression and prognostic difference between Lin28A and Lin28B in oral squamous cell carcinoma (OSCC). Expression level was assessed by immunohistochemistry and staining location was confirmed by immunofluorescence. Prognostic values were analysed and compared by the Kaplan–Meier analysis and uni and multivariate Cox regression models. Besides, in vitro cell assays and in vivo nude mice xenograft were used to demonstrate the influence of increased Lin28B expression in OSCC. Lin28A and Lin28B expression increased in OSCC, and co-expression of Lin28A and Lin28B showed no significant association with patient prognosis. Kaplan–Meier analysis showed that patients with high Lin28B but not Lin28A expression had lower overall survival (OS) rates than those with low Lin28B expression. Further Univariate analysis showed that patients with increased Lin28B expression had shorter disease-free survival (DFS) and shorter OS, while multivariate analysis showed Lin28B overexpression with TNM stage predicted poor prognosis in patients with OSCC. Besides, stable expressing Lin28B in oral cancer cells promoted cell migration, invasion, colony formation, in vivo proliferation and increased the expression of cancer suppressor miRNA let-7 targeted genes IL-6, HMGA2, the EMT markers Snail and Twist, the angiogenesis inducer VEGF, and the apoptosis inhibitor Survivin. These combined results indicate that Lin28B is a novel marker for predicting prognosis in patients with OSCC and may be a therapeutic target.     

Drosha mediates destabilization of Lin28 mRNA targets

Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.     

Drosha mediates destabilization of Lin28 mRNA targets

Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.     

Overexpression of Lin28 Decreases the Chemosensitivity of Gastric Cancer Cells to Oxaliplatin,Paclitaxel, Doxorubicin,and Fluorouracil in Part via microRNA-107

Higher Lin28 expression is associated with worse pathologic tumor responses in locally advanced gastric cancer patients undergoing neoadjuvant chemotherapy. However, the characteristics of Lin28 and its mechanism of action in chemotherapy resistance is still unclear. In this study, we found that transfection of Lin28 into gastric cancer cells (MKN45 and MKN28) increased their resistance to the chemo-drugs oxaliplatin (OXA), paclitaxel (PTX), doxorubicin (ADM), and fluorouracil (5-Fu) compared with gastric cancer cells transfected with a control vector. When knockdown Lin28 expression by Lin28 small interfering RNA (siRNA) was evaluated in vitro, we found that the resistance to chemo-drugs was reduced. Furthermore, we found that Lin28 up-regulates C-myc and P-gp and down-regulates Cylin D1. Finally, we found that miR-107 is a target microRNA of Lin28 and that it participates in the mechanism of chemotherapy resistance. Our results suggest that the Lin28/miR-107 pathway could be one of many signaling pathways regulated by Lin28 and associated with gastric cancer chemo-resistance.     

A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs

Type I collagen is composed of two α1(I) polypeptides and one α2(I) polypeptide and is the most abundant protein in the human body. Expression of type I collagen is primarily controlled at the level of mRNA stability and translation. Coordinated translation of α(I) and α2(I) mRNAs is necessary for efficient folding of the corresponding peptides into the collagen heterotrimer. In the 5' untranslated region (5' UTR), collagen mRNAs have a unique 5' stem-loop structure (5' SL). La ribonucleoprotein domain family member 6 (LARP6) is the protein that binds 5' SL with high affinity and specificity and coordinates their translation. Here we show that RNA helicase A (RHA) is tethered to the 5' SL of collagen mRNAs by interaction with the C-terminal domain of LARP6. In vivo, collagen mRNAs immunoprecipitate with RHA in an LARP6-dependent manner. Knockdown of RHA prevents formation of polysomes on collagen mRNAs and dramatically reduces synthesis of collagen protein, without affecting the level of the mRNAs. A reporter mRNA with collagen 5' SL is translated three times more efficiently in the presence of RHA than the same reporter without the 5' SL, indicating that the 5' SL is the cis-acting element conferring the regulation. During activation of quiescent cells into collagen-producing cells, expression of RHA is highly up-regulated. We postulate that RHA is recruited to the 5' UTR of collagen mRNAs by LARP6 to facilitate their translation. Thus, RHA has been discovered as a critical factor for synthesis of the most abundant protein in the human body.     

Lin28 gene and mammalian puberty

Lin28a and Lin28b, homologs of the Caenorhabditis elegans Lin28 gene, play important roles in cell pluripotency, reprogramming, and tumorigenicity. Recently, genome‐wide association and transgenic studies showed that Lin28a and/or Lin28b gene were involved in the onset of mammalian puberty, the stage representing the attainment of reproduction capacity; however, the detailed mechanism of these genes in mammalian puberty remains largely unknown. The present paper reviews the research progress on the roles of Lin28a/b genes in the onset of mammalian puberty by analyzing the results coming from gene expression patterns, mutations, and transgenic studies, and put forward possible pathways for further studies on their roles in animal reproduction.     

An RNA-binding Protein,Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.     

Lin28a up‐regulation is associated with the formation of restenosis via promoting proliferation and migration of vascular smooth muscle cells

To explore the potential role of Lin28a in the development of restenosis after percutaneous transluminal angioplasty, double‐balloon injury surgery and mono‐balloon injury surgery were used to establish restenosis and atherosclerosis models, respectively, so as to better distinguish restenosis from atherosclerotic lesions. Immunohistochemical analysis revealed that significantly higher expression of Lin28a was observed in the iliac arteries of restenosis plaques than that of atherosclerosis plaques. Immunofluorescence studies showed the colocalization of Lin28a with α‐smooth muscle actin in restenosis plaques, rather than in atherosclerosis plaques, which suggested that Lin28a might be related to the unique behaviour of vascular smooth muscle cells (VSMCs) in restenosis. To further confirm above hypothesis, Lin28a expression was up‐regulated by transfection of Lenti‐Lin28a and inhibited by Lenti‐Lin28a‐shRNA transfection in cultured VSMCs, and then the proliferation and migration capability of VSMCs were detected by EdU and Transwell assays, respectively. Results showed that the proliferation and migration of VSMCs were significantly increased in accordance with the up‐regulation of Lin28a expression, while above behaviours of VSMCs were significantly suppressed after inhibiting the expression of Lin28a. In conclusion, the up‐regulation of Lin28a exerts its modulatory effect on VSMCs’ proliferation and migration, which may play a critical role in contributing to pathological formation of restenosis.