Orsolya Galamb, Alexandra Kalm�r, Anna Sebesty�n, Titanilla Dank�, Csilla Kriston, Istv�n F?ri, P�ter Holl�si, Istv�n Csabai, Barnab�s Wichmann, Tibor Kren�cs, Barbara Kinga Bart�k, Zs�fia Brigitta Nagy, S�ra Zsigrai, G�bor Barna, Zsolt Tulassay, P�ter Igaz & B�la Moln�r
| DOI: 10.1007/s12253-020-00800-8
Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p?
Taieb, M;Ghannoum, D;Barré, L;Ouzzine, M;
PMID: 37296099 | DOI: 10.1038/s41419-023-05875-0
Genetic mutations in the Xylt1 gene are associated with Desbuquois dysplasia type II syndrome characterized by sever prenatal and postnatal short stature. However, the specific role of XylT-I in the growth plate is not completely understood. Here, we show that XylT-I is expressed and critical for the synthesis of proteoglycans in resting and proliferative but not in hypertrophic chondrocytes in the growth plate. We found that loss of XylT-I induces hypertrophic phenotype-like of chondrocytes associated with reduced interterritorial matrix. Mechanistically, deletion of XylT-I impairs the synthesis of long glycosaminoglycan chains leading to the formation of proteoglycans with shorter glycosaminoglycan chains. Histological and Second Harmonic Generation microscopy analysis revealed that deletion of XylT-I accelerated chondrocyte maturation and prevents chondrocytes columnar organization and arrangement in parallel of collagen fibers in the growth plate, suggesting that XylT-I controls chondrocyte maturation and matrix organization. Intriguingly, loss of XylT-I induced at embryonic stage E18.5 the migration of progenitor cells from the perichondrium next to the groove of Ranvier into the central part of epiphysis of E18.5 embryos. These cells characterized by higher expression of glycosaminoglycans exhibit circular organization then undergo hypertrophy and death creating a circular structure at the secondary ossification center location. Our study revealed an uncovered role of XylT-I in the synthesis of proteoglycans and provides evidence that the structure of glycosaminoglycan chains of proteoglycans controls chondrocyte maturation and matrix organization.
Annals of Gastroenterological Surgery
Ichihara, M;Takahashi, H;Nishida, N;Ivan, C;Okuzaki, D;Yokoyama, Y;Ohtsuka, M;Miyoshi, N;Uemura, M;Tanaka, S;Calin, G;Mori, M;Doki, Y;Eguchi, H;Yamamoto, H;
| DOI: 10.1002/ags3.12649
Background Studies have shown that cancer stemness and the endoplasmic reticulum (ER) stress response are inversely regulated in colorectal cancer (CRC), but the mechanism has not been fully clarified. Long noncoding RNAs (lncRNAs) play key roles in cancer progression and metastasis. In this study we investigated lncRNA 01534 (LINC01534) as a possible modulator between cancer stemness and ER stress response. Methods In vitro experiments using CRC cell lines were performed to explore a possible role of LINC01534. The expression of LINC01534 in clinical CRC samples was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization. Results Silencing LINC01534 led to suppression of cell proliferation, invasiveness, and cell cycle progression at the G2-M phase, and promoted apoptosis. Moreover, we found that silencing LINC01534 suppressed cancer stemness, while it activated the ER stress response, especially through the PERK/eIF2α signaling pathway. In situ hybridization revealed LINC01534 was expressed in tumor cells and upregulated in CRC tissues compared with normal epithelium. A survival survey indicated that high LINC01534 expression was significantly associated with shorter overall survival in 187 CRC patients. Conclusion This is the first report on LINC01534 in human cancer. Our findings suggest that LINC01534 may be an important modulator of the maintenance of cancer stemness and suppression of the ER stress response, and that it could be a novel prognostic factor in CRC.
Proceedings of the National Academy of Sciences of the United States of America
Tu, Z;Hu, Y;Raizada, D;Bassal, MA;Tenen, DG;Karnoub, AE;
PMID: 36269860 | DOI: 10.1073/pnas.2203180119
The phosphoinositide 3-kinase (PI3K) pathway represents the most hyperactivated oncogenic pathway in triple-negative breast cancer (TNBC), a highly aggressive tumor subtype encompassing ∼15% of breast cancers and which possesses no targeted therapeutics. Despite critical contributions of its signaling arms to disease pathogenesis, PI3K pathway inhibitors have not achieved expected clinical responses in TNBC, owing largely to a still-incomplete understanding of the compensatory cascades that operate downstream of PI3K. Here, we investigated the contributions of long noncoding RNAs (lncRNAs) to PI3K activities in clinical and experimental TNBC and discovered a prominent role for LINC01133 as a PI3K-AKT signaling effector. We found that LINC01133 exerted protumorigenic roles in TNBC and that it governed a previously undescribed mTOR Complex 2 (mTORC2)-dependent pathway that activated AKT in a PI3K-independent manner. Mechanistically, LINC01133 induced the expression of the mTORC2 component PROTOR1/PRR5 by competitively coupling away its negative messenger RNA (mRNA) regulator, the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1). PROTOR1/PRR5 in turn was sufficient and necessary for LINC01133-triggered functions, casting previously unappreciated roles for this Rictor-binding protein in cellular signaling and growth. Notably, LINC01133 antagonism undermined cellular growth, and we show that the LINC01133-PROTOR1/PRR5 pathway was tightly associated with TNBC poor patient survival. Altogether, our findings uncovered a lncRNA-driven signaling shunt that acts as a critical determinant of malignancy downstream of the PI3K pathway and as a potential RNA therapeutic target in clinical TNBC management.
Journal of the Endocrine Society
Parekh, V;Sun, H;Chen, M;Weinstein, L;Agarwal, S;
| DOI: 10.1210/jendso/bvac141
Maternally expressed gene 3 (MEG3) is a long non-coding RNA (lncRNA) that has been implicated as a tumor suppressor. The expression of MEG3 RNA is downregulated in various human tumors, including pituitary adenoma and pancreatic islet tumors due to MEG3 gene deletion or DNA hypermethylation. Mouse models with conventional germline deletion of Meg3 have shown that Meg3 is essential for perinatal or postnatal development and survival. However, a direct role of Meg3 loss in tumorigenesis has not been shown. To observe a causal relationship between Meg3 loss and tumorigenesis, we have generated a mouse model with conditional deletion of Meg3 mediated by the RIP-Cre transgene which initiated Meg3 deletion in pancreatic islet β-cells and anterior pituitary. Meg3 loss did not lead to the development of islet tumors. Interestingly, RIP-Cre mediated Meg3 loss led to the development of an enlarged pituitary. The genes in the Meg3 region are transcribed together as a 210 kb RNA that is processed into Meg3 and other transcripts. Whether these tandem transcripts play a functional role in the growth of pancreatic endocrine cells and pituitary cells remains to be determined. Our mouse model shows that Meg3 loss leads to hyperplasia in the pituitary and not in pancreatic islets, thus serving as a valuable model to study pathways associated with pituitary cell proliferation and function. Future mouse models with specific inactivation of Meg3 alone or other transcripts in the Meg3 polycistron are warranted to study tissue-specific effects on initiating neoplasia and tumor development.
Huang, X;Yu, J;Lai, S;Li, Z;Qu, F;Fu, X;Li, Q;Zhong, X;Zhang, D;Li, H;
PMID: 35918619 | DOI: 10.1007/s10528-022-10255-y
Abnormal expression of long non-coding RNAs (lncRNAs) is involved in many pathological processes of cancers. However, the role of lncRNA LINC00052 in breast cancer progression is still unclear. Here, LINC00052 expression was detected by in situ hybridization and quantitative real-time PCR assays. Cell Counting Kit-8, wound healing, and transwell assays were used to investigate changes in the proliferation, migration, and invasion of breast cancer cells. MiR-548p was found associated with LINC00052 or Notch2 by RNA pull-down, dual-luciferase reporter, and qRT-PCR assays. The effect of LINC00052 on lung metastasis was explored through in vivo experiments. High LINC00052 expression was observed in breast cancer tissues and cells. LINC00052 silencing inhibited the proliferation, migration, and invasion of MCF7 cells, and LINC00052 overexpression produced the opposite results. MiR-548p, a target gene of LINC00052, partially rescued the effects of LINC00052 on proliferation, migration, and invasion of MCF7. Notch2 was the target of miR-548p and LINC00052 could promote Notch2 expression. Moreover, the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a downstream factor of Notch2, was increased by LINC00052, and a Pyk2 mutant could inhibit the cell migration and invasion induced by LINC00052 overexpression in MDA-MB-468 cells, which was similar to the function of the miR-548p mimic. We further demonstrated that LINC00052 exacerbated the metastases of breast cancer cells in vivo. Our research demonstrated that LINC00052 is highly expressed in breast cancer and promotes breast cancer proliferation, migration, and invasion via the miR-548p/Notch2/Pyk2 axis. LINC00052 could serve as a potential therapeutic target for breast cancer.
Journal of biomedical science
Yan, RL;Luan, CL;Liao, CC;Liu, LH;Chen, FY;Chen, HY;Chen, RH;
PMID: 35538574 | DOI: 10.1186/s12929-022-00815-0
Autophagy plays important roles in cell homeostasis and protein quality control. Long non-coding RNAs (lncRNAs) have been revealed as an emerging class of autophagy regulators, but the majority of them function in regulating the expression of autophagy-related genes. LncRNAs that directly act on the core autophagic proteins remain to be explored.Immunofluorescence staining and Western blotting were used to evaluate the function of BCRP3 in autophagy and aggrephagy. RNA immunoprecipitation and in vitro RNA-protein binding assay were used to evaluate the interaction of BCRP3 with its target proteins. Phosphatidylinositol 3-phosphate ELISA assay was used to quantify the enzymatic activity of VPS34 complex. qRT-PCR analysis was used to determine BCRP3 expression under stresses, whereas mass spectrometry and Gene Ontology analyses were employed to evaluate the effect of BCRP3 deficiency on proteome changes.We identified lncRNA BCRP3 as a positive regulator of autophagy. BCRP3 was mainly localized in the cytoplasm and bound VPS34 complex to increase its enzymatic activity. In response to proteotoxicity induced by proteasome inhibition or oxidative stress, BCRP3 was upregulated to promote aggrephagy, thereby facilitating the clearance of ubiquitinated protein aggregates. Proteomics analysis revealed that BCRP3 deficiency under proteotoxicity resulted in a preferential accumulation of proteins acting in growth inhibition, cell death, apoptosis, and Smad signaling. Accordingly, BCRP3 deficiency in proteotoxic cells compromised cell proliferation and survival, which was mediated in part through the upregulation of TGF-β/Smad2 pathway.Our study identifies BCRP3 as an RNA activator of the VPS34 complex and a key role of BCRP3-mediated aggrephagy in protein quality control and selective degradation of growth and survival inhibitors to maintain cell fitness.
Evidence-based complementary and alternative medicine : eCAM
Zhang, S;Song, S;Cui, W;Liu, X;Sun, Z;
PMID: 35027936 | DOI: 10.1155/2022/8504601
Intervertebral disc degeneration (IDD) contributes to cervical and lumbar diseases. Long noncoding RNAs (lncRNAs) are implicated in IDD. This study explored the mechanism of lncRNA HOTAIR in IDD.Normal and degenerative nucleus pulposus (NP) cells were isolated from NP tissues obtained in intervertebral disc surgery. Cell morphology was observed by immunocytochemistry staining and toluidine blue staining. NP cell markers were detected by RT-qPCR. Proliferation was detected by MTT assay. Autophagy-related proteins were detected by Western blot. Autophagosome was observed by monodansylcadaverine fluorescence staining. Apoptosis was detected by TUNEL staining and flow cytometry. si-HOTAIR and/or miR-148a inhibitor was introduced into degenerative NP cells. Binding relationships among HOTAIR, miR-148a, and PTEN were predicted and verified by dual-luciferase reporter assay and RNA pull-down. Finally, IDD rat models were established. Rat caudal intervertebral discs were assessed by HE staining. Expressions of HOTAIR, miR-148a, and PTEN were determined by RT-qPCR.HOTAIR was highly expressed in degenerative NP cells (p < 0.05). si-HOTAIR inhibited degenerative NP cell apoptosis and autophagy (p < 0.05). HOTAIR upregulated PTEN as a sponge of miR-148a. miR-148a was poorly expressed in degenerative NP cells. miR-148a deficiency partially reversed the inhibition of si-HOTAIR on degenerative NP cell autophagy and apoptosis (all p < 0.05). In vivo assay confirmed that si-HOTAIR impeded autophagy and apoptosis in intervertebral disc tissues, thus improving pathological injury in IDD rats (all p < 0.05).LncRNA HOTAIR promoted NP cell autophagy and apoptosis via promoting PTEN expression as a ceRNA of miR-148a in IDD.
Journal of Dermatological Science
Xian, J;Shang, M;Dai, Y;Wang, Q;Long, X;Li, J;Cai, Y;Xia, C;Peng, X;
| DOI: 10.1016/j.jdermsci.2021.11.007
Background Psoriasis is a chronic, complicated, and recurrent inflammatory skin disease. However, the precise molecular mechanisms remain largely elusive and the present treatment is unsatisfactory. Objective This study aimed to unravel the functions of long noncoding RNA (lncRNA) AGAP2-AS1 and its biological mechanism in psoriasis pathogenesis, hinting for the new therapeutic targets in psoriasis. Methods The expression of AGAP2-AS1 in the skin tissue of psoriasis patients and healthy controls were detected by qRT-PCR and RNAscope™. Cell Counting Kit‑8 (CCK8) and clone formation assays were utilized to assess proliferation. Methylated RNA immunoprecipitation (MeRIP) was performed to detect the N6-methyladenosine (m6A) modification. RNA immunoprecipitation (RIP) was used to detect the interaction of AGAP2-AS1 with YTH domain family 2(YTHDF2). The relationships among AGAP2-AS1, miR-424-5p and AKT3 were examined by dual-luciferase reporter assay and RIP assay. Results We found that AGAP2-AS1 level was upregulated in the skin tissue of psoriasis patients than that of healthy controls and AGAP2-AS1 could promote proliferation and inhibit apoptosis of keratinocytes. Methyltransferase like 3(METTL3)-mediated m6A modification suppressed the expression of AGAP2-AS1 via YTHDF2-dependent AGAP2-AS1 stability. Thus, downregulation of METTL3 resulted in the upregulation of AGAP2-AS1 in psoriasis. AGAP2-AS1 functioned as a competitive endogenous RNA by sponging miR-424-5p to upregulate AKT3, activate AKT/mTOR pathway, as well as promote cell proliferation in keratinocytes. Conclusion AGAP2-AS1 is upregulated in the skin tissue of psoriasis patients and m6A methylation was involved in its upregulation. AGAP2-AS1 promotes keratinocyte proliferation through miR-424-5p/AKT/mTOR axis and may be a promising target for psoriasis therapy.
Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1
Proceedings of the National Academy of Sciences of the United States of America
Liu, H;Xu, J;Yang, Y;Wang, X;Wu, E;Majerciak, V;Zhang, T;Steenbergen, RDM;Wang, HK;Banerjee, NS;Li, Y;Lu, W;Meyers, C;Zhu, J;Xie, X;Chow, LT;Zheng, ZM;
PMID: 33436409 | DOI: 10.1073/pnas.2014195118
Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.
Jiang Z, Slater CM, Zhou Y, Devarajan K, Ruth KJ, Li Y, Cai KQ, Daly M, Chen X.
PMID: 28558830 | DOI: 10.1186/s13058-017-0853-2
Abstract
BACKGROUND:
Recent genome-wide profiling by sequencing and distinctive chromatin signatures has identified thousands of long non-coding RNA (lncRNA) species (>200 nt). LncRNAs have emerged as important regulators of gene expression, involving in both developmental and pathological processes. While altered expression of lncRNAs has been observed in breast cancer development, their roles in breast cancer progression and metastasis are still poorly understood.
METHODS:
To identify novel breast cancer-associated lncRNA candidates, we employed a high-density SNP array-based approach to uncover intergenic lncRNA genes that are aberrantly expressed in breast cancer. We first evaluated the potential value as a breast cancer prognostic biomarker for one breast cancer-associated lncRNA, LincIN, using a breast cancer cohort retrieved from The Cancer Genome Atlas (TCGA) Data Portal. Then we characterized the role of LincIN in breast cancer progression and metastasis by in vitro invasion assay and a mouse tail vein injection metastasis model. To study the action of LincIN, we identified LincIN-interacting protein partner(s) by RNA pull-down experiments followed with protein identification by mass spectrometry.
RESULTS:
High levels of LincIN expression are frequently observed in tumors compared to adjacent normal tissues, and are strongly associated with aggressive breast cancer. Importantly, analysis of TCGA data further suggest that high expression of LincIN is associated with poor overall survival in patients with breast cancer (P = 0.044 and P = 0.011 after adjustment for age). The functional experiments demonstrate that knockdown of LincIN inhibits tumor cell migration and invasion in vitro, which is supported by the results of transcriptome analysis in the LincIN-knockdown cells. Furthermore, knockdown of LincIN diminishes lung metastasis in a mouse tail vein injection model. We also identified a LincIN-binding protein, NF90, through which overexpression of LincIN may repress p21 protein expression by inhibiting its translation, and upregulation of p21 by LincIN knockdown may be associated with less aggressive metastasis phenotypes.
CONCLUSIONS:
Our studies provide clear evidence to support LincIN as a new regulator of tumor progression-metastasis at both transcriptional and translational levels and as a promising prognostic biomarker for breast cancer.
Am J Respir Crit Care Med.
Savary G, Dewaeles E, Diazzi S, Buscot M, Nottet N, Fassy J, Courcot E, Henaoui IS, Lemaire J, Martis N, Van der Hauwaert C, Pons N, Magnone V, Leroy S, Hofman V, Plantier L, Lebrigand K, Paquet A, Lino Cardenas CL, Vassaux G, Hofman P, Günther A, Crestani B, Wallaert B, Rezzonico R, Brousseau T, Glowacki F, Bellusci S, Perrais M, Broly F, Barbry P, Marquette CH, Cauffiez C, Mari B, Pottier N.
PMID: 30964696 | DOI: 10.1164/rccm.201807-1237OC
Abstract
RATIONALE:
Given the paucity of effective treatments for Idiopathic Pulmonary Fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. Transforming growth factor β (TGF-β) is the main pro-fibrotic factor, but its inhibition is associated with severe side effects due to its pleiotropic role.
OBJECTIVES:
We hypothesized that downstream non-coding effectors of TGF-β in fibroblasts may represent new effective therapeutic targets whose modulation may be well-tolerated.
METHODS:
We investigated the whole non-coding fraction of TGF-β-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblast. Differential expression of the long non-coding RNA DNM3OS and its associated miRNAs was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis.
MEASUREMENTS AND MAIN RESULTS:
We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-β-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e. miR-199a-5p/3p and miR-214-3p), which influence both SMAD and non-SMAD components of TGF-β signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis.
CONCLUSION:
Pharmacological approaches aiming at interfering with DNM3OS may represent new effective therapeutic strategies in IPF.
