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LncRNA MALAT1/miR‑26a/26b/ST8SIA4 axis mediates cell invasion and migration in breast cancer cell lines.

ABSTRACT: Metastasis‑associated lung adenocarcinoma transcript 1 (MALAT1) is a long non‑coding RNA that is overexpressed in various human cancers, including breast cancer. Evidence has associated the function of the α‑2,8‑sialyltransferase (ST8SIA) family with breast cancer. The present study aimed to investigate the potential roles of MALAT1 in breast cancer development and progression using analyses of both breast cancer tissues and cell lines. The mRNA levels of MALAT1, microRNA (miR)‑26a/26b and ST8SIA4 were detected by reverse transcription‑quantitative PCR (RT‑qPCR) and the protein level of ST8SIA4 was assessed by western blot analysis. Cell proliferation, invasion and migration were detected by CCK‑8, wound healing and Transwell assays, respectively. Interactions between MALAT1 and miR‑26a/26b were assessed using fluorescence in situ hybridization, RNA immunoprecipitation and luciferase reporter assays. Herein, different levels of MALAT1 were primarily observed in human breast cancer samples and cells. Upregulated MALAT1 was a crucial predictor of poor breast cancer prognosis. Altered MALAT1 modulated cell progression in breast cancer. Moreover, miR‑26a/26b was confirmed as a direct regulator of MALAT1, and ST8SIA4 was predicted as a target of miR‑26a/26b. Functional analysis in human breast cancer cell lines demonstrated that MALAT1 modulated breast cancer cell tumorigenicity by acting as a competing endogenous lncRNA (ceRNA) to regulate ST8SIA4 levels by sponging miR‑26a/26b. The identification of the MALAT1/miR‑26a/26b/ST8SIA4 axis which contributes to breast cancer progression may constitute a potential new therapeutic target.

SUBMITTER: Wang N

PROVIDER: S-EPMC8273684 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Sialylation is one of the altered glycosylation patterns associated with cancer progression. In this study, we investigated the N-glycan profiles of breast cancer patients and cell lines to reveal sialylation associated with breast cancer progression, and provided new evidences of miRNA-mediated sialylation. MALDI-TOF MS analysis revealed that N-glycans found in breast cancer tissues and breast cancer cell MDA-MB-231 featured increased levels of sialylation compared with adjacent tissues and normal breast epithelial cell MCF-10A. The expressional profiles of 20 sialyltransferase genes were then analyzed and found significantly different comparing breast cancer samples with adjacent tissues, and two breast cancer cell lines MDA-MB-231 and MCF-7 with different metastatic potential and MCF-10A cells. Tumor tissues and highly metastatic breast cancer cell line MDA-MB-231 exhibited higher levels of ST8SIA4. Knocking down ST8SIA4 in breast cancer cell lines significantly inhibited their malignant behaviors including cell proliferation and invasion in a sialyltransferase-dependent manner. By applying bioinformatic approaches for the prediction of miRNA targeting 3'-UTR of ST8SIA4, we identified ST8SIA4 as one of the miR-26a/26b-targeted genes. Further data analysis revealed the inversely related expression of ST8SIA4 and miR-26a/26b in breast cancer cells, tumor tissues and corresponding adjacent tissues. The ability of miR-26a/26b to interact specifically with and regulate the 3'-UTR of ST8SIA4 was demonstrated via a luciferase reporter assay. The forced expression of miR-26a/26b was able to induce a decrease of ST8SIA4 level and also to affect breast cancer cells progression, while altered expression of ST8SIA4 in breast cancer cells modulated progression upon transfection with miR-26a/26b mimics or inhibiter. Taken together, these results indicate that changes in the glycosylation patterns and sialylation levels may be useful markers of the progression of breast cancer, as well as miR-26a/26b may be widely involved in the regulation of sialylation machinery by targeting ST8SIA4.

Project description:BackgroundPrevious studies have revealed the key functions of N6-methyladenosine (m6A) modification in breast cancer (BC). MALAT1 as a highly m6A modified lncRNA associated with cancer development and metastasis, but the functional relevance of m6A methyltransferase and MALAT1 in BC is still unknown. Here, our study investigated the effects of the novel m6A methyltransferase METTL3 on epithelial-mesenchymal transition (EMT) in BC via the MALAT1/miR-26b/HMGA2 axis.MethodsFirstly, we collected clinical BC samples and cultured BC cells, and detected mRNA and protein levels in the human samples and human cell lines by RT-qPCR and Western blot, respectively. Then, the binding of MALAT1 and miR-26b and the targeting relationship between miR-26b and HMGA2 were examined by dual-luciferase assay. Moreover, the binding of MALAT1 and miR-26b was tested by RNA pull down and RNA immunoprecipitation (RIP) assays. Methylated-RNA immunoprecipitation (Me-RIP) was used to detect the m6A modification level of MALAT1. The interaction of METTL3 and MALAT1 was detected by photoactivatable ribonucleoside-crosslinking immunoprecipitation (PAR-CLIP). Finally, effects on invasion and migration were detected by Transwell.ResultsIn BC, the level of miR-26b was consistently low, while the levels of METTL3, MALAT1 and HMGA2 were high. Further experiments showed that METTL3 up-regulated MALAT1 expression by modulating the m6A modification of MALAT1, and that MALAT1 could promote the expression of HMGA2 by sponging miR-26b. In BC cells, we found that silencing METTL3 could inhibit EMT and tumor cell invasion by suppressing MALAT1. Furthermore, MALAT1 mediated miR-26b to target HMGA2 and promote EMT, migration, and invasion. In summary, METTL3 promoted tumorigenesis of BC via the MALAT1/miR-26b/HMGA2 axis.ConclusionsSilencing METTL3 down-regulate MALAT1 and HMGA2 by sponging miR-26b, and finally inhibit EMT, migration and invasion in BC, providing a theoretical basis for clinical treatment of BC.

Project description:Macrophages (Mφ) are long-lived myeloid cells that can polarize towards the proinflammatory M1 or proresolving M2 phenotype to control diverse biological processes such as inflammation, tissue damage, and regeneration. Noncoding RNA are a class of nonprotein-coding transcriptome with numerous interdependent biological roles; however, their functional interaction in the regulation of Mφ polarization and immune responses remain unclear. Here, we show antagonistic relationship between lncRNA (MALAT1) and microRNA (miR-30b) in shaping macrophage polarization and immune functions. MALAT1 expression displays a time-dependent induction during Mφ differentiation and, upon challenge with TLR4 agonist (E. coli LPS). MALAT1 knockdown promoted the expression of M2Mφ markers without affecting M1Mφ markers, suggesting that MALAT1 favors the M1 phenotype by suppressing M2 differentiation. Compared to the control, MALAT1 knockdown resulted in reduced antigen uptake and processing, bacterial phagocytosis, and bactericidal activity, strongly supporting its critical role in regulating innate immune functions in Mφ. Consistent with this, MALAT1 knockdown showed impaired cytokine secretion upon challenge with LPS. Importantly, MALAT1 exhibit an antagonistic expression pattern with all five members of the miR-30 family during M2 Mφ differentiation. Dual-luciferase assays validated a novel sequence on MALAT1 that interacts with miR-30b, a microRNA that promotes the M2 phenotype. Phagocytosis and antigen processing assays unequivocally demonstrated that MALAT1 and miR-30b are functionally antagonistic. Concurrent MALAT1 knockdown and miR-30b overexpression exhibited the most significant attenuation in both assays. In human subjects with periodontal disease and murine model of ligature-induced periodontitis, we observed higher levels of MALAT1, M1Mφ markers and downregulation of miR-30b expression in gingival tissues suggesting a pro-inflammatory function of MALAT1 in vivo. Overall, we unraveled the role of MALAT1 in Mφ polarization and delineated the underlying mechanism of its regulation by involving MALAT-1-driven miR-30b sequestration.

Project description:Background The ocular inflammatory microenvironment has been reported to be closely associated with the occurrence and progression of highly myopic cataract (HMC). Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) could alter the biological properties of mammalian cells by modulating the expression of inflammatory mediators; therefore, it may contribute to the development of HMC. Objective To investigate the function of MALAT1 in the inflammatory response, proliferation, migration, and epithelial-mesenchymal transition (EMT) of inflammatory and injured human lens epithelial cells (HLECs) and to reveal the underlying molecular signals. Methods Patients with HMC and age-related cataract (ARC) with an axial length of more than 26 mm were selected, and the anterior capsular tissue was obtained during cataract surgery. TNF-α (20 ng/mL) was chosen to induce inflammatory damage in HLECs to simulate the inflammatory microenvironment in HMC eyes. Specific siRNAs, inhibitors, and mimics were used to suppress or enhance the functions of MALAT1 and miR-26a-5p. RT-qPCR and Western blot analysis were performed to measure gene and protein expression, respectively. Results The expression of MALAT1 and the inflammatory mediators IL-6, MMP-2, and MMP-9 were significantly higher in HMC anterior capsule tissues than in ARC. TNF-α treatment increased the expression of MALAT1, while it also promoted the proliferation, migration, and EMT of HLECs. MALAT1 interference decreased the expression of IL-6 and MMP-2 and inhibited the aforementioned processes. Furthermore, MALAT1 negatively regulated the expression of miR-26a-5p and then promoted TET1 expression. TET1 was identified as a direct target of miR-26a-5p, and the promoting effect of MALAT1 on TET1 expression could be reversed by miR-26a-5p mimics. Conclusion The inflammatory environment and MALAT1 expression could be reciprocally induced in HLECs. MALAT1 may act as a ceRNA via the “sponge” miR-26a-5p and target TET1 to regulate the inflammatory response, proliferation, migration, and EMT processes in HLECs.

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LncRNA MALAT1/miR‑26a/26b/ST8SIA4 axis mediates cell invasion and migration in breast cancer cell lines.

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