MIR210HG is highly expressed in breast cancer
By analyzing lncRNA MIR210HG expression profile in TCGA-BRCA datasets, we confirmed that MIR210HG was highly expressed in breast cancer tissues compared to normal tissues (Fig. 1A and B). In addition, high MIR210HG expression also indicated advanced pathological stages (Fig. 1C). In Invasive Ductal Carcinoma (IDC) and Invasive lobular Carcinoma (ILC), MIR210HG showed higher expression level compared to normal tissues (Fig. 1D). Then we performed analysis in three independent GEO datasets (GSE70905, GSE65194 and GSE65212) to further confirm MIR210HG expression profiles. High expression of MIR210HG was observed in breast cancer in all selected datasets (Fig. 1E). Next, q-PCR analysis was performed in 5 breast cancer cell lines including MCF-7, SK-BR-3, MDA-MB-231, MDA-MB-435 and MDA-MB-436. The results revealed that MIR210HG commonly had a significant high level in all 5 breast cancer cell lines compared to the normal breast epithelium cell 76 N-F2V (Fig. 1F). Moreover, we also tested MIR210HG expression in tissues from 10 breast cancer patients. In line with the results of cell lines, MIR210HG was observed to have a significant high expression level in breast cancer tissues compared to the adjacent parts (Fig. 1G). Of note, pan-cancer analysis indicated MIR210HG was highly expressed in most cancer types (Fig. 1H), suggesting that MIR210HG is an oncogene and promising target for cancer therapy. Collectively, MIR210HG is an oncogenic lncRNA, which is possibly involved in breast cancer progression.
MIR210HG promotes proliferation of breast cancer
Since MIR210HG was validated highly expressed in breast cancer, we continuously clarified MIR210HG functions. Firstly, MIR210HG was knocked down by siRNA or overexpressed by inducible vector in MCF-7 and MDA-MB-231 cells. MIR210HG overexpression cells were named MCF-7MIR210HG−OV and 231MIR210HG−OV respectively. Subsequently, MTT assay was employed to evaluate proliferation rates of breast cancer cells. Silencing MIR210HG repressed proliferation of breast cancer cells (Fig. 2A), whereas DOX induced MIR210HG increased breast cancer cell proliferation (Fig. 2B). Colony formation assay revealed that the colony forming capacity of breast cancer cells was prohibited by MIR210HG siRNA (Fig. 2C). Conversely, ectopic MIR210HG enhanced the colony forming capacity of breast cancer cells (Fig. 2D). In addition, we stained human breast cancer tissues with Ki-67, which indicated a high Ki-67 level in breast cancer tissues compared to adjacent tissues (Fig. 2E). Furthermore, in line with the in vitro results, in vivo analysis showed tumor growth was repressed by MIR210HG siRNA but induced by MIR210HG overexpression (Fig. 2F), evidenced by tumor weight and volume changes. Taken together, MIR210HG promotes breast cancer proliferation.
MIR210HG promotes metastasis of breast cancer
In following step, we determined the function of MIR210HG in migration and invasion of breast cancer cells. Wound-healing assay results indicated that MIR210HG knocking down significantly repressed cell migration, whereas ectopic MIR210HG induced migration ability of MCF-7MIR210HG−OV cell (Fig. 3A). In Transwell assay, silencing MIR210HG resulted in a lower invasion ability evidenced by less stained MCF-7 cells at the bottom of the chamber. Conversely, DOX induced MIR210HG promoted MCF-7MIR210HG−OV cells to penetrate matrigel, indicating an enhanced invasion ability (Fig. 3B). Since Epithelial Mesenchymal Transition (EMT) is the main cause of migration and invasion, we investigated the EMT markers regulated by MIR210HG potentially. Q-PCR analysis showed that epithelial marker E-cadherin was increased while mesenchymal marker vimentin was repressed when MIR210HG was knocked down. MIR210HG overexpression resulted in a prohibited E-cadherin level but induced vimentin expression (Fig. 3C). Western blot results also showed similar results (Fig. 3D). Therefore, MIR210HG induces migration and invasion of breast cancer cells by regulating EMT process.
miR-210 mediates MIR210HG function
As MIR210HG is the host gene of miR-210, it is interesting to clarify whether miR-210 is required for MIR210HG function. By analyzing TCGA-BRCA datasets, miR-210 was confirmed to be highly expressed in breast cancer (Fig. 4A and B). In addition, high miR-210 expression indicated a poor overall survival rate and a poor distant metastasis free survival rate in breast cancer (Fig. 4C). Indeed, the expression of miR-210 was positively correlated to MIR210HG expression in breast cancer (Fig. 4D), suggesting an interaction in chain between miR-210 and MIR210HG. Furthermore, in five breast cancer cell lines, miR-210 was validated to have high expression levels compared to the normal breast epithelium cell 76 N-F2V (Fig. 4E). Similarly, high miR-210 was also confirmed in breast cancer patients (Fig. 4F). Next, to demonstrate miR-210 function, miR-210 was knocked down achieved by a specific miR-210 inhibitor in both MCF-7MIR210HG−OV and 231MIR210HG−OV cells (Fig. 4G). Interestingly, miR-210 inhibition largely abrogated proliferation rate which was enhanced by MIR210HG overexpression (Fig. 4H). In addition, colony formation capacity elevated by MIR210HG was also repressed by miR-210 inhibition (Fig. 4I). Similarly, miR-210 inhibitor prohibited invasion ability of MCF-7MIR210HG−OV, which was induced by MIR210HG (Fig. 4J). Collectively, miR-210 is an effector of MIR210HG and mediates MIR210HG function in breast cancer progression.
MIR210HG is induced by IGF2BP1 mediated m6A modification
Accumulating evidences imply that m6A modification is one of the important epigenetic regulations on lncRNA functions. Therefore, it is interesting to investigate whether MIR210HG can be regulated through m6A modification. Through bioinformatics analysis, there were four potential m6A modification sites were searched (chr11:565778, chr11:567257, chr11:567410, chr11:567484), and m6A reader IGF2BP1 was predicted to bind and recognize the sites (Fig. 5A). Indeed, MIR210HG was enriched within the m6A sites in both MCF-7 and MDA-MB-231 cells (Fig. 5B). RIP results indicated that IGF2BP1 directly bind MIR210HG (Fig. 5C). Above results suggested that IGF2BP1 interacted with MIR210HG through m6A modification. Since IGF2BP1 functions an RNA binding protein, next we evaluated the stability of MIR210HG with ectopic IGF2BP1 and silenced IGF2BP1 (Fig. 5D and E). By overexpressing IGF2BP1 and blocking new RNA synthesis, the degradation of MIR210HG was largely repressed (Fig. 5F). Conversely, silencing IGF2BP1 induced the degradation of MIR210HG (Fig. 5G). Therefore, MIR210HG is regulated by IGF2BP1 through both m6A modification and transcript stabilization. Since previous studies identified that three known mRNA stabilizers including ELAV-like RNA binding protein 1 (ELAVL1; also known as HuR), matrin 3 (MATR3), and poly(A)-binding protein cytoplasmic 1 (PABPC1), are co-factors of IGF2BPs in enhancing stability of mRNA [16], here we also examined whether the three factors are involved in MIR210HG stabilization. In breast cancer cell lines, the expression of ELAVL1 was up-regulated (Fig. 5H), indicating that ELAVL1 may serve as a co-factor of IGF2BP1 to stabilize MIR210HG. Interestingly, silencing ELAVL1 induced the degradation of MIR210HG (Fig. 5I). In addition, silencing ELAVL1 largely abrogated the effect of ectopic IGF2BP1 on maintaining MIR210HG transcript (Fig. 5J). Furthermore, ELAVL1 was enriched in IGF2BP1 specific immunoprecipitation (Fig. 5K). Therefore, ELAVL1 is a co-factor of IGF2BP1, which contributes to the stabilization of MIR210HG.
From analysis of TCGA-BRCA datasets, IGF2BP1 was confirmed to be highly expressed in breast cancer (Fig. 6A and B). Furthermore, high IGF2BP1 expression indicated a poor relapse survival rate (Fig. 6C). Q-PCR and western blot analysis indicated that IGF2BP1 was highly expressed in breast cancer cells compared to normal cell (Fig. 6D and E), implying that IGF2BP1 was positively correlated to breast cancer. In breast cancer patients, IGF2BP1 was also validated had a higher expression compared to adjacent normal tissues (Fig. 6F and G). Smaller tumors were observed in xenograft model injected with MDA-MB-231 cells transfected IGF2BP1 siRNA (Fig. 6H). Silencing IGF2BP1 significantly repressed MIR210HG expression in MCF-7 and MDA-MB-231 cells (Fig. 6I). In conclusion, IGF2BP1 induces MIR210HG through m6A modification, through which IGF2BP1 functions as an oncogene in breast cancer.
MYCN regulates MIR210HG via IGF2BP1
MYC family has been identified playing critical roles in tumorigenesis by serving as transcription factor, which activates numerous oncogenes by directly binding E-box. In addition, previous studies showed MYC is also regulated by m6A modification. Here, we focused on the transcription ability of MYC family on m6A modification regulators. From Cistrome DB tool, we confirmed that there was one E-box binding motif (CACGTG) under the MYCN enriched peaks in IGF2BP1 promoter (Fig. 7A), which indicated MYCN is a potential transcription factor of IGF2BP1. Clinical relevance analysis revealed that MYCN was up-regulated in tumor tissues (Fig. 7B and C). In addition, the expression of IGF2BP1 was identified positively correlated to MYCN expression in breast cancer (Fig. 7D). Therefore, combining previous studies, MYCN is an oncogene in breast cancer. Next, we performed q-ChIP analysis to validate the target between MYCN and IGF2BP1. Indeed, IGF2BP1 was enriched in the MYCN occupancy site (Fig. 7E). Then luciferase reporter assay was performed following the indicated mutation strategy in IGF2BP1 promoter (Fig. 7F). Mutation in binding site resulted in a significantly repressed luciferase activity (Fig. 7G) in generated MYCN overexpression MCF-7 cell with inducible vector (MCF-7MYCN−HA−OV). q-PCR analysis showed DOX induced MYCN increased IGF2BP1 expression (Fig. 7H) in a time dependent manner. The similar results can also be observed in western blot analysis (Fig. 7I). Of note, ectopic MYCN expression resulted in increased tumor volume and weight in xenograft model but IGF2BP1 silencing largely abrogated this effect (Fig. 7J–L). In summary, MYCN functions as a transcription factor of IGF2BP1, which induces IGF2BP1 expression and promotes breast cancer progression.
Since we confirmed that IGF2BP1 is a direct target of MYCN, next, the potential regulation between MYCN and MIR210HG was investigated. q-PCR analysis showed that MYCN activation increased MIR210HG expression (Fig. 7M). To investigate whether IGF2BP1 mediated the function of MYCN on MIR210HG, IGF2BP1 was knocked down achieved by siRNA pool in MCF-7MYCN−HA−OV cell (Fig. 7N). MYCN activation induced MIR210HG expression but this effect was largely abrogated by IGF2BP1 knocking down (Fig. 7O). The similar results can also be observed in miR-210 expression pattern (Fig. 7O). In addition, clinical relevance data showed that MIR210HG expression was positively correlated to MYCN expression in breast cancer (Fig. 7P). Therefore, both miR-210 and MIR210HG are regulated by MYCN, which is mediated by IGF2BP1.