Long Non-Coding RNA HOTAIRM1 Promotes Proliferation and Inhibits Apoptosis of Glioma Cells by Regulating the miR-873-5p/ZEB2 Axis

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Long Non-Coding RNA HOTAIRM1 Promotes Proliferation and Inhibits Apoptosis of Glioma Cells by Regulating the miR-873-5p/ZEB2 Axis

Glioblastoma, the most aggressive form of glioma, is a malignant brain tumor with a poor prognosis and high mortality rate. Despite advances in clinical treatments such as surgical resection, radiotherapy, and chemotherapy, the recurrence rate remains high, and drug resistance and treatment complications persist. Therefore, understanding the molecular mechanisms underlying glioblastoma initiation and progression is crucial for developing novel therapeutic strategies. Recent research has highlighted the roles of non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in regulating gene expression and influencing cancer progression. This study investigates the role of miR-873-5p in glioblastoma and explores the regulatory axis involving lncRNA HOTAIRM1 and the transcription factor ZEB2.

Background and Significance

Gliomas account for 80% of primary malignant brain tumors, with glioblastoma (Grade IV glioma) being the most aggressive and prevalent. Current treatments are insufficient due to the tumor’s invasive nature and resistance to therapy. Non-coding RNAs, particularly miRNAs and lncRNAs, have emerged as critical regulators of gene expression in cancer. MiRNAs, small non-coding RNAs of 18-25 nucleotides, regulate gene expression by binding to complementary sites in the 3′-untranslated regions (UTRs) of target mRNAs, leading to mRNA degradation or translational inhibition. LncRNAs, on the other hand, can act as competing endogenous RNAs (ceRNAs), sponging miRNAs and preventing them from binding to their target mRNAs, thereby modulating gene expression.

The transcription factor ZEB2 is known to promote epithelial-mesenchymal transition (EMT), a process associated with cancer metastasis and progression. In glioblastoma, ZEB2 has been implicated in cell proliferation, migration, and apoptosis. This study aims to elucidate the role of miR-873-5p in glioblastoma and its interaction with lncRNA HOTAIRM1 and ZEB2, providing insights into the molecular mechanisms driving glioblastoma progression.

Methods

Clinical Samples and Cell Culture

The study analyzed 20 glioblastoma tissue samples and 10 normal brain tissue samples collected from Zhejiang Tongde Hospital. Normal brain tissues were obtained from adjacent non-tumor regions of glioblastoma patients. The glioblastoma cell lines U87, LN-229, U-251, and A172, along with normal human astrocytes (HA), were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics.

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from tissues and cell lines using TRIzol reagent. Complementary DNA (cDNA) was synthesized using a High Capacity cDNA Reverse Transcription Kit, and qRT-PCR was performed to measure the expression levels of miR-873-5p, ZEB2, and HOTAIRM1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as endogenous controls for mRNA and miRNA expression, respectively.

Cell Transfection and Functional Assays

MiR-873-5p mimics, inhibitors, and mock controls were transfected into U87 cells using Lipofectamine 2000. The effects of miR-873-5p on cell proliferation, migration, and apoptosis were assessed using the Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and flow cytometry, respectively. Additionally, the expression of ZEB2 and HOTAIRM1 was modulated using pcDNA vectors and shRNA constructs.

Luciferase Reporter Assay and RNA Immunoprecipitation (RIP)

The interaction between miR-873-5p and the 3′-UTR of ZEB2 or HOTAIRM1 was examined using luciferase reporter assays. RNA immunoprecipitation was performed to confirm the binding of HOTAIRM1 and miR-873-5p to Argonaute 2 (Ago2), a key component of the RNA-induced silencing complex (RISC).

Western Blotting

Protein levels of ZEB2, Cyclin A1, Cyclin D1, Bcl-2, and cleaved Caspase-3 were analyzed using Western blotting. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE before being transferred to PVDF membranes. Specific primary antibodies were used to detect target proteins, and tubulin or GAPDH served as loading controls.

Statistical Analysis

Data were analyzed using GraphPad Prism software. Differences between groups were assessed using Student’s t-test or the Kruskal-Wallis test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant.

Results

MiR-873-5p is Downregulated in Glioblastoma

Analysis of the GEO dataset GSE103228 revealed that miR-873-5p was significantly downregulated in glioblastoma tissues compared to normal brain tissues. This finding was confirmed in clinical samples, where miR-873-5p expression was lower in glioblastoma tissues (0.378 ± 0.114) than in normal tissues (0.762 ± 0.231). Similarly, miR-873-5p levels were reduced in glioblastoma cell lines, with the lowest expression observed in U87 cells.

MiR-873-5p Inhibits Cell Proliferation and Migration and Promotes Apoptosis

Overexpression of miR-873-5p in U87 cells significantly inhibited cell proliferation and migration while promoting apoptosis. Conversely, inhibition of miR-873-5p had the opposite effect, enhancing cell growth and migration and reducing apoptosis. These findings suggest that miR-873-5p acts as a tumor suppressor in glioblastoma.

ZEB2 is a Target of MiR-873-5p

Bioinformatics analysis predicted that ZEB2 is a target of miR-873-5p. Luciferase reporter assays confirmed that miR-873-5p binds to the 3′-UTR of ZEB2 mRNA, leading to its downregulation. Overexpression of miR-873-5p reduced ZEB2 expression, while inhibition of miR-873-5p increased ZEB2 levels. Reintroduction of ZEB2 in miR-873-5p-overexpressing cells reversed the effects on cell proliferation, migration, and apoptosis, indicating that ZEB2 mediates the tumor-suppressive function of miR-873-5p.

HOTAIRM1 Promotes Glioblastoma Progression

The lncRNA HOTAIRM1 was found to be upregulated in glioblastoma and negatively correlated with patient survival. HOTAIRM1 overexpression increased ZEB2 expression and promoted cell proliferation and migration while inhibiting apoptosis. Conversely, knockdown of HOTAIRM1 reduced ZEB2 levels and suppressed tumor progression. These effects were counteracted by miR-873-5p inhibition, suggesting that HOTAIRM1 acts as a ceRNA, sponging miR-873-5p and preventing it from targeting ZEB2.

HOTAIRM1 Sponges MiR-873-5p to Activate ZEB2

Luciferase reporter assays and RNA immunoprecipitation confirmed that HOTAIRM1 directly interacts with miR-873-5p. Overexpression of HOTAIRM1 reduced miR-873-5p levels and increased ZEB2 expression, while knockdown of HOTAIRM1 had the opposite effect. These findings indicate that HOTAIRM1 promotes ZEB2 expression by sequestering miR-873-5p, thereby enhancing glioblastoma progression.

Discussion

This study identifies a novel regulatory axis involving lncRNA HOTAIRM1, miR-873-5p, and ZEB2 in glioblastoma. MiR-873-5p acts as a tumor suppressor by inhibiting ZEB2 expression, thereby reducing cell proliferation and migration and promoting apoptosis. In contrast, HOTAIRM1 functions as an oncogene by sponging miR-873-5p, leading to increased ZEB2 expression and enhanced tumor progression. These findings provide new insights into the molecular mechanisms driving glioblastoma and highlight potential therapeutic targets for this aggressive cancer.

The role of miR-873-5p in cancer appears to be context-dependent. While it has been reported to promote lung cancer progression, it acts as a tumor suppressor in colon cancer by inhibiting the TUSC3/AKT pathway. In glioblastoma, our study demonstrates that miR-873-5p suppresses tumor progression by targeting ZEB2, a transcription factor involved in EMT and cancer metastasis.

The lncRNA HOTAIRM1 has previously been implicated in myeloid cell development and cancer progression. In colorectal cancer, HOTAIRM1 acts as a tumor suppressor, while in gastric cancer, it inhibits tumor progression by regulating the miR-17-5p/PTEN axis. In glioblastoma, our findings reveal that HOTAIRM1 promotes tumor progression by sponging miR-873-5p and activating ZEB2 expression.

The identification of the HOTAIRM1/miR-873-5p/ZEB2 axis in glioblastoma provides a potential therapeutic strategy for targeting this aggressive cancer. Inhibition of HOTAIRM1 or restoration of miR-873-5p expression could suppress ZEB2-mediated tumor progression and improve patient outcomes. Further studies are needed to explore the therapeutic potential of targeting this regulatory axis in glioblastoma.

Conclusion

This study uncovers a novel regulatory axis involving lncRNA HOTAIRM1, miR-873-5p, and ZEB2 in glioblastoma progression. MiR-873-5p acts as a tumor suppressor by inhibiting ZEB2 expression, while HOTAIRM1 promotes tumor progression by sponging miR-873-5p and activating ZEB2. These findings provide new insights into the molecular mechanisms driving glioblastoma and highlight potential therapeutic targets for this aggressive cancer. Future research should focus on developing strategies to modulate this regulatory axis for the treatment of glioblastoma.

doi.org/10.1097/CM9.0000000000000615

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