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FHL2 interacts with LDHA to promote glioma cell proliferation
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Abstract:
Objective: To investigate the effects of four-and-a-half LIM domains 2 (FHL2) on glioma cell proliferation and its molecular mechanisms. Methods: The relationship between FHL2 mRNA expression levels in gliomas and patient prognosis was analyzed using TCGA and CGGA databases. The protein expression levels of FHL2 in collected human glioma tissue samples and human glioma cell lines (U87, T98G, U251, SNB19, GSC23, A172, LN229, G267), and astrocyte NHA were analyzed using Western blot (WB). Lentiviral vecors were used to construct U87 cells with stable FHL2 knockdown and SNB19 cells overexpressing FHL2, namely U87-shGFP, U87-shFHL2-1#, U87-shFHL2-4#, and SNB19-3flag, SNB19-3flag-FHL2 groups. The effects of FHL2 knockdown and overexpression on cell proliferation were assessed using CCK-8 assays and colony formation assay. Coimmunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC/MS) were employed to identify proteins interacting with FHL2 in glioma cells, and Co-IP and immunofluorescence were used to verify their binding and co-localization. Changes in intracellular lactate production and lactate dehydrogenase (LDH) activity following FHL2 knockdown and overexpression were measured using a microplate reader. WB was used to analyze the protein expression levels of FHL2, LDHA, and p-LDHA in normal brain tissues and glioma tissues, as well as their relationships. The small molecule inhibitor of LDHA, AT-101, was used to treat SNB19 cells overexpressing FHL2. The role of FHL2 in glioma lactate metabolism and the potential therapeutic effect of AT-101 in glioma were validated using CCK-8 assays and colorimetric assays with a microplate reader. Results: Co-IP and LC/MS analyses revealed an interaction between FHL2 and LDHA in glioma cells. Overexpression of FHL2 increased LDHA activity and lactate production (all P < 0.001), thereby promoting glioma cell proliferation (P < 0.001 or P < 0.001). Conversely, knockdown of FHL2 reduced LDHA activity and lactate production (P < 0.001, P < 0.05) and inhibited cell growth (P < 0.01). AT101 inhibited LDHA activity and significantly suppressed FHL2-induced glioma cell proliferation while restoring phosphorylated LDHA (Y10) levels (P < 0.01, P < 0.001). Conclusion: FHL2 interacts with LDHA protein, and FHL2 promotes LDHA activity and lactic acid production by activating the expression of p-LDHA (Y10), thus facilitating the proliferation of glioma cells. Targeting this interaction may become a potential strategy for treating gliomas.
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