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Author Spotlight: RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells
Published on: June 16, 2023
[Mechanism of lncRNA-SRLR induced invasion and metastasis in U2OS osteosarcoma cells]
1Department of Orthopaedics, Pingdingshan First People's Hospital, Pingdingshan 467000, China.
Abstract:
Objective: To explore the potential mechanism of sorafenib resistance associated long non-coding RNA (lncRNA-SRLR) promoted invasion and metastasis in U2OS osteosarcoma cells. Methods: We transfected U2OS cells with negative control lentivirus (LV-NC) or lncRNA-SRLR overexpressed lentivirus (LV-over/SRLR) particles. LV-NC and LV-over/SRLR stable transfected cells (U20S/NC and U20S/SRLR) were selected by primary cell culture medium containing puromycin. The mRNA expressions of lncRNA-SRLR and procollagen-lysine, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of lncRNA-SRLR on the invasion of U2OS cells were determined by wound-healing assay and Transwell migration assay. The effect of SRLR on the interleukin-6 (IL-6) secretion of U2OS cells was evaluated by enzyme-linked immunosorbent assay (ELISA) analysis. The subcellular distribution of SRLR in U2OS cells was detected by fluorescence in situ hybridization (FISH) analysis.The expression of PLOD2 in cells was detected by immunofluorescence (IF). The expressions of PLOD2 and focal adhesion kinase (FAK)/signal transducer and activator of transcription 3 (STAT3) signal pathway related proteins in U2OS/NC and U2OS/SRLR cells were detected by western blotting. Results: qRT-PCR assay showed that mRNA expressions of lncRNA-SRLR and PLOD2 in U2OS/SRLR cells were (3 964.97±0.05) and (2.77±0.11), respectively, significantly higher than those in U2OS/NC cells (P<0.001 or P<0.01). The results of wound-healing and Transwell migration assay showed that over-expression of SRLR markedly promoted the invasion ability of U2OS cells (P<0.05). The result of ELISA analysis showed that the IL-6 secretions in U2OS/NC or U2OS/SRLR cells were (125.38±11.22) pg/ml or (119.97±13.43) pg/ml, without statistical significance (P>0.05). The subcellular distribution assay revealed that lncRNA-SRLR is predominately located in the nucleus. The result of IF showed that compared with U2OS/NC cells, the expression of PLOD2 was up-regulated in U2OS/SRLR cells. The result of western blotting showed that over-expression of SRLR significantly increased the expression levels of PLOD2, phosphorylation (p)-FAK and p-STAT3 in U2OS cells (P<0.01). Conclusion: lncRNA-SRLR promotes invasion and metastasis of osteosarcoma by activating PLOD2-FAK/STAT3 signal axis.
Insights
This study reveals that the long non-coding RNA SRLR promotes osteosarcoma cell invasion and metastasis by activating the PLOD2-FAK/STAT3 signaling pathway, offering potential therapeutic targets for sorafenib resistance.
Area of Science:
- Molecular Biology
- Oncology
- Cell Biology
Background:
- Sorafenib resistance is a significant challenge in osteosarcoma treatment.
- Long non-coding RNAs (lncRNAs) are increasingly recognized for their roles in cancer progression.
- Understanding the mechanisms underlying drug resistance and metastasis is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the role of sorafenib resistance-associated long non-coding RNA (lncRNA-SRLR) in promoting invasion and metastasis in U2OS osteosarcoma cells.
- To elucidate the molecular mechanism by which lncRNA-SRLR contributes to osteosarcoma progression.
Main Methods:
- U2OS cells were transfected with lentivirus to overexpress lncRNA-SRLR.
- Quantitative real-time PCR (qRT-PCR), wound-healing assays, Transwell migration assays, ELISA, fluorescence in situ hybridization (FISH), immunofluorescence (IF), and western blotting were employed.
- Expression levels of lncRNA-SRLR, PLOD2, and key proteins in the FAK/STAT3 pathway were analyzed.
Main Results:
- Overexpression of lncRNA-SRLR significantly increased the mRNA and protein expression of PLOD2 in U2OS cells.
- lncRNA-SRLR overexpression markedly enhanced the invasion and migration abilities of U2OS cells.
- lncRNA-SRLR activation led to increased phosphorylation of FAK and STAT3, suggesting activation of the PLOD2-FAK/STAT3 signaling axis.
Conclusions:
- lncRNA-SRLR plays a critical role in promoting the invasion and metastasis of osteosarcoma cells.
- The mechanism involves the activation of the PLOD2-FAK/STAT3 signaling pathway.
- Targeting lncRNA-SRLR or the PLOD2-FAK/STAT3 axis may represent a novel therapeutic strategy for overcoming sorafenib resistance in osteosarcoma.
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