Silencing Novel Long Non-Coding RNA FKBP9P1 Represses Malignant Progression and Inhibits PI3K/AKT Signaling of Head and Neck Squamous Cell Carcinoma In Vitro
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is associated with a poor prognosis. The survival rate of HNSCC patients is further compromised by the tendency of tumors to invade surrounding tissues and metastasize to cervical lymph nodes. Despite advances in treatment modalities, including surgery, chemotherapy, and radiotherapy, the 5-year overall survival rate remains disappointingly low, ranging from 25% to 60%. This underscores the urgent need for novel therapeutic strategies that move beyond traditional treatments to biomarker-guided approaches based on the genetic differences of tumors. However, few biomarkers are currently used in clinical practice for HNSCC, highlighting the necessity to identify new molecular markers and explore their underlying mechanisms in HNSCC progression.
Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides that lack protein-coding potential. These molecules have emerged as key players in the regulation of various biological processes, including cancer progression. The aberrant expression of lncRNAs has been implicated in the diagnosis and prognosis of numerous human cancers. FKBP prolyl isomerase 9 pseudogene 1 (FKBP9P1) is a newly identified lncRNA located on chromosome 7p11.2. Previous studies have shown that FKBP9P1 is highly expressed in HNSCC tissues compared to adjacent normal tissues. However, the functional significance and molecular mechanisms of FKBP9P1 in HNSCC remain poorly understood.
This study aimed to elucidate the role of FKBP9P1 in HNSCC progression and to explore its potential as a diagnostic and prognostic biomarker. The research was conducted using a combination of clinical samples and in vitro experiments to assess the expression, functional impact, and underlying mechanisms of FKBP9P1 in HNSCC.
The study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University, and all patients provided written informed consent. A total of 114 HNSCC patients who underwent surgical treatment between 2011 and 2015 were enrolled. Tumor tissues and adjacent normal tissues were collected, with one portion used for quantitative real-time polymerase chain reaction (qRT-PCR) and the other for histopathological evaluation. All tumor tissues were confirmed as HNSCC, and adjacent normal tissues were confirmed as tumor-free based on histopathological examination. Patients had not received chemotherapy or radiotherapy prior to surgery.
Follow-up was conducted every 3 months for the first year, every 6 months for the second to fifth years, and annually thereafter. Routine endoscopy and imaging studies were performed to monitor disease progression. Overall survival (OS) was defined as the time from surgery to death or the last follow-up, while disease-free survival (DFS) was defined as the time from surgery to metastasis or relapse. The last follow-up date was December 2018.
Human HNSCC cell lines (FaDu, Cal-27, SCC4, and SCC9) and the human immortalized keratinocyte cell line HaCaT (normal control) were cultured under standard conditions. For FKBP9P1 knockdown, Cal-27 and SCC9 cells were transfected with lentiviral vectors expressing sh-FKBP9P1-1, sh-FKBP9P1-2, or a normal control (sh-NC). Stable cell lines were selected using puromycin, and knockdown efficiency was confirmed by qRT-PCR.
Total RNA was extracted from tissues and cell lines using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit. qRT-PCR was performed using SYBR Green fluorescence, and FKBP9P1 expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative mRNA expression was calculated using the 2^ΔΔCt method.
Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay, where cells were plated in 96-well plates and absorbance was measured at 450 nm over 5 days. Colony formation assays were performed by seeding cells in 6-well plates and staining colonies with crystal violet after 14 days. Wound healing assays were conducted by creating a linear wound in a confluent cell monolayer and measuring wound width at 0 and 48 hours. Trans-well migration and invasion assays were performed using chambers coated with or without Matrigel, and migrated or invaded cells were stained and counted.
Western blotting was used to detect the expression of proteins related to the PI3K/AKT signaling pathway. Total protein was extracted, separated by gel electrophoresis, and transferred to membranes. Antibodies against PI3K, phosphorylated PI3K (p-PI3K), AKT, phosphorylated AKT (p-AKT), and GAPDH were used for detection.
Statistical analyses were performed using SPSS and GraphPad Prism software. The Chi-squared test was used to assess the association between FKBP9P1 expression and clinicopathological features. Survival analyses were conducted using the Kaplan-Meier method and log-rank test. Comparisons between groups were analyzed using Student’s t-test or one-way ANOVA with Bonferroni post hoc tests. A P-value of less than 0.05 was considered statistically significant.
FKBP9P1 expression was significantly up-regulated in HNSCC tissues compared to adjacent normal tissues (tumor vs. normal, 1.914 vs. 0.957, t = 7.746, P < 0.001). Similarly, FKBP9P1 expression was higher in HNSCC cell lines (FaDu, Cal-27, SCC4, and SCC9) than in the HaCaT control cell line (all P < 0.01). These findings suggest that FKBP9P1 may play an oncogenic role in HNSCC.
Patients were divided into high and low FKBP9P1 expression groups based on the median expression level (1.677). High FKBP9P1 expression was associated with advanced T stage (P = 0.022), advanced N stage (P = 0.036), advanced clinical stage (P = 0.018), and poor prognosis (OS, P = 0.002; DFS, P < 0.001). These results indicate that FKBP9P1 could serve as a potential prognostic biomarker for HNSCC.
Knockdown of FKBP9P1 using shRNAs (sh-FKBP9P1-1 and sh-FKBP9P1-2) significantly reduced FKBP9P1 expression in Cal-27 and SCC9 cells (all P < 0.01). CCK-8 and colony formation assays demonstrated that FKBP9P1 knockdown inhibited cell proliferation and colony-forming ability (all P < 0.01). Wound healing and trans-well assays showed that FKBP9P1 knockdown impaired cell migration and invasion (all P < 0.01). These findings suggest that FKBP9P1 promotes HNSCC progression by enhancing cell proliferation, migration, and invasion.
Western blotting revealed that FKBP9P1 knockdown suppressed the phosphorylation of PI3K and AKT, while total PI3K and AKT levels remained unchanged. This indicates that FKBP9P1 knockdown inhibits the PI3K/AKT signaling pathway, which may be the underlying mechanism by which FKBP9P1 promotes HNSCC progression.
This study provides evidence that FKBP9P1 is up-regulated in HNSCC and is associated with tumor progression and poor survival. Silencing FKBP9P1 represses HNSCC cell proliferation, migration, and invasion, and inhibits the PI3K/AKT signaling pathway. These findings highlight the potential of FKBP9P1 as a novel diagnostic biomarker and therapeutic target in HNSCC. Further research is needed to elucidate the detailed mechanisms by which FKBP9P1 modulates the PI3K/AKT pathway and to explore its potential in clinical applications.
doi.org/10.1097/CM9.0000000000000933
Was this helpful?
0 / 0