Abstract
The implication of the let-7 family in cancer development is multifaceted. The family acts as tumor suppressor miRNA although overexpression of let-7 has also been described in many types of cancer, including head and neck squamous cell carcinoma (HNSCC). The aim of this study includes whether different expression levels of let-7d has an influence on chemo- and radiosensitivity. FaDu cell line models with a gradually increased level of let-7d (models from A to E) were generated with the lentiviral system. Expression levels of pluripotency, chemo-radioresistance/apoptosis, and targets of mRNAs were analyzed by real-time reverse transcription-PCR (qRT-PCR). Radiosensitivity was analyzed using a clonogenic assay after irradiation. Response to cisplatin, 5-FU, doxorubicin, and paclitaxel was done with MTT assay. Statistically significant decrease of K-RAS (p = 0.0369) and CASPASE3 (p = 0.0342) were observed with the growing expression level of let-7d. Cisplatin, 5-FU and doxorubicin caused similar decreased of cell survival with the increase of let-7d level (p = 0.004, post-trend p = 0.046; p = 0.004, post trend p = 0.0005 and p<0.0001, post trend p = 0.0001, respectively). All models were resistant to paclitaxel, irrespective of let-7d expression levels. Only two of the generated models (A and C) were radiosensitive (p = 0.0002). Conclusion: the above results indicated that the level of let-7d expression is an important factor for cell response to irradiation and chemotherapeutics.
Highlights
Hypopharynx cancer is an uncommon cancer of the head and neck area
Two FaDu cell lines were created with a single cassette of let-7d (FaDu-7d) and with a double cassette of let-7d (FaDu-7d7d)
It may be assumed that a lack of substantial changes in expression levels of studied mRNAs was caused by high levels between-model variation
Summary
The FaDu cell line and its variants were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4.5 g/L, Sigma), supplemented with 8.85% (v/v) of fetal bovine serum (Sigma), 1.77 mM L-glutamine (PAA), 0.885% (v/v) MEM non-essential amino acid solution (PAA), 0.885% (v/v) penicillin-streptomicin (PAA), and 8.85 mM HEPES (Sigma). The 293T cell line used for the production of lentiviral vectors was cultured in DMEM with high glucose (4.5 g/L) medium (Sigma) supplemented with 8.85% (v/v) fetal bovine serum (Sigma) and 20 mg gentamicin (Kirka). The mycoplasma detection tests (Minerva) were performed routinely during cell line culturing. All cells used in the experiments were the same ones used in the 15th splitting. Statistical analysis was performed with the use of Prism v. The Bonferroni correction was used for estimation of post-test significance
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