Research Article: Different levels of let-7d expression modulate response of FaDu cells to irradiation and chemotherapeutics

Date Published: June 30, 2017

Publisher: Public Library of Science

Author(s): Katarzyna Monika Lamperska, Tomasz Kolenda, Anna Teresiak, Anna Kowalik, Marta Kruszyna-Mochalska, Weronika Jackowiak, Renata Bliźniak, Weronika Przybyła, Marta Kapałczyńska, Piotr Kozlowski, Aamir Ahmad.

http://doi.org/10.1371/journal.pone.0180265

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.

Partial Text

Hypopharynx cancer is an uncommon cancer of the head and neck area. It occurs both in males and females, primarily aged between 50 and 60 years. In most cases, hypopharyngeal cancer is of a mucosal squamous cell origin, preceded by various precancerous lesions. Hypopharyngeal cancer does not usually have early symptoms, along with a high ability to metastasize, thus causing the lowest survival rate of all head and neck cancers. The treatment approach is based on surgery and postoperative radiotherapy. These advanced, unresectable cancers are treated by radiation and/or chemotherapy. Personalization of therapy might improve patients’ survival, but it is necessary to find biomarkers describing radio- and chemosensitivity for this kind of tumor. One promising group of biomarkers is miRNAs.

The role of let-7 in cancer development and diagnostics has already been described. Currently, it is considered to be a therapeutic application, based on the restoration of the normal let-7 level. This approach is recommended for those particular cancers, which show underexpression of miRNA. On the other hand, let-7 may be also be overexpressed, which indicates that let-7 does not play an only suppressor function, but can also act as oncomiR. This notion, among others, was supported by experiments on liver cells showing that chronic overexpression of let-7 caused liver damage, degeneration, and finally cancer [43]. Overexpression of let-7 may be induced by different mechanisms: from regulation of let-7 cluster transcription via changes in maturation machinery to global deregulation of cell pathways. Firstly, the effect of overexpressed let-7d was showed by Chang et al [44] demonstrating that let-7d negatively modulated TWIST and SNAIL expression. However, target mRNA analysis in this work revealed statistically significant result only for K-RAS and CASPASE3. It may be assumed that a lack of substantial changes in expression levels of studied mRNAs was caused by high levels between-model variation. On the other hand, in most cases, we observed a negative or close to 0 slope, which suggested that different let-7d levels inhibited expression of the examined mRNAs. Another explanation of our results relates to the very low level of let-7d expression in FaDu cells, in fact, generated expression of let-7d in the models was still too low to see substantial changes in analyzed mRNA expression. This notion is in line with the observation that when model A was excluded, a statistically significant trend was observed for CASPASE3, ATM, K-RAS, N-RAS, HMGA1, and ARID3A. This may suggest that more noticeable differences for the investigated genes expression may be observed for higher levels of let-7d than presented in model A and the effect of let-7d overexpression ought to be analyzed in models with its expression higher than in model E. Thirdly, let-7d belongs to the family including 10–13 members [27], sharing common mRNAs targets but having their own specific characteristics. Some authors postulate that the overall level of the let-7 family is important for its biological effect [45]. We analyzed the influence of let-7d expression, but it is possible that not all of its levels harmonized with those of other members of the family. The specific FaDu genetic context influenced our data.

The determination of individual miRNA expression changes can be an important marker of successful patient treatment. We focused on let-7d, known as miRNA suppressor molecule, for which overexpression was described in HNSCC. In other studies both over and underexpression of let-7d in different cancer types were also observed. In fact, the actual level of let-7d differ substantially between cancer samples and cannot be simply classified either as a high (overexpression) or low (underexpression). Therefore in our study, we focused on the question, whether incrementally different levels of let-7d influence chemo- and radiosensitivity. Generally, increased levels of let-7d enhanced cells sensitivity to chemotherapeutics. Only two of the investigated models showed an effect after irradiation. On the other hand, growing level of let-7d expression caused a declining tendency in the expression of the studied genes but it does not let to connect these specific radio- and chemoresistant let-7d levels with changes of expression of investigated mRNAs. Summarizing, we found that level of let-7d was of significance for cell response to irradiation and chemotherapeutics.

 

Source:

http://doi.org/10.1371/journal.pone.0180265

 

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