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Evaluation of expression levels of NFATc2 and PPARG genes two effector elements of WNT pathway in non-small cell lung carcinoma
Kaveh Motamediyan1, Venus Zafari2, Soghra Bornehdeli1, Ayşe Caner2, Milad Asadi2, Shahryar Hashemzadeh3, Mohammad Reza Firozi1, Mortaza Raeisi4
1 Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Basic Oncology of Health, Institute of Ege University, Izmir, Turkey
3 Tuberculosis and Lung Disease Research Center; Department of General and Vascular Surgery, Imam Reza Hospital, Tabriz University of Medical Sciences, Tabriz, Iran
4 Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| Date of Submission | 02-Jun-2022 |
| Date of Acceptance | 15-Mar-2023 |
| Date of Web Publication | 20-Jul-2023 |
Correspondence Address:
Dr. Mortaza Raeisi
Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Daneshgah Street, Tabriz
Iran
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/abr.abr_185_22
Background: There is an emergency need in discovering an efficient profile of molecular biomarkers for early diagnosis of Non-small cell lung cancer (NSCLC). Transcription factors as important groups of regulators that are able to adjust the cell cycles have attracted the attention of most researchers recently. NFATc2 and PPARG are two important factors that have been selected for this project to assess their potential for being a biomarker for NSCLC.
Materials and Methods: Here in this study, 50 NSCLC patients were included. During bronchoscopy, which was their routine diagnostic approach, we collected tumoral and marginal normal tissues. After the extraction of the total RNA from the tissues, cDNA was synthesized, and the transcriptional level of NFATc2 and PPARG was examined by quantitative real-time PCR. Subsequently, the data were analyzed by proper statistical analyses.
Results: The mRNA expression of NFATc2 and PPARG were down-regulated in biopsy tissues of NSCLC patients compared with their pair marginal tissues (Pvalues were 0.0011 and <0.0001 respectively). Moreover, both of them had significant AUC (area under the curve) in the ROC curve analysis (0.65 for NFATc2 and 0.81 for PPARG, Pvalue <0.05).
Conclusion: It appears that mRNA expression of NFATc2 and PPARG possesses the potential to be regarded as a diagnostic or prognostic biomarker for NSCLC.
Keywords: Biomarker, PPARG, NFATc2, NSCLC
How to cite this article:
Motamediyan K, Zafari V, Bornehdeli S, Caner A, Asadi M, Hashemzadeh S, Firozi MR, Raeisi M. Evaluation of expression levels of NFATc2 and PPARG genes two effector elements of WNT pathway in non-small cell lung carcinoma. Adv Biomed Res 2023;12:184 |
How to cite this URL:
Motamediyan K, Zafari V, Bornehdeli S, Caner A, Asadi M, Hashemzadeh S, Firozi MR, Raeisi M. Evaluation of expression levels of NFATc2 and PPARG genes two effector elements of WNT pathway in non-small cell lung carcinoma. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:184. Available from: https://www.advbiores.net/text.asp?2023/12/1/184/382064 |
| Introduction | |  |
Lung cancer is one of the deadliest malignancies worldwide. Despite the significant advances in the control and therapy of lung cancer, it is on the rise globally. Tobacco smoking accounts for the main risk factor for lung cancer and outweighs all other factors that lead to this malignancy. Lung cancer has different histological subtypes; Non-small cell lung cancer (NSCLC) is responsible for about 85% of all cases all around the world, and lung adenocarcinoma is the most common subgroup of NSCLC. The 5-year survival of lung cancer patients is dramatically low so it doesn't reach 19.4 even after surgical intervention.[1],[2] Successful treatment and good prognosis of malign cancers depend on early detection and enhanced screening programs, which require a valid profile of molecular markers. Molecular alterations occur prior to all visual symptoms, even histological changes. So, it makes sense to invest in discovering new efficient biomarkers.[3]
NFAT family encompasses transcription factors that play critical roles in carcinogenesis.[4] This family includes four members, i.e. NFATc1 (also known as NFATc or NFAT2), NFATc2 (also known as NFATp or NFAT1), NFATc3 (also known as NFATx or NFAT4), NFATc4 (also known as NFAT3), and the more distantly related NFAT5 (also known as TonEBP or OREBP).[5] The members of the NFAT family transcription factors are involved in the modulation of diverse cellular processes like cell invasion, proliferation, survival, migration, and angiogenesis. Different members of the NFAT family are constitutively triggered and overexpressed in diverse tumor cells, where they are able to trigger a downstream pathways that plays critical functions in the progression of tumor behaviors. That notwithstanding, different members of the NFAT family members are involved in the distinct steps of tumorigenesis and different cellular processes.[6] Recent studies indicated that NFAT promotes metastasis and angiogenesis in lung cancer.[7] In another study, the expression of the NFAT1/2/4/5 in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) was reported to be downregulated, whereas NFAT3 mRNA expression was upregulated. Also, they suggested that the NFAT family members divulged different degrees of aberrant expressions, advocating that NFATs might be therapeutic targets for NSCLC patients.[8] In another study, the researcher found that abnormal expression of the NFATc1 and NFATc3 might be involved in the incidence of NSCLC as well as Brain metastases through modulating IL-11, CDH5, and CCL2.[9]
Peroxisome proliferator-activated receptor gamma (PPARG) is an important ligand-activated factor and one of the nuclear hormone receptor superfamily members, which are expressed in different types of body tissues. This molecule is proposed as one of the potential therapeutic targets of various cancers. PPARG is involved in the differentiation of adipocytes, metabolism of lipids, utilization of peripheral glucose, and also inflammatory response. The regulatory roles of PPARs in inflammation are by means of controlling prostaglandins and Leukotrienes. Both two isoforms of this gene, PPARG1, and PPARG2, function as tumor suppressors in different types of malignancies such as prostate cancer.[10],[11]
A bulk of research indicates that PPAR-γ is involved in the carcinogenesis as well as the progression of several tumor types, and its dysregulation has been implicated with cell proliferation, differentiation, and resistance to apoptosis. Recent studies indicated that the level of PPAR-γ protein expression was associated with the histopathological steps of tumors as well as molecules related to apoptosis, implying its possible implication in the biological approaches underlying the carcinogenic alterations of the lung tissue.[12] Also, in another study researchers found that PPAR-γ protein was more significantly detected in squamous cell lung carcinoma than in lung adenocarcinoma.[13] Considering the critical function of these two transcription factors, NFATc2 and PPARG, in cancer-related pathways, in this study we aimed to the assessment of the potential of these genes as biomarkers for non-small cell lung cancer.
| Materials and Methods | | |
Study population
A total of fifty NSCLC tissue samples and their paired marginal normal tissues were collected in this study during bronchoscopy. All the sample collection process was done from March to July 2018 in the Imam Reza Hospital affiliated with the Tabriz University of Medical Sciences. Patients with any experience of chemo- or radiotherapy, and also unwilling subjects were ruled out from the study. The prepared tissues were immediately immersed into RNase later solution (Qiagen, Hilden, Germany) and were stored at -80°C for further experiments. This study was confirmed by the Ethical Committee of Tabriz University of Medical Sciences and all of the participants had signed the written informed consent before the sampling process (Confirmation Code is IR.TBZMED.REC.1397.133. [Table 1] depicts the baseline specifications of the study participants.
RNA isolation and cDNA synthesis
The total RNA extraction process from tissue samples was done by RiboEx reagent (Gene All biotechnology, Seoul, Korea) in accordance with the manufacturer's instructions. After quality and quantity control of the extracted RNA using gel electrophoresis and Nanodrop OneC Spectrophotometer (Thermo Scientific™, USA), the cDNA synthesis was performed by means of 2× RT-PCR Pre-Mix (Taq) Universal cDNA synthesis kit (BioFACT TM, Seoul, South Korea) based on the manual of the kit.
Real-time PCR
Measuring the NFATc2 and PPARG mRNA expression levels was conducted by means of qRT-PCR using Step One™ qRT-PCR system (Applied Biosystems, Foster City, CA, USA) and Real Q plus Master mix Green (Ampliqon, Denmark). Primers were designed by Oligo 7 software (Molecular Biology Insights. Inc., Cascade, Co, USA) and were checked using Basic Local Alignment Search Tool (BLAST) tool at National Center for Biotechnology Information (NCBI). Each reaction mixture contained a total volume of 20 μl (10 μl master mix, 2 μl of cDNA (5 ng/ml), 1 μl of the primer mix (10 pmol/ml), and 7 μl H2O). The transcript level of the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the housekeeping gene. The light cycler temperature conditions were as follows: 94°C for 10 min, the 40 amplification cycles of 94°C for 15 sec, the annealing temperature for 30 sec, and 72°C for 30 sec for the extension. [Table 2] shows the primer sequences and annealing temperatures.
Statistical analysis
To determine the normality of the scale data distribution, the Kolmogorov-Smirnov test was run. To compare the expression level of target genes between groups, an unpaired t-test was run. All quantitative data were expressed as mean ± standard deviation (SD) and P values less than 0.05 were considered as the significance level for analytical statistics results. Graph Pad Prism 6 (Graph Pad Software Inc. San Diego, CA, USA) was used to conduct statistical comparisons.
| Results | | |
Down-regulation of both NFATc2 and PPARG in tumor tissues
According to the analyzed results of qRT-PCR, the expression level of both NFATc2 and PPARG genes was significantly downregulated in tumor samples compared to adjacent normal tissues (Pvalue for NFATc2 = 0.0011 and Pvalue for PPARG <0.0001) [Figure 1]. In addition, the expression level of NFATc2 and PPARG were significantly correlated with metastasis and lymph-nod invasion [Table 3]. | Figure 1: The relative expression level of NFATc2 (a) and PPARG (b) in NSCLC tissues in comparison to normal marginal tissues. (*: the sign of significance) (Pvalue** =0.0011, Pvalue **** <0.0001)
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 | Table 3: NFATc2 and PPARG expression correlation with cilinocopathological Features
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The potential of NFATc2 and PPARG as a biomarker
Receiver operating characteristics (ROC) were traced for both NFATc2 and PPARG genes. The specificity and sensitivity of this molecule were assessed by the area under the curve (AUC) calculation. NFATc2 had an AUC of 0.65 and PPARG had an AUC of 0.81. Both values were significant (P < 0.05) [Figure 2]. | Figure 2: The ROC curve analyze of the NFATc2 (a) and PPARG (b) expression. The Area under curve (AUC) for NFATc2 is 0.65 and for PPARG is 0.81. Both of values are significant (Pvalue < 0.05)
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| Discussion | | |
A biomarker is determined as a factor that is assessed and evaluated as a sign of natural biological processes, pathogenic processes, therapeutic responses, or drug resistance.[9],[14] Considering the asymptomatic progress of lung cancer, and the detection of cases at an advanced stage, we need to find new efficient markers to be used for initial screening and informing us of the onset or recurrence of lung cancer.
Transcription factors, which function as the main regulatory elements of cell cycles, can potentially be used as cancer biomarkers. NFATc2 and PPARG are two important transcription factors that have involvement in the carcinogenesis phenomenon, and their expression level as an indicator have been analyzed in the current study. Our results showed that the mRNA expression level of NFATc2 and PPARG genes was significantly reduced in tumor tissues in comparison with the normal adjacent tissues, suggesting the tumor suppressor role of these transcription factors in the carcinogenesis process.
NFATc2 is one of the protein types its transcriptional activity is adjusted through a cycle of dephosphorylation (by the calcium-dependent phosphatase calcineurin) and re-phosphorylation (potentially by glycogen synthase kinase-3) processes.[10],[15] This molecule plays a role in the last steps of the non-canonical WNT signaling pathway, which is critically involved in the tumorigenesis of several cancers.[11],[16] The result of our study about NFATc2 is contrary to most previous studies, which have indicated the up-regulation of this molecule in malignancies. The major of previous studies had introduced the NFATc2 as an oncogene which promotes the metastasis, invasion, proliferation, and survival of cancer cells. For instance, Lang T et al. reported that NFATc2 promotes the stemness of colorectal cancer (CRC) cells by means of regulating downstream targets.[12],[16] Also, NFATc2 was demonstrated to promote angiogenesis by inducing vascular endothelial growth factor (VEGF) gene expression.[13],[17] In contrast, Maxeiner et al. whose work was on lung adenocarcinoma too, showed the downregulation of NFATc2 in tumor biopsy samples in comparison to healthy control participants.[14],[18] Because of this controversy, we aimed to investigate the expression of this gene in lung cancer tissues compared with its normal marginal tissues and found a similar result to the Maxeiner H project. Moreover, according to our results, alteration of NFATc2 expression has a high significant potential to be exerted as a biomarker for NSCLC diagnosis. In another research, Zafari et al.[19] found that NFATc2 was upregulated in the tumor tissues in relation to the normal marginal tissues, while PPARG expression level was not significantly differed between the tumor tissues and marginal normal tissue. Also, they suggested that NFATc2 might be exerted as an early diagnostic or prognostic biomarker for CRC as well as a therapeutic target.[19] Also, Lang et al.[16] reported that NFATc2 stimulates the stemness of CRC stem cells by AJUBA-mediated activation of the Hippo/YAP signaling pathway. They also revealed that NFATc2 might be a novel therapeutic target for CRC stem cells and suggested that YAP inhibitors could be an effective therapeutic tool for treating human CRC due to the dysregulation of NFATc2 activation.
PPARG, the second transcriptional factor that we analyzed in NSCLC, initially was thought to have a role just in fatty acids metabolisms. However, later a regulatory role of it has discovered in other important cell processes. Villa et al. showed that PPARG was downregulated in CRC and plays a critical function in modulating the proliferation of different tumor types like CRC[15],[16],[17],[18] which is in line with our study.[20] Moreover, according to the study of Goldstein et al., PPARG has sufficient competency to be the therapeutic target of bladder cancer.[19],[21] Also Yaghoubizadeh et al.[22] reported that PPARα and PPARδ were upregulated while PPARγ was downregulated in CRC tumor tissues, and these aberrant expressions were correlated with cancer development and poor disease prognosis. On the other hand, there are some reports of PPARG upregulation in certain cancer types. For example, Yang et al. in their study on HER2-positive breast cancer found the upregulation of PPARG and suggested that PPARG is a suitable therapeutic target for HER2-positive breast cancer.[20],[23] These observations indicate that PPARG is able to serve as an oncogene or tumor suppressor or according to the tissue type, cellular context, and genetic predisposition of a case.[21],[24]
In this study, considering normal margin tissues of the same participants as the control group cause to elimination of some usual biases like race, individual differences, and also the effect of external factors. Due to the limited budget amount, we could not confirm our claims in protein level, so we suggest for future projects assess the molecular changes of these genes at the protein level, as well as with their target genes.
| Conclusion | | |
In summary, NFATc2 and PPARG are two vital transcription factors that have notable functions during the initiation and development of cancers, especially lung cancer. Down-regulation of these genes in NSCLC tissues compared with normal marginal tissues proposed the tumor suppressor effect of them in NSCLC. They possess a high and valuable potential to be a diagnostic or prognostic biomarker for NSCLC. We hope that a profound acknowledgment of molecular alterations, like what we have done in this study, helps to design novel and more efficient drugs too.
Ethics approval
This study was confirmed by the Ethics Committee of Tabriz University of Medical Sciences as a research project.
Financial support and sponsorship
The study has been funded as a research grant, by the Ministry of Health and Medical Education, Deputy of Research and Technology (Grant number: 700/98, 2015.03.14 [1394/12/24]) from the Iran Ministry of Health.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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