Introduction
Classification of different stages of the tumor is one of the most important steps in colorectal cancer (CRC). Investigating the relationship between different gene expressions in patients with CRC and clinical features such as the stage of the disease can estimate the survival and prognostic status of patients and identify patients who are at higher risk for recurrence. PBK is a mitogen-activated protein kinase (MAPKK) among MEK1/2 and MEK7 and can phosphorylate P38, JNK, and ERK in many cellular functions [9]. In addition, PBK/TOPK plays an important role in inflammation, cell apoptosis, and cell cycle regulation. A high PBK/TOPK expression contributes to tumor growth, proliferation, and metastasis [10]. E2F is a group of genes that encode the transcription factor family and regulate genes involved in cell proliferation, differentiation, and apoptosis [12]. In this study, the expression level of genes in the early and advanced stages of CRC has been investigated based on laboratory data and the TCGA (The Cancer Genome Atlas) database.
Materials and Methods
To perform this study, tumor tissue was sampled from 32 patients with CRC at different stages. After surgery, tissue samples were placed in RNALater solution (Behnogen, Iran) and transferred to the Biotechnology Research Center of Islamic Azad University, Shahrekord Branch. In this study, Trisol (Invitrogen, USA) was used to extract total RNA according to the protocol, and the extracted RNA was evaluated qualitatively and quantitatively. The treated RNAs were used in the presence of DNaseІ enzyme for 30 minutes at 37°C to remove possible contamination of the extracted RNA with genomic DNA. Finally, for cDNA synthesis, the kit of Yekta Tajhiz Azma Company and a random Hexamer primer were used. In the present study, the RT-qPCR technique was used to quantitatively measure the expression level of the desired genes. Also, based on RNASeq data extracted from TCGA and clinical information of the samples, the stage information of each sample was determined, and the samples were classified into two groups: stage I+II (n=268) and stage III+IV (n=199). The expression of the two genes was compared between these two groups.
Results
In this study, the expression levels of PKB and E2F7 in different stages of CRC in tumor tissues were analyzed using the RT-qPCR technique and ΔCt method. The results showed that the expression level of these genes changed significantly in different stages of the disease (Figure 1). As can be seen, the expression levels of PBK and E2F7 in stage I+II are 1.3 and 2.5 times higher than in the III+IV  stage, respectively (Figure 1). 
Also, the expression levels of PKB and E2F7 in colorectal tumor tissues were compared between stage I+II and stage III+IV based on the stage information and expression extracted from the TCGA database. The results showed that both genes in the early stages of the disease show a significant increase in expression (Figure 2).  The results from the TCGA database, based on the ROC curve diagram, showed PBK and E2F7 markers with areas below the surface of the diagram AUC=0.7035 and AUC=0.8323, respectively, as significant genes in isolation of these two populations (Figure 3). Discussion 
The study of molecular genetics and the expression level of determining genes at different stages are useful for further understanding the pathogenesis and early detection of CRC. Therefore, detecting the expression level of PBK and E2F7 genes in the early and advanced stages of CRC with RT-qPCR, as well as confirming the results by transcriptome data set (TCGA), may help in early diagnosis and planning effective therapies. PBK and E2F7 genes have been considered biomarkers in various cancers. Therefore, in this study, for the first time, PBK and E2F7 genes were selected as genes with diagnostic potential in early CRC. The results of studies on gastric cancer showed that nuclear expression of PBK was significantly associated with increased invasion and lymph node metastasis.
On the other hand, the results of the Kaplan-Meier survival analysis showed that higher expression of the PBK gene reduces the survival of patients. These results indicate that PBK increases carcinogenicity and metastasis of gastric cancer and acts as a potential biomarker for the prognosis of the disease. The analysis of laboratory data showed that the expression level of the PBK gene was higher in the early stages compared to the advanced stages. The results of transcriptome data also showed that the expression level of this gene in the early stages is higher than in the advanced stages.
The findings of this study showed that PBK and E2F7 genes have higher expression levels in the early stages than in the advanced stages. These results suggest that PBK and E2F7 genes can be used as biomarkers in the early stages. However, these findings need to be confirmed by further studies. In addition, the level of expression of candidate genes in the early stages can be well distinguished from the advanced stages. Therefore, more molecular studies are needed to provide a deeper picture of the function of PBK and E2F7 in CRC.

Ethical Considerations
Compliance with ethical guidelines
This research was approved by the ethics committee of Islamic Azad University, Shahrekord Branch with code IR.IAU.SHK.REC.1399.022.

Funding
This article is taken from the PhD thesis of the Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University and was done with the support of the Research and Technology Vice-Chancellor of Shahrekord Azad University.

Authors' contributions
Study design and conceptualization and methodology: Reza Hashemi and Maryam Peymani; Data mining, formal analysis and review: Reza Hashemi; Supervision, validation, visualization and final approval: Maryam Peymani and Kamran Qaidi; Interpretation of obtained information: Maryam Peymani; sample collection: Hana Safar; Article writing, revision and editing: Reza Hashemi; All authors read and approved the final manuscript.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors thank and appreciate all the people who helped in collecting blood samples in this research.

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