Evaluation of the Chemo-Radiotherapy Effect on the Level of Expression of miR-374 Gene Expression in Rectal Cancer Samples

Background: There are several methods for treatment of rectal cancer including surgery, radiation therapy and chemotherapy. Despite all the treatment approaches for cancer, still one of the major challenges in the treatment of cancers is defect in early diagnosis. miRNAs can be used as markers of diagnosis and treatment of cancers. This study will be investigated the effect of radiotherapy on the expression level of miR-374 gene and two its target genes in WNT pathway in Samples of rectal cancer treated with radiotherapy. Methods: In previous study, we predicted the miR-374 that target main genes of the WNT signal transduction pathway using bioinformatics analysis. The RNA extraction and cDNA synthesis were performed on 25 patients with rectal cancer in three periods before and after radiotherapy treatment. The expression levels of miR-374, GSK-3β and APC were evaluated by using qRT-PCR. The amplicon products were sent to sequencing by Macrogene Company. Finally, expression data were evaluated with demographic feathers and signicantly of data were evaluated using specic software. Results: The results sequencing conrmed properly amplication reaction. Quantitative RT-PCR revealed signicant down-regulation of miR-374 (0.63 folds) and up-regulation of APC (1.12 folds) and GSK-3β ( 1.22 folds), on patients with rectal cancer after radiotherapy treatment compared with before starting radiotherapy interestingly. The alterations of expression levels of miR-374 gene were critically changed with passing a more time after radiotherapy treatment and related to tumor grading. Conclusion: Our results revealed change in the expression of miR-374 and its two target genes, in patients with rectal cancer after radiotherapy. The evaluation of aforementioned genes in present study can be used as a marker for radiotherapy treatment. In other words nding a reliable diagnostic tumor marker would be critical in more effective therapy of cancer.


Introduction
Colorectal cancer (CRC) is one of the most common cancers worldwide with high percentage of mortality rate. Types of known colorectal cancers based of originating include adenocarcinoma; a type that originates from mucus-producing cells, Carcinoid tumors; a type derived from speci c hormone-producing cells, Gastrointestinal stromal tumors (GISTs); Lymphomas originate from immune cells present in the colorectal tissue, and Sarcomas originate from blood vessel cells. Also a type that originates from the intestinal wall-speci c cells called interstitial cells of Cajal (1,2). Genetic causes of this heterogeneous disease are changes in several genes. Generally, genes involved in CRC are classi ed into several groups, including oncogenes and proto-oncogenes such as transcription factors, growth factors and their receptors and tumor suppressor genes such as Rb, APC and involved genes in metastasis, repair and apoptosis process. The products of each of these genes are present in different signalling pathways, including the p53, TGFB, JAK / STAT, PI3K and WNT pathways (3)(4)(5)(6)(7)(8). There are several key genes in each signalling pathway. One of the important components in the WNT pathway is beta-catenin which is normally surrounded and degraded by scaffolds from APC, GSK3B, Axin2, etc. After degradation was unable to enter the nucleus and activate proliferative genes such as C-myc, cyclinD, and so on. Deregulation of genes causing by mutations in the cancer process lead to survival of beta-catenin, and expression of proliferative genes (9)(10)(11)(12). One of the regulatory RNA molecules involved in new oncology topics are miRNAs. Today the role of miRNAs as diagnostic biomarkers is considered by researches (13). Peter Jo et al. in 2017 showed that presented miRNAs in the blood plasma of patients with rectal cancers can be used as biomarkers of response to treatment (12). Today, routine biomarkers for rectal cancer tumors used in chemotherapy regimens include KRAS, BRAF, MSI and SMAD4. Common treatments for colorectal cancer include rectal surgery, radiation therapy, chemotherapy and immunotherapy. Each of these methods has its side effects. Despite all the treatment methods for cancer, it is still one of the major challenges in the treatment of cancers is a defect in early diagnosis. This study will be investigated the effect of radiotherapy on the expression level of miR-374 gene and two its target gene in wnt-pathway in Samples of rectal cancer treated with radiotherapy.

Collecting of cancer samples
In this study, 2 ml blood samples from patients with rectal cancer referred to Khansari Hospital, Arak, Iran (2018-2020) were collected the day before radiotherapy and in the rst, third and fth weeks after starting radiation therapy. All patients were treated with radiation as part of curative treatment using a linear accelerator (Elekta, precise) with a daily dose of 1.8 Gy, (dose per fraction: 1.8 Gy). The radiation treatment dose was 45 Gy with 5.4 Gy boost (to a total of 50.4 Gy) for all patients. Radiotherapy was done as a 3D-conformal protocol with 18 MV photon beams (5 days per week).
The number of samples studied was estimated to be 25 cases. These patients also received Folfox. This study was approved by the Ethics Committee of Arak University of Medical Sciences with Code of Ethics (IR.ARAKMU.REC.1397.52).

Evaluation of coding nucleotide sequence of miR-374 in tumoral samples
To ensure conservation of the miR-374 coding region and con rmation of amplicons, its coding sequence was examined in tumor and non-tumor samples using sequencing method. template sample was used as a negative control of the replication reaction. Figure 1 shows the binding site of the used primers. The sequencing of samples was performed with ABIbiosystem xl100 Macrogen Company, South Korea. Statistical analyzes were performed with bioedit and chromas software.
Assessment of gene expression in samples using real-time PCR The expression levels of miRNAs and their target genes were evaluated by quantitative real-time PCR. on Blood samples were performed RNA extraction using RNX kit (Sinaclone, Iran) and then cDNA synthesis using mmulv enzyme (YTA, Iran), random hexamer and stem-loop primers from previous study (data not published). cDNAs were used as templates in a real-time PCR reaction with Master Mix SYBR green (YTA, Iran) and 10 pmol of forward and reverse primers in protocols with annealing temperature 54 degrees centigrade in Roche PCR. Also GAPDH and SNORD47 (U47) used as internal control.

Analysis of statistical results
Expression analysis data in this study were performed using Excel 2007 and GraphPad 7.0 software and the differences between the groups were considered statistically signi cant at less than 0.05. Sequencing results were also analyzed with Bioedit alignment tool and Chrome software.

Results
Demographic data from the collected samples are shown in the following table.  28  72  56  44  85  15  68  32  33  40  27  54  54  27  60 hsa-miR-374 predicted as miRNA target of key WNT signalling pathway receptor genes in a previous study using bioinformatics (data not published).
Evaluation of miR-374 precursor coding sequence was performed in tumor and non-tumor samples after DNA genomic extraction and ampli cation. The size of bands of products (291 bp) in electrophoresis showed the correctness of the ampli cation reaction.    The expression of miR-374, APC and GSK3ß genes after the rst week, third week and fth week after radiotherapy in the studied samples are shown as colum form in Fig. 3 and Fig. 4 respectively using two-ways Anova analyis. Figure 3: Evaluation of expression of miR-374 genes before and after radiotherapy, Two-way Anova, P < 0.05 Figure 4: Evaluation of expression of studied genes before and after radiotherapy, Two-way Anova, P < 0.05 The analysis of the changes in expression of the studied genes and some demographic features of the affected individuals (age, grade and differentiation) showed a positive corralation (pearson r, p = 0.0005, r = 0.08) between increase in BMI, age and increase in tumor grade (decrease in differentiation) with the fold-change of expression of miR-374. Figure 1 up: Schematic overview of primers to amplify the miRNA precursor region. down: 291 base-pairs of products on 1.3% agarose gel Results of sequencing products sequenced in the studied samples using software Bioedit. The sequences are shown in comparison with precursor sequence.

Figure 3
Evaluation of expression of miR-374 genes before and after radiotherapy, Two-way Anova, P<0.05