Background: and validated with the reference gene when compared

Background: Colorectal cancer (CRC) is one of the most common malignancies types in the world wild with high frequencies and mortality. JS-2 is a novel gene found at 5p15.2, which was formerly identified in esophageal cancer patients. To date, several limited investigations have been carried out on the role of JS-2 in CRC. The purpose of this study was performed to be ascertained the gene expression changes of JS-2 in the patients with CRC and correlate these to the clinicopathological features.

Methods: We appraised mRNA levels in fifty fresh invasive colorectal tissues adenocarcinoma and matched adjacent non-neoplastic tissue using real-time polymerase chain reaction. JS-2 expression was also assessed in two colorectal tissue types and validated with the reference gene when compared to clinical features.

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Results: Our investigation showed that the mRNA expression level of JS2 hasn’t remarkably changed in the CRC patients. However, the findings revealed that there are no statistically significant differences in relative expression levels for JS-2 between different colorectal tissues (P = 0.067). The mRNA expression level of JS-2 was not different between the colon and rectal adenocarcinoma (p = 0.096).

Conclusions: In the present investigation,  we have acknowledged that changes in mRNA levels of JS-2 cannot be practiced as a biomarker in the diagnosis and prognosis of CRC.

Keywords: CRC- Gene Expression- JS-2 Gene –  Colorectal Cancer

 

 

 

Introduction

Colorectal cancer (CRC) is the third world’s malignancies, which ordinarily have a higher frequencies and mortality rates. Approximately 50% of lately diagnosed cases estimate for a metastatic stage, which is correlated with high death 1. The pathogenesis of the CRC is an intricate procedure, tightly managed through various regulatory mechanisms such as gene rearrangements, chromatin remodeling, epigenetic modifications, genetic variations and gene expression changes 2. Previously has also been described that many oncogenes and tumor suppressor genes (TSG) are nearly correlated to the molecular pathogenesis of CRC, notwithstanding the entire knowledge of the molecular and genetic basis for the tumorigenesis of CRC yet remained broadly unexplained 3. Furthermore, it will be of the highest significance to recognize the novel molecular biomarkers for distinguishing the molecular carcinogenic mechanisms of the CRC. This is significant not only for the conceivable increase of the popular curative protocols, while also for the prognosis and early detection of CRC disease 3. An earlier investigation of esophageal squamous cell carcinoma (ESCC) tissue with employing comparative genomic hybridization (CGH) technique revealed that the gene copy number increasing in the region of 5p15.2 is a prevalent issue 4. The findings disclosed two novel transcripts which have not been studied earlier in ESCC and other malignancies, and they are found 5′ upstream to the Delta-catenin gene 2. JS-2 is one of the novel genes, which was assessed using real-time polymerase chain reaction (Real Time-PCR) method and findings revealed that the JS-2 gene overexpressed in the nearly 18% of tissues from of the ESCC 3. JS-2  encodes a unique protein consisting of 233 amino acids, which was not identified sequence amino acid according to either recognized protein sequences in SwissProt (http://au.expasy.org/sprot/). Also, overexpression of JS-2 was remarked in the ESCC and CRC cancerous tissues in a limited number of investigations, future considerations are needed to entirely recognize the percentage of gene expression changes for JS-2 in human carcinoma. Moreover, notwithstanding the identification of the gene in a region specifically for amplification in cancer, the role of gene expression alterations to JS-2 has not been completely approved in the other human malignancies. In this investigation, we aimed to measure the alterations mRNA expression of JS-2 level in cancerous and non-cancerous tissues of CRC patients. This is the first report to analyze the gene expression changes JS-2 in North Western Iranian with CRC patients.  

 

Materials and Methods

Specimens selection

For this study, we have analyzed a total of fifty fresh of colorectal adenocarcinoma and adjacent non-neoplastic tissues, which were obtained from of the patients undergoing colorectal surgery in the Imam Reza Hospital, Medical University of Tabriz, Iran, between April 2015 and March 2016. In our study, exclusion criteria consisting of the subjects who underwent the chemotherapy and radiotherapy regime before the surgery. The pathological board re-assessed all specimens and verified grades and classification according to the World Health Organization (WHO) criteria 5. Ethics and Human Rights Committee in Pathology center approved the current research and the informed consents were signed by all participants.

 

RNA extraction procedures

All tissue specimens were deposited in RNAlater solution (Qiagen, Germany) at -80°C until RNA extraction. Total RNA was purified from of tissue specimens using TRIzol (Molecular Research Center Inc., Cincinnati, OH, USA) reagent, then RNA was treated with RNase-free DNase I (Thermo Scientific, USA), according to the manufacturer’s instructions. To ascertain the quality and quantity of RNA extracted, we were utilizing 1% agarose gel electrophoresis and NanoDrop® ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, Denmark), respectively.

 

cDNA Synthesis

Reverse transcription reactions were done using 1 ?g total RNA in a final reaction volume of 20 ?l. RNA was converted to cDNA using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas- Canada) with random Hexamer primer, according to the manufacturer protocol. All cDNA samples were separately diluted to 50 ng/?L, in order to obtain uniform concentration specimen for quantitative real-time PCR experiments.   

 

Quantitative Real-time –PCR

Real-time PCR was carried out in the triplicate form using specific primer pairs shown in Table 1 and  Syber Green-I dye in AccuPower® 2X GreenStarTM qPCR Master Mix (Bioneer, Korea) through the Rotor-GeneTM 6000 systems (Corbett Research, Australia) according to the manufacturer guidance. GAPDH was applied as a housekeeping gene for normalization, and a no sample was used as a no-template control (NTC). In all reaction, we included 150 ng cDNA, master mix 2X, ROX dye 50X, and 10 pmols2w of each primer pair for SJ-2 and GAPDH in a final volume of 25 ?l. Real Time-PCR conditions were begun with an initial denaturation step (94 °C, 5 min) was followed by 35 cycles of denaturation (94 °C, 10 s) and annealing temperatures  (59°C for SJ-2 and 62 °C for GAPDH) for 10s, followed extension at 72°C for the 20s. Standard curves were scheming to apply serially diluted cDNA and the expression levels of SJ-2 in the samples were normalized to the mean expression of the Housekeeping gene GAPDH.

 

Statistical analysis

The statistical interpretation was done with SPSS ver. 22.0 (SPSS Inc., Chicago, IL, USA).  To compare the mRNA expression level of JS-2 gene between cancerous tissues subjects with CRC and non-cancerous adjacent tissues, the results were assessed using the Mann-Whitney U-test.  Statistical examinations between the mRNA expression level of the JS-2 gene and each clinical features were done by using the Kruskal-Wallis test. Ordinarily, P<0.05 was considered statistically significant.   Results mRNA expression levels of JS-2 in CRC tissues The mean age of patients (18 men; 22 women) was 50.38 ± 6.12 years (ranging between 45 and 74 years). In order to evaluate whether the significant differences in the individuals of patients with CRC, we were appraised the mRNA expression level of JS-2 in neoplastic and adjacent non-neoplastic tissues. Former to statistical interpretation, the findings obtained from of JS-2  mRNA expression were normalized to the Housekeeping gene, GAPDH. In the expression level of JS-2, findings revealed that there are no statistically significant differences in relative expression levels for JS-2 between cancerous and adjacent non-cancerous colorectal tissues (P = 0.067) Fig 1.  In adjacent non-cancerous tissues, 74 % (37 of 50) of cases showed in the normal range of JS-2 mRNA, 26 % (13of 50) exhibited decreased or increased level. In cancerous adenocarcinoma tissue, 80 % (40 of 50) of cases were in normal range level of JS-2 mRNA, 10 % (5 of 50) showed decreased expression and   10 % (5 of 50) were displayed increased levels. The JS-2 mRNA expression seems to be greater in men compared to the women (p= 0.001). In addition, the mRNA expression level of JS-2 was not different between the colon and rectal adenocarcinoma (p = 0.096). JS-2 mRNA expression was not increased in adenocarcinoma between T3 and T4 pathological stages compared with the T1 and T2 pathological stages  (P =0.852), Table 2. Moreover, our findings were not shown the relationship between the JS-2 mRNA expression patterns and age, sexuality, pathological grade and size of a tumor.   Discussion This is the first investigation to identifying patterns mRNA expressions of JS-2 gene in Iranian patients suffering from of CRC. Our findings obtained conflicted of the results was reported in the preliminary investigation, which has been disclosed that there are significant alterations in pattern expression of the JS-2 in adenocarcinoma and adenoma tissues in comparison to marginal colorectal tissues 3. According to our results, alteration in pattern expression of JS-2 cannot be implicated in the pathogenesis of CRC. Notwithstanding, previous investigations have been described that the presence of an additional segment at chromosomal region 5p15.33, the area comprising the JS-2 gene, in the various malignancies such as bladder, thyroid, lung, and ESCC 6- 9.  In the previous report, Fatima et al. who had been investigated the mRNA expression levels of JS-2 on twenty cases of ESCC announced that fourteen and nine percent of cases revealed overexpression and decreased expression patterns, respectively 10. According to the findings reported by Lam et al. which were assessed one hundred sixteen specimens of colorectal carcinoma tissues, the expression of JS-2 was decreased in many of cancerous specimens when matched to non-cancerous tissues 3. The earlier results indicated that JS-2 was down-regulated and may play a significant role in the pathogenesis of CRC as an oncogene, whereas would not have to be disclosed a potential capacity as a tumor suppressor gene (TSG) 3, 10. Previously, Bohm et al. have been described that multiple TSG on 5p13-12, which were implicated in carcinogenesis of solid tumors could be inactivated 11.  Although, the findings of several investigations have been revealed that the JS-2 gene involved in the progress of different malignancies, while the precision of the roles in  CRC carcinogenesis remained unclear 6-9. In the current investigation, JS-2 changes were not linked to the pathological staging and other clinical features of CRC. However, our findings confronting with to those results published regarding the JS-2 gene expression alterations, which may be implicated in CRC pathogenesis. One possible explanation for inconsistent our results compared with the previous findings may be a result of small sample size or different in tissues, which were analyzed. Taken together, our findings revealed that we cannot recognize variations expression patterns of the JS-2 gene in the progression of CRC. In the present investigation, we have acknowledged that changes in mRNA levels of JS-2 cannot be practiced as a biomarker in the diagnosis and prognosis of CRC. However, further investigations are required to elucidate the possible roles of JS-2 in the pathogenesis of CRC.   Acknowledgments We are grateful to the Imam Reza Hospital, the Medical University of Tabriz for providing well-characterized patients with CRC.   Funding: This study was not funded by any of the individuals or institutes researcher.    Conflicted of Interested: Negin Akbarih declares that she has no conflict of interest.  Saeid Ghorbian declares that he has no conflict of interest. Ethical Approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed Consent: Informed consent was obtained from all individual participants included in the study.