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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 4  |  Page : 1313-1317

Evaluation of hypoxia inducible factor-1 alpha gene expression in colorectal cancer stages of Iranian patients


1 Clinical Biochemistry and Nutrition Department, Zanjan University of Medical Sciences, Zanjan, Iran
2 Medical Biotechnology Department, Zanjan University of Medical Sciences, Zanjan, Iran
3 Department of Tissue Engineering and Regenerative Medicine, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Iran
4 National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Date of Web Publication7-Feb-2017

Correspondence Address:
Mojtaba Fathi
Clinical Biochemistry and Nutrition Department, Cancer Gene Therapy Research Center, Zanjan Univercity of Medical Sciences, Zanjan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.199542

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 > Abstract 

Aim of Study: Colorectal cancer (CRC) is the fourth most prevalent cancer globally. Several factors have roles in cancer establishment. One of the most important factors is hypoxia that induces hypoxia inducible factor-1 (HIF-1). The HIF-1 alpha overexpressed in hypoxia conditions and plays a pivotal role in carcinogenesis features. In this study, we aimed to examine the efficiency of HIF-1 alpha gene expression at mRNA and protein's level for CRC diagnosing and staging.
Materials and Methods: In this study, the cases included into 75 cancer specimens in different stages (Group 2 = Stage 1, Group 3 = Stage 2, and Group 4 = Stage 3, 4) and ten normal specimens as control (Group 1). Real-time reverse transcription-polymerase chain reaction and immunohistochemistry (IHC) were performed for measuring gene expression at RNA and protein's level, respectively. The raw data were analyzed in the SPSS20 software.
Results: HIF-1 alpha gene expression rate (2ΔΔCT) and ΔCT values were significantly higher increased in Group 4 in compare to control (P < 0.001). Other cancer groups (2 and 3) had greater ΔCT values than control, but it was not statistically significant. Moreover, the rate of HIF-1 alpha gene expression (2ΔΔCT) was increased with cancer stages. According to the IHC results, there was a positive relationship between CRC stages and HIF-1 alpha protein expression (P < 0.05).
Conclusions: HIF-1 alpha gene expression increased in earlier up to metastasis stages of CRC, but the assessment of HIF-1 alpha gene expression has not important role in the diagnosis of cancer in early stages and classification of carcinoma because the increasing of HIF-1 alpha gene expression is not significant in early cancer stages.

Keywords: Cancer and diagnosis, colorectal, hypoxia inducible factor-1 alpha gene


How to cite this article:
Mansour RN, Enderami SE, Ardeshirylajimi A, Fooladsaz K, Fathi M, Ganji SM. Evaluation of hypoxia inducible factor-1 alpha gene expression in colorectal cancer stages of Iranian patients. J Can Res Ther 2016;12:1313-7

How to cite this URL:
Mansour RN, Enderami SE, Ardeshirylajimi A, Fooladsaz K, Fathi M, Ganji SM. Evaluation of hypoxia inducible factor-1 alpha gene expression in colorectal cancer stages of Iranian patients. J Can Res Ther [serial online] 2016 [cited 2020 Oct 25];12:1313-7. Available from: https://www.cancerjournal.net/text.asp?2016/12/4/1313/199542


 > Introduction Top


Colorectal cancer (CRC) is the second leading cause of death and the third prevalent cancer in developed countries.[1] Several factors have a role in cancer establishment. One of the most important factors is hypoxia inducible factor-1 (HIF-1). For instance, the genes that have an important role in cancer cell survival, when a normal cell transforms into tumor cells, demonstrate some metastatic feature.[2] When normal cells turn to tumoral cells, the amount of tumoral cells becomes increase and takes some distances from vessels; it leads to hypoxia and making some problems with feeding cells. Followed by, the metastatic and invasive features were demonstrated in these cells.[3] One-way that classifies tumor cell growth is based on cancer cell stages. In the first stage, the tumor cell growth established up to the inner layer. In the second stage, the tumor cells go out of muscle layer, but the lymphatic nodes do not get involved. In the third stage, the lymphatic nodes involve tumor cells, and in fourth stages, the tumor cells broadcast with blood circulation and metastasized to other tissue.[4] HIF-1 is a heterodimeric helix-loop-helix transcription factor that consists of two subunits: HIF-1 alpha and HIF-1 beta,[5] the gene expression of HIF-1 beta is permanent, but HIF-1 alpha depends on cell hypoxic condition. The HIF-1 alpha has much more important role of the HIF-1 beta. When the two subunits were separated, both of them have lost their activities (HIF-1 alpha and HIF-1 beta). The HIF-1 protein degradation is with Von Hippel–Lindau tumor suppressor protein, and the transcriptional factor was lost when O2 concentration is normal.[6] When two subunits bind together, the HIF-1 transcription factor becomes active and transcript some genes and caused by upregulate and downregulate themes. Commonly these gene's role has important effects in hypoxia cell survive. Some of these genes are vascular endothelial growth factor (VEGF),[7] glucose transporter-1 and -3, lactate dehydrogenase-A, carbonic anhydrase 9,[8] heme oxygenase,[9] inducible nitric oxide synthase,[10] glycolytic pathway enzymes,[11] and erythropoietin [12] that have an important role in glucose metabolisms and angiogenesis.[13] The HIF-1 alpha response to the some of the genetic alterations with the operation of oncogenes and tumor suppressor genes.[14] The expression of HIF-1 alpha was increased in both pathological and physiological statements that due to hypoxia, the physiologic hypoxia can cause by living in mountain and erythropoietin secretion [15] or vigorous exercise.[16] The pathological condition such as obesity,[17] smoking,[18] anemia,[19] malignance, and all circumstances that induce hypoxia.[20] The lifestyle is the common part of tumor genesis and few studies about HIF-1 alpha mention this fact. A number of studies about CRC were done in Tehran, but they were not about HIF-1 alpha gene expression in different stages. HIF-1 alpha protein has been recognized by immunohistochemistry (IHC) in neoplastic cells; for instance, lung, prostate, breast and colon carcinoma, and HIF-1 alpha gene expression also have been detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) in some malignancies.

In this study, we aimed to recognize the HIF-1 alpha role in staging and treatment as a tumor marker.


 > Materials and Methods Top


Patient selection

Seventy-five tumoral and ten normal biopsy specimens were snap-frozen in liquid nitrogen and stored at −80°C up to use for real-time RT-PCR and 46 formalin-fixed, paraffin-embedded (FFPE) tissues for IHC analysis. Tumoral tissues were obtained from four stages (Stage 1, Stage 2, and Stage 3 + metastasis). Tumoral and normal specimens were obtained from patients who had undergone diagnosis surgery at Tehran Imam Khomeini Hospital. Specimen was divided into four groups, Group 1 included normal specimen, Group 2 included Stage 1 CRC specimen, Group 3 included Stage 2 CRC specimen, and Group 4 included Stage 3 and metastasis specimen assembled. The specimens have a number of demographic data such as age, sex, hemoglobin amount, and patient's smoking or nonsmoking. Pathological reports and clinical histories were at the time of surgery and patient's staging was according to tumor-node-metastasis. None of the patients or normal tissues had not been received any remedy (chemotherapy, drug remedy, or radiotherapy) and they had not been diagnosed hereditary nonpolyposis CRC or Crohn's disease. The human tissue samples were approved by the Medical Ethics Committee of tumor bank of Iran. Informed consents from the patients were obtained for the study of the resection specimens. The classification of groups was: group 1 = control, Group 2 = Stage 1, Group 3 = Stage 2, and group 4 = metastasis + Stage 3. Control group specimen was obtained from normal tissues.

Real-time reverse transcription-polymerase chain reaction

Total RNA was extracted from human CRC tissue and normal tissues, with Easy Blue solution buffer (Intron Biotechnology Co, #Cat 17061) according to kit protocol. Two pairs of primer were designed with oligo7 and gene runner software and blasted with www.pubmed.com for the sequence HIF-1 alpha forward primer: 5′-GATGTGGTTGTATTCGTG-3′ and reverse primer: 5′-ATCTCCTGCTTCTTTAGTC-3′, for beta-actin forward primer 5′-CTTGATGTCACGGACGATT-3′, and for reverse primer was 5′-CACGGCATTGTCACCAACT-3′, after cDNA synthesis used quantitative real-time polymerase chain reaction in 85 specimens. Our aim of this test was to evaluate that the expression levels of HIF-1 alpha in colorectal stages could be appropriate as an independent predictor of CRC patients outcome. In this assay, the primer was designed exon–exon junction to avoid probably other DNA contamination. HIF-1 alpha gene expression was assessed triplicate in diluted 25 µL reaction, contain 12.5 µL SYBER Green Master Mix TaKaRa #KRR820Q, 5 ng of total RNA (concentration = 1000 ng/µL), 0.5 µL for each primer, 5.5 µL water, and 1 µL ROX. Thermal cycling program included: 3 min for initial step, and forty cycles of 5 s at 95°C, 31 s for annealing step at 57°C (for HIF-1 alpha primer) and 58°C (for internal gene primer). For normalized in gene level, we checked four internal genes and totally chose beta-actin gene as an internal control because it showed the lowest expression in several types, normal and colorectal, of specimens. For each specimen, we calculated a cycle threshold (CT) using rotor-gene corbet-6000 series device and software. This condition used for all reactions and negative control specimen to determine nonspecific amplification, we also used concentration serial dilution of a positive control specimen for both target and internal control genes and calculated the slope of yielded diagram, and in the following, the efficiency number of each gene was obtained. The initial quantitative of cDNA measured by NanoDrop device and achieved cDNA concentration. Finally, the expression of HIF-1 alpha mRNA species was analyzed by ΔΔCT method.[21] Furthermore, PCR products were checked on 2.5% agarose gel containing ethidium bromide with TBE 1× and photographed.

Protein assay

For protein assay, used IHC examination that executed on FFPE tissues cut into 4 µm thick slices. Obtained sections were deparaffinized, rehydrated then washed slides in 3% H2O2. Antigen retrieved was done with microwave method in 0.1M of citrate buffer; blocking stage was done by incubating tissues with 5% of bovine serum albumin (BSA) at 4°C overnight. After removal BSA, sections were incubated with diluted HIF-1 alpha's antibody (Abcam Anti-HIF-1-alpha antibody [1A3] (ab113642)) for 1 h in room temperature. The sections were incubated with diluted (1:200 in 1% of phosphate buffered saline [PBS]) and biotinylated with secondary antibody (Dako Corporation, Carpinteria, CA, USA) for 30 min in room temperature and washed with PBS. Antibodies sitting bonds were visualized with diaminobenzidine (DAB, Sigma, St Louis, USA) and contrasted with hematoxylin. Finally analyzed obtained picture of an optical microscope with ImageJ software and classified the obtained data.[22]

Statistical analysis

In this study, we used of SPSS version 20 software (IBM, New York, NY, USA) to statistical analysis, specimen parametric condition checked with Kolmogorov–Smirnov test; our date were parametric then we tested one-way ANOVA for quantitative data and followed post hoc Tukey's test to demonstrate significance between groups. Qualitative data significance and relations checked with Chi-square test. For real-time PCR analysis, we used ΔCT and 2ΔΔCT formulas. For analyzed the pictures obtained from IHC, we used ImageJ software and classified percentage of software in 0–25% = +1, 25–50% = 2+, 75–100% = 3+.


 > Results Top


Quantitative and qualitative assay of mRNA extraction

We checked mRNA qualitative with NanoDrop device, all mRNA concentration of specimens was more than 1000 ng/µL and the 260/280 OD ratio was on 1.8–2, the integration of mRNA molecule assessed with 1.5% agarose gel electrophoresis [Figure 1].
Figure 1: 1.5% agarose gel electrophoresis total RNA: demonstrate extracted mRAN qualify

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HIF-1 alpha and beta-actin reverse transcription-polymerase chain reaction

To know the quality of designed primers and find the best annealing temperature, we checked cDNA specimen with RT-PCR and followed the PCR product on 1.5% agarose gel electrophoresis. Some of the specimens have been shown in [Figure 2]a for beta-actin primer and in [Figure 2]b for HIF-1 alpha primer. The data from RT-PCR shown in [Figure 2] are extensile to all specimens, as a result the HIF-1 alpha gene expression increases with tumoral cancer stages.
Figure 2: Beta-actin primer for some specimens in normal and tumoral stages (161 bp) ladder 50 bp: the beta-actin gene expression was the same in all stages (a), hypoxia inducible factor-1 alpha primer for some specimens: 1: Metastasis specimen, 2: Stage 3 specimen, 3: Stage 2 specimen, 4: Stage 1 specimen and 5 is normal specimen, (149 bp) ladder 100 bp (b)

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HIF-1 alpha gene expression assessed with real-time polymerase chain reaction

HIF-1 alpha gene expression was significantly increased totally in CRC stages compare with normal samples (P < 0.001). However, the increasing between groups was not significant. The gene expression between groups 1 and 2, 1 and 3, and 2 and 3 was not significant but between groups 1 and 4 (P < 0.001), 2 and 4 (P < 0.001), and 3 and 4 (P < 0.005) was increased significantly. It is demonstrated in [Figure 3].
Figure 3: Hypoxia inducible factor-1 alpha gene expression according to ΔCT/groups: The gene expression of hypoxia inducible factor-1 alpha increase with cancer stages

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The ratio of gene expression was calculated by 2ΔΔCT and demonstrated that HIF-1 alpha gene expression was increased by 1.95-fold in Stage 1 compare with normal, Stage 2 in compare with normal specimen increased by 6.70-fold, Stage 3 and metastasis specimen increased by 15.11-fold in compare with normal specimen. It is necessary to say efficiency number for HIF-1 alpha and beta-actin genes was 2.

The hemoglobin amount of patients was measured and compared with HIF-1 alpha gene expression. It demonstrates that the amounts of hemoglobin are increased with HIF-1 alpha gene expression significantly (P < 0.001).

HIF-1 alpha gene expression in protein level

The result of IHC showed with photograph obtained from optical microscope with × 400 magnification and demonstrates that HIF-1 alpha gene expression was increased significantly in CRC stages compare with normal specimen [Figure 4].
Figure 4: The immunohistochemistry result: photograph obtained immunohistochemistry with ocular microscope, ×400, (a) normal tissue microarray, (b) Stage 1 tissue microarray, (c) Stage 2 tissue microarray, (d) Stage 3 tissue microarray, (e) negative control tissue microarray, the brown color demonstrate DAB sediment, the gene expression increased in protein level with cancer stage increasing (a up to d)

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Stage 1: 100% 1+, Stage 2: 48% 1 + and 52% 2+, Stage 3: 11% 1+, 85% 2+, 4% 3+, and Stage 4: 47% 2 + and 57% 3+.


 > Discussion Top


The aim of this study was to know the ability of HIF-1 alpha gene to determine cancer staging and recognize the primary stage of CRC. To achieve this end, we checked HIF-1 alpha gene expression in the level of protein and mRNA. We found that HIF-1 alpha gene expression was increased in the levels of protein and mRNA significantly, however, in the level of mRNA has increased generally significant, but in comparison, normal specimen with Stage 1 and 2 was not significant, and between stage 3 and metastatic specimen was increased significantly. Even, the HIF-1 alpha gene expression in the level of protein has increased significantly. According to the above-mentioned results, HIF-1 alpha has an important role in hypoxia condition. The decreasing of cellular oxygen pressure leads to hypoxia and the HIF-1 alpha gene expression in many tumoral cells like Lung, liver esophagus and neck-head cancers. As a result of increasing the HIF-1 alpha gene expression, the angiogenesis phenomenon was increased and increases cancer cell survival. As shown in results, in these cancers and other type of cancers when tumors get a bigger size, the HIF-1 alpha gene expression becomes increased. In CRC like other solid tumors, with increasing hypoxia, the HIF-1 alpha gene expression becomes intensified. Some studies have mentioned this matter. A study that was done on four kinds of CRC cell lines and fresh tissues demonstrate that HIF-1 alpha and VEGF genes' expression become increased, but the HIF-1 alpha is neither a good prognostic factor and nor for determining the quality of microvessel density.[23] In another study, the HIF-1 and HIF-2 mix expressions assay demonstrate that HIF-2 gene expression at protein level has a more important role than HIF-1 alpha in cancer staging in CRC.[24] This study was done on protein level and genes. The result of another study that was done with the tumoral tissue of CRC indicated that VEGF is better than HIF-1 alpha in staging and use as a tumor marker. This study was on protein expression level,[25] but another study that was done on CRC tissue and assay, both proteins and mRNA level of HIF-1 alpha and 13 other genes related to HIF-1 alpha, conformed with the previous study, and indicate that although the increasing of HIF-1 alpha gene expression in cancer stage is significant, the result of Kaplan–Meier test showed HIF-1 alpha is not appropriate gene in patient survival and has not usage on primary level diagnosing.[26] In our study, we investigate that although HIF-1 alpha gene expression level of mRNA in CRC stages was increased generally significantly, in pairwise group compare, the increasing between groups was not significantly, in details, the HIF-1 alpha gene expression was increased significantly between normal specimen and Stage 3 + metastasis but was not significant between normal specimen and Stage 1, Stage 2. From the past and other studies, we can realize that the HIF-1 alpha protein increased in different pathways and different conditions. Therefore, this expression depends on the patient's condition and their lifestyle, such as the body mass index,[27] smoking,[28] altitude life location,[29] and heavy exercises.[30]


 > Conclusions Top


In accordance with this study, it can seem that the HIF-1 alpha has a key role in many biological pathways. These pathways become active in pathological or physiological conditions that lead to hypoxia. Therefore, the HIF-1 alpha gene expression is not in CRC specifically.

The main conclusion to be drawn from this discussion is that the HIF-1 alpha is not an appropriate gene for diagnosing and staging of the CRC. Our suggestion is that it is better that use the assessment of HIF-1 alpha with the other gene expression profiling of CRC to confirm the stages of colorectal.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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