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 » Introduction
 »  Materials and Me...
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 » Discussion
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ORIGINAL ARTICLE
Year : 2014  |  Volume : 62  |  Issue : 1  |  Page : 53-56

Relative quantitative expression of hypoxia-inducible factor 1 alpha messenger ribonucleic acid in recurrent craniopharyngiomas


Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China

Date of Submission22-Jul-2013
Date of Decision26-Jul-2013
Date of Acceptance02-Feb-2014
Date of Web Publication7-Mar-2014

Correspondence Address:
Jianguo Xu
Department of Neurosurgery, West China Hospital, Sichuan University, 37 Guoxue Street, Chengdu, Sichuan, 610041
China
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Source of Support: This work was supported by Grant No. 3097382 and No. 81172411 from National Natural Science Foundation of China and China Medical Board of New York, Conflict of Interest: None


DOI: 10.4103/0028-3886.128291

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 » Abstract 

Background: Craniopharyngioma is a benign tumor, but recurrences are common and prognosis is poor. The pathologic mechanism underlying the high recurrence is still unknown. Aims: We hypothesized that there are hypoxic microenvironments within craniopharyngiomas and hypoxia inducible factor-1 alpha (HIF-1α) and its related genes are largely expressed in recurrent craniopharyngiomas. Materials and Methods: A total of 19 patients with craniopharyngiomas have been identified. The relative quantitative expressions of HIF-1α, vascular endothelial growth factor (VEGF) and carbonic adhydrase 9 (CA9) messenger ribonucleic acid (mRNA) of craniopharyngioma tissues were detected by real time reverse transcription polymerase chain reaction. Results: HIF-1α and VEGF mRNA was significantly up-regulated in recurrent craniopharyngiomas. Mean expression levels (recurrent craniopharyngiomas compared with non-recurrent craniopharyngiomas, as normalized to expression of β-actin) were 3.09 versus 0.75 (P = 0.001) for HIF-1α, 1.07 versus 0.32 (P = 0.000) for VEGF, 1.21 versus 1.93 (P = 0.503) for CA9. In craniopharyngiomas, the expression of VEGF showed a significant correlation with HIF-1α (r = 0.836, P = 0.000). Conclusion: There were hypoxic microenvironments within craniopharyngiomas. Therefore, preventing the tumor cells from adapting to the hypoxic conditions may be an effective way to obviate the relapse of craniopharyngioma.


Keywords: Craniopharyngioma, hypoxia inducible factor-1 alpha, real time reverse transcription polymerase chain reaction, recurrent craniopharyngioma


How to cite this article:
Liu H, Liu Z, Li J, Li Q, You C, Xu J. Relative quantitative expression of hypoxia-inducible factor 1 alpha messenger ribonucleic acid in recurrent craniopharyngiomas. Neurol India 2014;62:53-6

How to cite this URL:
Liu H, Liu Z, Li J, Li Q, You C, Xu J. Relative quantitative expression of hypoxia-inducible factor 1 alpha messenger ribonucleic acid in recurrent craniopharyngiomas. Neurol India [serial online] 2014 [cited 2019 Sep 19];62:53-6. Available from: http://www.neurologyindia.com/text.asp?2014/62/1/53/128291



 » Introduction Top


Craniopharyngioma, the most common intracranial tumor in children, is histologically benign tumor and is histologically divided into two groups: Adamantinomatous type and squamous papillary type and the transitional forms are included in adamantinomatous type. [1] It is difficult to do total resection of the tumor as it often has adherences to the surrounding vital structures. Even if complete resection has been done, the risk for recurrence is high, the 10-year recurrence rate can be up to 95%. [2] Unfortunately, the pathogenic mechanisms involved in the recurrence and progression of craniopharyngioma are still unknown.

Tumor growth is multifactorial processes that require an optimal microenvironment. [3] Various studies have shown that the formation of new blood vessels, the angiogenesis, plays a significant role in tumor progression. [4] Possibly the cellular adaptation to hypoxia is a key factor of angiogenesis under hypoxic microenvironment within craniopharyngioma.

Hypoxia inducible factor-1 alpha (HIF-1α), which is one of the hypoxia inducible factor-1 subunits, [5] plays a vital role in tumor cells adaptation to hypoxic microenvironment by regulating its downstream genes, including vascular endothelial growth factor (VEGF) and carbonic adhydrase 9 (CA9). [6] which have been discussed as molecular markers of tumor hypoxic microenvironments. [7] As reported recently, Zhong et al., have found that HIF-1α may play a pivotal role for tumor progression. [8] Birner et al. reported that the over expression of HIF-1α was significantly correlation to poor prognosis in oligodendrogliomas. [9] However, to the best of our knowledge, there is no data on whether the expression of HIF-1α messenger ribonucleic acid (mRNA) is up-regulated in recurrent craniopharyngiomas. The objective of our study was to investigate the expression of HIF-1α and hypoxic related genes, including VEGF and CA9 by real time reverse transcription polymerase chain reaction (qRT-PCR) in recurrent and non-recurrent craniopharyngiomas. Furthermore, the correlations between the expression of HIF-1α and VEGF, CA9 mRNA in craniopharyngiomas were also evaluated.


 » Materials and Methods Top


Tumor specimens were obtained from 6 patients with recurrent craniopharyngiomas and 13 patients with primary craniopharyngiomas from Department of Neurosurgery of West China Hospital between January 2009 and March 2012. All patients and/or their legal surrogates signed a written consent form at the time of entry. The protocols were authorized by Ethics Committee of Sichuan University.

RNA subtraction and real time RT-PCR

Total RNA was isolated from the tumor tissues by a GenElute Mammalian Total RNA miniprep kit (Sigma-Aldrich, St. Louis, MO, USA) followed by the protocols. The procedure of RT-PCR was performed as the manufactures' instruction as described. Part of the RNA was firstly reverse transcribed to complementary deoxyribonucleic acid (cDNA) with RevertAid First Strand cDNA Synthesis Kit (Fermentas, St. Louis, MO, USA). And then the PCR was performed with SsoAdvanced SYBR Green Supermix kits ([BIO-RAD, Fukuoka, Japan]. Briefly, The PCR primer used were 5`-TTACAGCAGCCAGACGATCA-3` [forward] and 5`-CCCTGCAGTAGGTTTCTGCT-3` [reversed] for HIF-1α [product size 233 bp], 5`-ACCCTCTCTGACACCCTGTG-3`and 5`-GGCTGGCTTCTCACATTCTC-3` [reversed] for CA9 (product size 342 bp), 5`-GCTACTGCCATCCAA TCGAG-3` and 5`-TGCATTCACATTTGTTGTGC-3` [reversed] for VEGF [product size 243 bp], [10] 5`-GGCATCCACGAAACTACTACCT-3` [forward] and 5`-TCCTGCTTGCTGATCCACAT-3` [reversed] for β-actin [product size 266 bp, designed by primer 5]). The PCR solution was mixed by 1 μl of template DNA, 5 μl of PCR Mixture and 3 μl of diethylpyrocarbonate treated water and each primer (0.5 μM). Processes of C1000 thermal cycler (BIO-RAD, Fukuoka, Japan) were set as follows: Initial denaturation at 95°C for 3 min, 32 cycles of amplification including a denaturation step at 95°C for 0.5 min, an annealing step at 59°C, 61°C and 63°C for 0.5 min for HIF-1α, CA9 and VEGF, an extension step at 72°C for 0.5 min and a final extension at 72°C for 10 min. When PCR was completed, 5 μl of the reaction mixture was subjected to electrophoresis as soon as possible. The PCR products were separated on a 2.0% agarose gel containing ethidium bromide and then observed under the Molecular Imager ChemiDoc XRS System (BIO-RAD, Fukuoka, Japan).

Statistics analyses

All of the statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, USA). The relative quantitative expression of HIF-1α, VEGF and CA9 mRNA, normalized to β-actin, were analyzed between recurrent and non-recurrent groups by Student's t-test. The correlation between the expression level of HIF-1α and CA9 or VEGF was tested by bivariate correlations. P < 0.05 was considered to be significant (two-tailed).


 » Results Top


Of the nineteen patients, the pathological diagnosis was adamantinomatous craniopharyngioma in 12 patients, of which 4 (33.3%) were recurrent tumors. The pathological diagnosis was squamous papillary craniopharyngioma in the remaining 7 patients, of which 2 (28.6%) were recurrent tumors. No significant difference in recurrence was found between adamantinomatous and squamous papillary craniopharyngioma (P = 1.000). The mean age of the adamantinomatous subtype and squamous papillary subtype was 36.41 ± 19.00 years and 44.14 ± 14.95 years respectively. The respective gender ratio was 8/4 and 4/3 (female/male). No significant different expressions of HIF-1α, CA9 and VEGF mRNA were noted between the adamantinomous and squamous papillary craniopharyngiomas (P = 0.332, P = 0.243, P = 0.142; respectively).The relative expression of HIF-1α, CA9 and VEGF mRNA of both recurrent and non-recurrent craniorharygiomas are given in [Table 1]. The relative quantitative expression of HIF-1, VEGF and CA9 mRNA in craniopharyngiomas were shown [Figure 1], [Figure 2], [Figure 3] respectively. The relative quantitative expression of HIF-1α and VEGF mRNA was significantly higher in recurrent groups compared with non-recurrent groups; however the relative expression of CA9 mRNA in recurrent group was slightly lower than non-recurrent group [Table 1]. A significant correlation between the expression of HIF-1α and VEGF mRNA was found in craniopharyngiomas (r = 0.836, P = 0.000), however, no significant correlations were found between the expression of CA9 and VEGF, CA9 and HIF-1α mRNA in craniopharyngiomas (P = 0.664, P = 0.927 respectively).
Figure 1: The fold hypoxia inducible factor-1 alpha messenger ribonucleic acid expression, as normalized to expression of β-actin, in recurrent or non-recurrent craniopharyngiomas through real time reverse-transcription polymerase chain reaction. R - Recurrent craniopharyngiomas, NR - Non-recurrent craniopharyngiomas

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Figure 2: The fold vascular endothelial growth factor messenger ribonucleic acid expression, as normalized to expression of β-actin, in recurrent or non-recurrent craniopharyngiomas through real time reverse-transcription polymerase chain reaction. R - Recurrent craniopharyngiomas, NR - Non-recurrent craniopharyngiomas

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Figure 3: The fold carbonic adhydrase 9 messenger ribonucleic acid expression, as normalized to expression of β-actin, in recurrent or nonrecurrent craniopharyngiomas through real time reverse-transcription polymerase chain reaction. R - Recurrent craniopharyngiomas; NR - Non-recurrent craniopharyngiomas

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Table 1: Mean (±standard deviation) relative expression of hypoxia inducible factor-1 alpha, carbonic adhydrase 9 and vascular endothelial growth factor messenger ribonucleic acid, as normalized to quantitative expression of β-actin, in tumor samples from recurrent or non-recurrent craniopharyngiomas through real time reverse-transcription polymerase chain reaction

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 » Discussion Top


World Health Organization Classification included craniopharyngiomas under benign tumor category. However, repeated recurrences and invasions to the adjacent vital structures of the intra/suprasellar regions are challenges for neurosurgeons. [11],[12] In our study, we found that the expression of HIF-1α mRNA was significantly higher in recurrent craniopharyngiomas, compared to non-recurrent craniopharyngiomas. Furthermore, there is a positively correlation between expression of HIF-1α and VEGF in craniopharyngiomas. The expression of HIF-1α protein was confirmed as an endogenous marker of hypoxia within tumor. [13],[14] Therefore, it could be concluded that there are hypoxia microenvironments within craniopharyngiomas. However, Proescholdt et al. recently reported HIF-1α expression was largely absent in craniopharyngioma cysts. [15] In addition, HIF-1α expression promotes tumor growth, angiogenesis and progression. [10],[12] Therefore, HIF-1α could be a marker for craniopharygnioma growth and progression.

Significant different expression levels of VEGF mRNA were also found between the recurrent and non-recurrent groups. This finding was inconsistent with the study that no correlation was found between the expression of VEGF and prognosis of craniopharyngioma. [16] A possible explanation was that angiogenesis may be an adaptation to hypoxic condition, stimulated by the HIF-1α pathways in craniopharyngioma and that could partially explain that VEGF mRNA was consistently upregulated in relation to the expression levels of HIF-1α mRNA in craniopharyngiomas. Similarly, Peter B found that the expression of HIF-1α had an impact on prognosis in oligodendroglioma, which had a hypoxia condition within the tumor tissues. [9]

CA9 mRNA was expressed in the majority of craniopharyngiomas and the relative quantitative expression of CA9 was even slightly lower in recurrent craniopharyngioma, compared to non-recurrent craniopharyngiomas. Our findings are consistent with Martin P who reported that the expression of CA9 protein was found in 85% of 20 craniopharyngioma cases, suggesting that CA9 may be considered to be a potential target for the adjuvant therapies in patients with craniopharyngioma. [15] There was no significant correlation between HIF-1α and CA9 in craniopharyngioma, which was not consistent with that CA9 could serve as an indicator of HIF-1-effective related radiosensitization in glioma. [17] The possible explanation was the proline degradation of HIF-1α was impaired and stabilizing the protein under hypoxia condition.


 » Conclusion Top


There are hypoxic microenvironments within craniopharyngiomas. Adapting to the hypoxic conditions, HIF-1α and VEGF mRNA are largely expressed in recurrent craniopharyngiomas, therefore, the HIF-1α may be considered to be a potential target for reducing craniopharyngioma recurrence. Nevertheless, it is difficult to conclude that the expression of HIF-1α has a predictive value for the recurrence of craniopharyngioma because the number of samples was limited in the retrospective study. Therefore, a random cohort prospective control trial was needed urgently to determine the value of HIF-1α in predicting craniopharyngioma recurrence.

 
 » References Top

1.Petito CK, DeGirolami U, Earle KM. Craniopharyngiomas: A clinical and pathological review. Cancer 1976;37:1944-52.  Back to cited text no. 1
    
2.Karavitaki N, Brufani C, Warner JT, Adams CB, Richards P, Ansorge O, et al. Craniopharyngiomas in children and adults: Systematic analysis of 121 cases with long-term follow-up. Clin Endocrinol (Oxf) 2005;62:397-409.  Back to cited text no. 2
    
3.Vidal S, Kovacs K, Lloyd RV, Meyer FB, Scheithauer BW. Angiogenesis in patients with craniopharyngiomas: Correlation with treatment and outcome. Cancer 2002;94:738-45.  Back to cited text no. 3
    
4.Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27-31.  Back to cited text no. 4
    
5.Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995;92:5510-4.  Back to cited text no. 5
    
6.Gillespie DL, Whang K, Ragel BT, Flynn JR, Kelly DA, Jensen RL. Silencing of hypoxia inducible factor-1alpha by RNA interference attenuates human glioma cell growth in vivo. Clin Cancer Res 2007;13:2441-8.  Back to cited text no. 6
    
7.Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721-32.  Back to cited text no. 7
    
8.Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, et al. Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and their metastases. Cancer Res 1999;59:5830-5.  Back to cited text no. 8
    
9.Birner P, Gatterbauer B, Oberhuber G, Schindl M, Rössler K, Prodinger A, et al. Expression of hypoxia-inducible factor-1 alpha in oligodendrogliomas: Its impact on prognosis and on neoangiogenesis. Cancer 2001;92:165-71.  Back to cited text no. 9
    
10.Eckerich C, Zapf S, Fillbrandt R, Loges S, Westphal M, Lamszus K. Hypoxia can induce c-Met expression in glioma cells and enhance SF/HGF-induced cell migration. Int J Cancer 2007;121:276-83.  Back to cited text no. 10
    
11.Barua KK, Ehara K, Kohmura E, Tamaki N. Treatment of recurrent craniopharyngiomas. Kobe J Med Sci 2003;49:123-32.  Back to cited text no. 11
    
12.Yaºargil MG, Curcic M, Kis M, Siegenthaler G, Teddy PJ, Roth P. Total removal of craniopharyngiomas. Approaches and long-term results in 144 patients. J Neurosurg 1990;73:3-11.  Back to cited text no. 12
    
13.Vordermark D, Brown JM. Evaluation of hypoxia-inducible factor-1alpha (HIF-1alpha) as an intrinsic marker of tumor hypoxia in U87 MG human glioblastoma: In vitro and xenograft studies. Int J Radiat Oncol Biol Phys 2003;56:1184-93.  Back to cited text no. 13
    
14.Vordermark D, Brown JM. Endogenous markers of tumor hypoxia predictors of clinical radiation resistance? Strahlenther Onkol 2003;179:801-11.  Back to cited text no. 14
    
15.Proescholdt M, Merrill M, Stoerr EM, Lohmeier A, Dietmaier W, Brawanski A. Expression of carbonic anhydrase IX in craniopharyngiomas. J Neurosurg 2011;115:796-801.  Back to cited text no. 15
    
16.Jianguo Xu, Sizhong Zhang, Chao You, Xiaojie Wang, Qiao Zhou. Microvascular density and vascular endothelial growth factor have little correlation with prognosis of craniopharyngioma. Surg Neurol 2006;66:S1,30-4.  Back to cited text no. 16
    
17.Said HM, Hagemann C, Staab A, Stojic J, Kühnel S, Vince GH, et al. Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1alpha in human glioma in vitro and in vivo. Radiother Oncol 2007;83:398-405.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]

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