|Year : 2015 | Volume
| Issue : 6 | Page : 183-187
Galanin is a novel epigenetic silenced functional tumor suppressor in renal cell carcinoma
Shengkun Sun1, Axiang Xu1, Guoqiang Yang1, Yingduan Cheng2
1 Department of Urology, Chinese PLA General Hospital, Beijing, China
2 Cipher Ground, North Brunswick, NJ, USA
|Date of Submission||15-Nov-2015|
|Date of Acceptance||15-Dec-2015|
|Date of Web Publication||30-Dec-2015|
Cipher Ground, 675 Rt. 1 South, North Brunswick, NJ 08902
Source of Support: None, Conflict of Interest: None
Aim: To assess the expression level and function of Galanin in renal cell carcinoma (RCC).
Methods: The expression level of Galanin in a series of RCC cell lines (769P, 786-O, A498, Caki-1, and ACHN) was studied. 786-O cells were exposed to pharmacological demethylation to assess the involvement of promoter methylation downregulation of Galanin level. Galanin normal expressed ACHN cell line was knockdown with siRNA and was further used to study its role in cell proliferation and invasion. The downstream targets were screened by real time-polymerase chain reaction.
Results: Galanin was downregulated in two (786-O and A498) out of five RCC cell lines. Pharmacological demethylation restored the Galanin expression to normal levels in 786-O cells suggesting of its downregulation through promoter methylation. Knockdown of Galanin resulted in increased proliferation and invasion ability of ACHN cells while increasing the expression of oncogenes MYC, CCND1, and FRA1.
Conclusion: The above results suggest that Galanin behaves as a tumor suppressor gene in RCC and may be of clinical importance as a potential biomarker and therapeutic target in renal cancer patients.
Keywords: Galanin, renal cell carcinoma, tumor suppressor gene
|How to cite this article:|
Sun S, Xu A, Yang G, Cheng Y. Galanin is a novel epigenetic silenced functional tumor suppressor in renal cell carcinoma. Cancer Transl Med 2015;1:183-7
|How to cite this URL:|
Sun S, Xu A, Yang G, Cheng Y. Galanin is a novel epigenetic silenced functional tumor suppressor in renal cell carcinoma. Cancer Transl Med [serial online] 2015 [cited 2019 May 25];1:183-7. Available from: http://www.cancertm.com/text.asp?2015/1/6/183/172861
Shengkun Sun and Axiang Xu have contributed equally to this article and were co-first authors
| Introduction|| |
Renal cell carcinoma (RCC) is one of the most malignant tumors affecting humans. In 2013, alone more than 350,000 people were diagnosed of this disease worldwide; with more than 140,000 deaths per year. With a male-to-female ratio of 1.5:1, the incidence rate of RCC is higher in men than in women. Looking at the age distribution, it shows that about 75% of patients are over 60 years old, reaching a plateau around 70–75 years while the disease is rare under 50 years of age. The risk factors for this disease include smoking tobacco, hypertension, and obesity. With the development and continuous progress in therapeutic strategies, the 5-year survival rates, from diagnosis, increased from 50% in 1975–1977 to 73% in 2003–2009.
The development of RCC from a normal cell is a complex and multi-step process with the involvement of multiple oncogenes, tumor suppressor genes (TSGs), and signaling transduction pathways., Disruption of normal genetic and epigenetic patterns will result in uncontrolled balance between cell proliferation and cell death, leading to tumorigenesis in vivo. Currently, it is well-accepted that epigenetic alterations precede genetic changes during tumorigenesis. Promoter methylated TSGs could function as diagnostic biomarker and therapeutic targets in cancer.,,,,, More and more promoter methylated TSGs have been reported to be involved in the tumorigenesis of RCC.,,, Such genes not only provide the novel insights for the tumorigenesis of this malignant disease but also serve as potential therapeutic targets for RCC.
Galanin is a 30 amino acid peptide widely expressed in the central and peripheral nervous system,, and signals through Galanin receptor 1 (GALR1), GALR2, and GALR3. It is involved in many physiological functions such as metabolism, feeding, and body weight control. There are few reports that suggest Galanin functions as a tumor suppressor by inhibiting the proliferation and inducing apoptosis in colon carcinoma cell line., The tumor suppressive function of Galanin and its receptors was also reported in head and neck squamous cell carcinoma (HNSCC), where Galanin and its receptors were silenced by DNA methylation which correlated to the poor prognosis of patients., Interestingly, the oncogenic role of Galanin was also reported in lung and pituitary cancer conditions., In this study, we evaluated the correlation of Galanin with RCC condition.
| Methods|| |
Cell culture and transfection
A series of RCC cell lines (769P, 786-O, A498, Caki-1, and ACHN) and an immortalized human embryonic kidney cell line - HEK293T were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified chamber maintained at 37°C and 5% CO2. Control siRNA and siGalanin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The ACHN cells were transfected with siRNA/siGalanin using RNAiMAX (Invitrogen, Eugene, OR, USA), following the manufacturer's protocol.
The protocol of 5-Aza-2'-deoxycytidine (Aza) treatment was performed as previously described., In brief, 786-O cells (1 × 105/mL) were allowed to grow overnight. The cell culture medium was then replaced with fresh medium containing 50 μmol/L Aza for every 24 h, for three consecutive days. Cells were then harvested for RNA extraction.
RNA extraction and semi-quantitative reverse transcription polymerase chain reaction
RNA extraction was performed using trizol reagent as per manufacturer's protocol (Invitrogen, Eugene, OR, USA). The cDNA was synthesized using Random hexamers and SuperScript-III (Invitrogen, Eugene, OR, USA). Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed for 32 cycles with hot-start Go-Taq, according to manufacturer's protocol (Promega, Madison, WI, USA).
Growth curve and invasion assay
ACHN cells at an appropriate density in six-well plates were transfected with control siRNA or siGalanin through RNAiMax. The cell numbers were counted at 0 h, 24 h, and 48 h after transfection. For invasion assay, 1 × 105 cells in serum-free medium were seeded to the upper chamber of each insert (BD Biosciences, San Jose, CA, USA) while the lower chamber was supplemented with medium containing 1% serum. After 48 h, the migrated cells on the lower surface of the membrane were fixed in methanol for 5 min and then stained with 0.1% crystal violet. After staining, cell numbers per microscopic image field were counted to compare the invasion ability. Each experiment was conducted in triplicate in three independent experiments.
Real time-polymerase chain reaction
All primers used are listed in [Table 1]. Quantitative real-time PCR was carried out with the iQ5 Multicolor real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) using real-time PCR Master Mix (SYBR Green). Each experiment was conducted in triplicate in three independent experiments.
Data are presented as mean ± standard deviation. Statistical assessments were carried out using Student's t test. P < 0.05 was considered statistically significant.
| Results|| |
Expression profile of Galanin in renal cell carcinoma cells
We first detected the expression profile of Galanin in a series of RCC cells and an immortalized human renal epithelial cell line HEK293T [Figure 1]a. By semi-quantitative RT-PCR, we found normal Galanin expression in 769P, Caki-1, ACHN, and HEK293T, but silenced in 786-O and A498 cell lines. This tumor specific silenced pattern suggested that Galanin is a potential candidate TSG for RCC.
|Figure 1. (a) Expression profile of Galanin in a series of renal cell carcinoma cells. GAPDH was used as an internal control. (b) Pharmacological demethylation restored the expression of Galanin in 786-O cells|
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Pharmacological demethylation restored Galanin expression in renal cell carcinoma cell
To check if promoter methylation played a role in the downregulation of Galanin, we treated Galanin silenced cells (786-O) with DNA methyltransferase inhibitor Aza. We found that Galanin expression was restored after 3 days of Aza treatment in 786-O cells suggesting the involvement of DNA methylation in the downregulation of Galanin [Figure 1]b.
Knockdown of Galanin increased renal cell carcinoma invasion and proliferation
The tumor-specific silencing of Galanin in RCC indicated that Galanin might function as a tumor suppressor in RCC. Here, we choose ACHN cell line which exhibited normal Galanin expression for functional study. We found that downregulation of Galanin greatly elevated the invasion ability of ACHN cells, which suggested that Galanin is a negative regulator of cell invasion in RCC [Figure 2]a and [Figure 2]b.
|Figure 2. (a and b) Knockdown of Galanin increased the cell invasion ability in ACHN cell. (c) Inhibition of Galanin increased cell proliferation rate in ACHN cells (**P < 0.01)|
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Galanin knockdown ACHN cells were further examined for their proliferation abilities. We counted the cell number at 0 h, 24 h, and 48 h after transfection. The results showed that the proliferation rate was significantly higher in siRNA treated group compared to control cells [Figure 2]c, indicating that Galanin could inhibit cellular proliferation in ACHN cells.
Galanin function as a tumor suppressor gene by regulating MYC, CCND1, and FRA1 expression
To test how Galanin affects this increased cell invasion and proliferation, we tested the mRNA levels of the following critical factors: MYC, CCND1, FRA1, PIK3CA, MDM2, JUN, and p57, which are associated with invasion, proliferation, or apoptosis. We found that knockdown of Galanin significantly increased mRNA levels of MYC [Figure 3]a, CCND1 [Figure 3]b, and FRA1 [Figure 3]c, with little effect on PIK3CA [Figure 3]e], MDM2 [Figure 3]f, JUN [Figure 3]g, and p57 [Figure 3]h; downregulation of Galanin [Figure 3]d was also confirmed by real-time PCR. This suggested that Galanin could regulate cell invasion and proliferation by modulating mRNA levels of MYC, CCND1, and FRA1.
|Figure 3. Real time polymerase chain reaction results of the screening of several critical factors in ACHN cells (**P < 0.01)|
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| Discussion|| |
Most RCCs are diagnosed in the advanced metastatic stage, resulting in a dramatic decrease in patient survival. The investigation of the underlying mechanism involved in its progression is critical for developing a better therapy for RCC patients. In this study, we found that Galanin is one of the silenced functional TSGs in RCC. Galanin is a 30 amino acid neuropeptide , which activates the GALR1, GALR2, and GALR3 and stimulates numerous signal transduction and integration pathways.,, There are several kinds of neuropeptides in humans, some of which have tumor suppressor functions with tumor specific hypermethylated pattern. For example, tachykinin-1 gene, which encodes the neuropeptides substance P, neurokinin A, and neuropeptide K and γ, was reported to be hypermethylated in esophageal squamous cell carcinoma and colon cancer and was associated with poor prognosis in such patients.,,Somatostatin was also found frequently methylated in human esophageal adenocarcinoma and colon cancer., The tumor-specific methylation pattern and tumor suppressive function of Galanin and its receptors were also reported in head and neck cancer., These suggested that neuropeptide genes could function as tumor suppressors and serve as potential biomarkers in multiple cancers.
In this study, we found Galanin was methylated in two of the assessed RCC cell lines but not in immortalized human embryonic kidney HEK293T cells. Downregulation of Galanin increased the invasion potential and proliferation rate of ACHN cells. All these results suggested that Galanin is a novel functional TSG in RCC. We further investigated how Galanin mediated the invasion and proliferation in RCC. We tested several critical factors which were involved in tumorigenesis. We found that knockdown of Galanin could significantly upregulate mRNA levels of MYC, CCND1, and FRA1. MYC is an oncogene that contributes to the genesis of multiple cancers. This oncoprotein could promote proliferation, cell survival, genetic instability, invasion, and angiogenesis of cancer cells including RCC.,, CCND1 is a critical cell cycle regulator which binds with cyclin-dependent kinases, and subsequently phosphorylates tumor suppressor protein Rb. Despite its well-known role in cell cycle regulation, the effect of CCND1 on cell migration was also reported.,, FRA1 (also named as FOSL1) is a member of FOS family. This family can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. FRA-1 can promote cell proliferation, invasiveness, and motility., Our data suggested that Galanin could regulate cell proliferation and invasion by targeting MYC, CCND1, and FRA1. Underlying molecular mechanisms of how Galanin regulates the expression of MYC, CCND1, and FRA1 in RCC cells needs further assessments.
In conclusion, we have identified Galanin as a potential TSG in RCC, which is silenced by DNA methylation. Galanin inhibits cell proliferation and invasion by targeting MYC, CCND1, and FRA1 oncogenes and could serve as a novel therapeutic target for treating RCC.
Financial support and sponsorship
This study was supported by a grant from the National Natural Science Foundation of China (No. 30600613).
Conflicts of interest
There are no conflicts of interest.
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