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Introduction

Lipogenic genes exert their biological effects through transcriptional regulation of their target genes, several of which are key regulatory genes involved in lipogenic metabolism (1). Sterol regulatory element-binding protein (SREBP)-1c is a key regulator of fatty acid metabolism and plays a pivotal role in the transcriptional regulation of different lipogenic genes that mediate lipid synthesis (2,3). Nuclear SREBP-1c preferentially binds to E-box motifs, thus enhancing transcription of genes required for saturated and unsaturated fatty acid and triglyceride biosynthesis. During this process, the lipogenic mRNAs for ATP citrate lyase (ACLY) and fatty acid synthase (FASN) are elevated (4,5), whereas the lipogenic mRNAs for carnitine O-palmitoyltransferase type I (CPT-I) are suppressed by SREBP-1c expression (6,7).

Cancer tissue proliferates by actively using the energy supplied by fatty acid metabolism. In cancer as well as normal cells, the upregulation of lipogenic enzymes is indispensable for fatty acid metabolism and high lipogenic gene expression is typical of a wide variety of cancers (8,9). SREBP-1c is a key transcription factor that affects cholesterol/lipid biosynthesis and uptake. miR-33b is embedded in SREBP-1 introns and targets several key regulators of cholesterol trafficking and of fatty acid/triglyceride homeostasis for post-transcriptional repression (10–12).

Although the mechanisms that underlie lipogenic gene overexpression in gastric cancer have not been elucidated, part of the lipogenic pathway is intensely expressed in metaplasia and in a subset of gastric adenocarcinomas that are characterized by disease progression, tumor aggressiveness and poor patient survival (13,14).

The aim of the present study was to investigate lipogenic gene expression in cancer and non-cancer tissues from gastric cancer patients using quantitative PCR (qPCR) analysis.

Materials and methods

mRNA quantification

Samples were obtained during surgical resection of gastric tissues from 34 Japanese gastric cancer patients (22 male and 12 female; median age, 69.6±10.5 years). Cancer and non-cancer tissues were investigated. Non-cancer tissue was sampled at >5 cm from the edge of each gastric cancer nodule. Samples were frozen in RNase Later (Ambion, Foster City, CA, USA) immediately after surgical resection and stored at −80°C until analysis. Written informed consent was obtained from each patient.

Total RNA was extracted from 10 mg of tissue using the Isogen II reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Complementary DNA (cDNA) was prepared by incubating DNase-treated total RNA (0.1 μg) with PrimeScript® II First Strand cDNA Synthesis kit (Takara Bio, Inc., Shiga, Japan). The qPCR reaction mixture was prepared using FastStart TaqMan® Probe Master (Rox) (Roche Applied Science, Mannheim, Germany) or Kapa Sybr® Fast qPCR Master mix (Kapa Biosystems, Inc., Woburn, MA, USA). Primers for amplifying the ACLY, FASN, CPT-I and SREBP-1 mRNAs and for miR-33b, are presented in Table I.

Table I Sequences of primers and probes.

Table I

Sequences of primers and probes.

Gene (primer/probe)	Sequence	Product size (bp)
ACLY
Forward	5′-GCTTGTTGGCGTGGATGAGAA-3′	138
Reverse	5′-ACTCCATCTTTGGTCACTACAA-3′
Probe	5′-ACACCTGTTGGTCCACGCCCCTGA-3′
CPT-I
Forward	5′-CGGCCGATGTTACGACAGGT-3′	190
Reverse	5′-GATGTCGCCTTTGCAGTGCC-3′
Probe	5′-ACTCCTGGGCAGATGCGCCGATCG-3′
FASN
Forward	5′-GTGTTTGAGGATGTGGTGCTGC-3′	185
Reverse	5′-CTTTCCGGGTGGTCGAAGAG-3′
Probe	5′-TGTCAGAGAACGGCAACCTGGTAGTGAGTG-3′
SREBP-1
Forward	5′-GGTAGGAGCCATGGATTGCACTT-3′	164
Reverse	5′-GAGGTGGAGACAAGCTGCCTG-3′
miR-33b
Forward	5′-TGTCAGGCAACCGTATTCACC-3′	74
1st reverse	5′-CATGCAGTGAGTTAGATGTAGAACGTGCATTGCTGT-3′
2nd reverse	5′-CATGCAGTGAGTTAGATGTAGAACGTG-3′
Probe	5′-CTGTTGCATTGCACCACTCACGG-3′

[i] ACLY, ATP citrate lyase; CPT-I, carnitine O-palmitoyltransferase type I; FASN, fatty acid synthase; SREBP-1, sterol regulatory element-binding protein 1.

PCR reactions comprised 45 cycles (at 95°C for 20 sec, at 60°C for 30 sec and at 72°C for 20 sec) with the CFX96 real-time PCR Detection system (Bio-Rad, Foster City, CA, USA). The first PCR reaction comprised 7 cycles (at 95°C for 10 sec, at 60°C for 15 sec and at 72°C for 10 sec) using FastStart TaqMan Probe Master (Rox) (Roche Applied Science). The second reaction comprised 45 cycles (at 95°C for 10 sec, at 60°C for 15 sec and at 72°C for 10 sec) using the CFX96 real-time PCR Detection system (Bio-Rad). The RNA samples were quantified by relating the PCR threshold cycles obtained from the cell line samples to the amplicon-specific standard curves. As normalization to the GAPDH housekeeping gene was inaccurate, the RNA expression levels were presented as the mRNA copy number per μg total RNA.

Statistical analysis

The samples in the experiments were tested in triplicate or quadruplicate. Data are expressed as means ± standard deviation. Differences between the mean values were evaluated using the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

The levels of CPT-1 mRNA in cancerous tissues were determined as 104.6±100.9 copies/μg total RNA using qPCR (Fig. 1) and were significantly higher in non-cancer tissues at 105.3±100.6 copies/μg total RNA (Student’s t-test; P<0.01). The levels of miR-33b in cancerous tissues were determined as 102.7±101.4 copies/μg total RNA and were significantly higher in non-cancerous tissues at 103.3±102.1 copies/μg total RNA (Student’s t-test; P<0.01).

Figure 1 Expression of lipogenic genes in patients with gastric cancer. T, tumor; N, non-tumor. qPCR analyses were performed as described in Materials and methods. RNA expression levels are presented as the number of RNA copies/μg of total RNA.

The levels of SREBP-1 mRNA in cancer and non-cancer tissues were determined as 104.5±101.1 and and 104.5±100.7 copies/μg total RNA, respectively (Fig. 1), using qPCR.

The levels of ACLY mRNA in cancer and non-cancer tissues were determined as 104.4±100.9 and 104.9±100.6 copies/μg total RNA, respectively, while the levels of FASN mRNA in cancer and non-cancer tissues were determined as 104.8±100.8 and 105.1±100.5 copies/μg total RNA, respectively. The differences for SREBP-1, ACLY and FASN were not statistically significant according to the Student’s t-test.

Discussion

Although lipogenesis is negligible in the majority of non-malignant adult tissues (15,16), it is upregulated in several tumors, rendering the investigation of endogenous lipogenesis a novel target for the prevention and/or treatment of cancer. This limited-size study demonstrated no statistically significant differences between the levels of mRNA for lipogenic genes and other clinicopathological characteristics, such as tumor size, degree of differentiation, tumor location, stage TNM and p53 mutation. However, CPT-1 mRNA and miR-33b expression were downregulated in the cancer tissues, suggesting that the downregulation of CPT-1 mRNA may be part of the mechanism responsible for SREBP upregulation and concords with the increased lipogenesis and lipogenic enzyme expression exhibited by a wide variety of cancers.

miR-33b mediates the transcription of its target genes, several of which are critical to lipogenesis and cholesterol metabolism (17,18), including SREBP-1c, ACLY and FASN, which increase fatty acid and triglyceride production (17,18). SREBP-1 is synthesized as an integral protein of endoplasmic reticulum membranes. At the nuclear level, mature SREBP-1 activates genes that encode FAS and other lipogenic enzymes by interacting with sterol response elements present in their promoter regions (19). Consistent with this hypothesis, SREBP expression was markedly stimulated by the inhibition of miR-33b expression, which may also result in increased fatty acid oxidation and reduced accumulation of fat in the liver stores. Considering the promising results of the use of anti-miRs in preclinical studies, miR-33b may become a viable therapeutic target in the future.

Our results provide a basis for more detailed studies on the regulation of SREBP activity and may assist in further investigations of miR-33b as a target of gastric cancer treatment. Although the reason for miR-33b downregulation in cancerous tissues is uncertain, the elucidation of its association with lipogenic genes may provide insight into gastric carcinogenesis and lead to the development of novel strategies for the genetic diagnosis of gastric cancer.

References