Gene chip-related genes in pancreatic cancer screening in the preliminary study
【Abstract】 Objective To investigate the gene chip
technology in screening for pancreatic cancer-related gene
expression in the role of the group. Method according to
micro-array of 4 096 kinds of full-length gene PCR product is
made BiostarH-40s based microarray expression microarray; using one-step extraction of pancreatic cancer and normal pancreatic tissue total RNA, using Oligotex mRNA Purification
of two kinds of organizations, centrifugal column The mRNA; by reverse transcription fluorescent molecule (Cy3/Cy5) incorporation of a chain of cDNA expression profile probe preparation, chip hybridization and washing tablet, using fluorescent scanner chip fluorescence signal images, analysis of pancreatic cancer and normal pancreatic tissue differentially expressed genes. Results 4096 genes in pancreatic cancer tissue and normal pancreatic tissue has 949 rooms (23.2%) differences in the expression of genes, which we
have identified nine upward and downward adjustment of 8 oncogenes and tumor suppressor genes may be related to the pancreas the pathogenesis of cancer, there is correlation. Conclusion cDNA microarray technology to screen out the abnormal expression of genes associated with pancreatic cancer group, their further research could help understand the pathogenesis of pancreatic cancer.
Key words pancreatic cancer, DNA microarray gene tumor suppressor gene
Pancreatic cancer is a highly malignant tumor with poor prognosis. Since the early non-specific clinical symptoms of
pancreatic cancer, is already found most of the middle and late, the 5-year survival rate of only 1% ~ 5%. In recent years, the incidence of pancreatic cancer showed an upward
trend year after year, due to poor treatment of advanced pancreatic cancer, early diagnosis would result in greater access to surgical resection in patients with opportunities to improve their survival rate. However, the existing check
imaging technology is still not achieve the purpose of early diagnosis of cancer, pancreatic cancer-related serological
markers still can not accurately reflect the tumor occurrence and development of the situation. Therefore, the tumor developed in recent years related to genetic testing technology for early diagnosis of pancreatic cancer offers a new way. In this study, gene chip technology, mainly through the detection of pancreatic cancer and normal pancreatic tissue of gene expression in the different groups, selected
genes for further study in pancreatic cancer occurrence and development of the role of providing clues.
1 Materials and methods
1.1 Clinical specimens
One cases of pathologically confirmed pancreatic cancer
specimens and adjacent control of fresh pancreatic tissue by General Surgery, Affiliated Hospital of Nantong University to provide. Specimens were cut, respectively, immediately after (10min in) will be taken by the organization back into the liquid nitrogen freezing.
1.2 Preparation of cDNA microarray
Carrier at both ends with universal primers to 4 096 full-length gene PCR amplification, the product length of 1 000 ~ 3 000bp, agarose gel electrophoresis, PCR quality control. Concentrated product as the target gene amplification
dissolved in spotting solution, with the Cartesian Pixsys7500 point-like instrument point in the silanized slide. Point-like
slides to 2h post-hydrated at room temperature, dry 30min, then 0.2% SDS, water, and 0.2% sodium borohydride treatment 10min, dry spare.
1.3 Probe Preparation
Optimized one-step extraction using conditional
pancreatic cancer and normal pancreatic tissue total RNA, the RNA precipitate was dissolved in RNase free of Milli Q
water, and measured D (260) / D (280), D (260) / D (230 ). According to Qiagen's Oligotex mRNA spin column operations
for RNA purification. In 50μl reverse transcription reaction system by adding 3μg mRNA, RT-cDNA probe synthesis and
purification, using Cy3 dUTP marked normal tissue mRNA,
using Cy5 dUTP cancer marker mRNA. Ethanol precipitation after Cy3 dUTP and Cy5 dUTP labeled probe mixture was
dissolved in 20μl 5 × SSC 0.2% SDS hybridization solution.
1.4 Hybridization and washing
The cDNA microarray and hybridization probes were doing 95 ? denaturation for 30 seconds and placed in hybridization chamber after 2min, immediately added to the gene chip probe, and with the hybrid capsule to be sealed, not to leave air
bubbles. Hybrid box at constant temperature 42 ? hybrid 18h.
Then open a cover slip, according to the order with 2 × SSC
0.2% SDS, 0.1% × SSC 0.2% SDS, 0.1% × SSC washing 10min,
room temperature drying.
1.5 difference in genetic testing and biological
ScanArray 4000 scanner chip, obtained Cy3/Cy5 image files through the planning grid to determine the scope of cross-
point, filtering background noise, extract the gene expression values of the fluorescence signal intensity. In order to avoid
systematic errors, experimental data for uniform treatment. Before homogenization processing based on the following two principles were selected to participate in effective gene homogenization treatment: (1) of the gene point of Cy3, Cy5
signal values were greater than 200, or one of more than 800; (2) the gene point Cy5 signal values / Cy3 signal values of the ratio between 0.1 to 10. Using GenePix Pro 3.0 image-
processing software to analyze two kinds of Cy3 and Cy5 fluorescent signal intensity and the ratio is greater than the ratio of selected genes 2 or less than 0.5 points, represented by the gene probe hybridization with the two kinds of time showed a greater differences, and the downward trend in line at a similar level.
2.1 Total RNA extraction and mRNA purification
From pancreatic cancer and normal pancreatic tissue total RNA extracted after agarose gel electrophoresis analysis of RNA, 8SrRNA and 28SrRNA band clear, both the ratio is about
1:2.5, indicating the extraction of pancreatic cancer and normal pancreatic tissue of the total RNA integrity of the good.
2.2 gene chip hybridization results
Normal pancreas tissue expression pattern (Cy3) scan results with pancreatic cancer tissue expression pattern (Cy5) scan through the computer data generated from the superimposed images (see Figure 1). The plan reflects each gene in different tissues abundance ratio, reflecting the cancer tissue and normal tissue between the gene expression
differences. Yellow spots on behalf of the abundance of gene expression close to the two organizations; red spots on behalf of the gene in pancreatic cancer tissues compared with normal pancreatic tissue is highly expressed (up trend); green dot
represents the gene in pancreatic cancer tissues compared with normal pancreatic tissue with low expression (downward trend). Reposted elsewhere in the paper for free download http://
Scatterplot distribution (see Figure 2) See the two chips
on the vast majority of genes in the expression of tissue abundance is the same, but there are a few differences in gene point (yellow), and its expression profile chip signal is greater than 2-fold difference in .
2.3 Bioinformatics analysis
Bioinformatics analysis of genes found differentially expressed genes accounted for the total number of 949 (4096) and 23.2%, which are proto-oncogenes and tumor suppressor gene 17 (0.42%), there are nine regulated expression (mean ratio value of 3.68) expression were down 8 (the average ratio value of 0.30) (Table 1).
Table 1 pancreatic cancer tissues abnormal oncogenes and tumor suppressor gene (abbreviated)
In fact, the incidence of cancer is a tumor-related gene
expression in most of disorder or tumor suppressor gene inactivation caused. Proto-oncogene is a growth factor
signaling system components, in malignant tumors to change often as the point mutation, amplification, chromosomal translocation, gene shear. Proto-oncogene expression in a
number of products on the cell membrane stability, receptor activity, signal transduction, enzyme activity and gene expression has significant regulatory role. Tumor suppressor genes are normal cellular genes, can induce differentiation,
apoptosis and regulation of cell growth and other functions, once the mutation and deletion, can lead to carcinogenesis. With pancreatic cancer have been found related to the
occurrence of the major tumor suppressor genes p53, p16, DPC4,
APC, DCC, RB, NM23 and so on. This study found that in pancreatic cancer tissue and normal pancreatic tissue were significantly differentially expressed genes, there are a lot of oncogenes and tumor suppressor genes may be related to the occurrence and development of pancreatic cancer has a close relationship.
Decreased expression of genes, 006698 BLCAP (Homo sapiens bladder cancer associated protein) gene, also known as bcl0, encoding the human bladder cancer associated protein. In 1999,
the gene first by differential screening of foreign scholars to use a different method from invasive bladder cancer was found in the filter. 2002 Gromova et al  the use of mRNA differential display was cloned into the gene, RT-PCR results
showed that the gene in non-invasive lesions overexpressed in
tumor progression that played a considerable role. BLCAP gene homology search of the discovery of several high homology genes associated with cell growth regulation, in view of BLCAP gene and structural similarity of these genes, and between
different species BLCAP gene sequences rather conservative, Presumably BLCAP genes regulating cell growth as a very important housekeeping genes, their functions may lead to imbalance in cell growth control, causing cell malignant
transformation . Number of studies have shown that the process of invasion in bladder cancer gene expression BLCAP down; while Fan et al  studies have shown that BLCAP antisense oligonucleotide on human osteosarcoma cell growth rate and colony-forming ability to significantly promote the effect, suggesting that BLCAP gene is a tumor suppressor gene in human osteosarcoma. This study showed that pancreatic cancer significantly down-regulated gene expression BLCAP, the gene may be the growth and metastasis of pancreatic cancer
also play a certain role.
007,295 breast cancer susceptibility gene (BRCAl gene), located in human chromosome 17q21, encoding 1863 amino acids. BRCAl as a tumor suppressor gene, through multiple signal transduction pathways inhibit cell proliferation, cell growth, induce apoptosis and DNA damage involved in cell repair and
maintain genome integrity is an important negative regulator of cell cycle. BRCAl has a role of tumor suppressor genes can be coordinated with a number of tumor suppressor genes,
inhibit tumor growth. BRCAl gene to a number of c myc, p53,
pRB, P300, E2F binding sites such as genes, activate or inhibit these genes can promote or inhibit DNA-damage repair.
The BRCAl gene mutation causes a BRCAl encoding a protein from
the nucleus into the cytoplasm, the loss of the ability to bind with the DNA. Currently considered BRCAl gene on cell growth and proliferation plays a negative regulatory role, its structure and function of the anomaly is an important reason
for malignant cells. At the same time, BRCAl gene regulation by estrogen, with tissue-specific, its expression is induced
mutations and low breast cancer, ovarian cancer, prostate cancer, one of the major [4 6]. In this study, the results,
BRCAl gene expression significantly down-regulated, is likely
to be an infinite proliferation of pancreatic cancer cells, one of the reasons.
Increased expression of genes, 001.91 thousand cathepsin
E gene (Cathepsin E gene), encoding a lysosomal acid protease, cathepsin family belonging to a member of. Azuma et al  studies have shown that patients with pancreatic duct adenocarcinoma pancreatic tissue can be detected in the expression of Cathepsin E and Cathepsin E pancreatic juice in a high positive rate, suggesting that Cathepsin E is an effective marker for diagnosis of pancreatic cancer. The current study of cathepsin E, fewer, but Cathepsin D in breast cancer, esophageal squamous cell carcinoma and gastric cancer
tissue of a high positive rate, and of guiding significance to the prognosis [8,9]. Cathepsin D on the basement membrane of a variety of components such as fibronectin, laminin, various types of collagen, protein mucopolysaccharide degradation of
the core protein, such as destruction of both role in the mechanism of infiltration and metastasis of malignant tumors play an important role in . In addition, Cathepsin B, D involved in the apoptosis of different models, indicating that
cathepsin process of signal transduction in apoptosis played an important role. The results of this study suggest that cathepsin E were significantly increased in the enzyme may be the process of invasion and metastasis of pancreatic cancer may play a role.
At present, the domestic and foreign-related genes in
pancreatic cancer study is a cancer gene or a possible tumor suppressor gene to cultured cells or transgenic animals, or of a particular gene knockout (knockout) to observe the physiological or pathological changes. However, due to the occurrence of cancer is a multi-factor, multi-step process, in
which there are at least two kinds or more than two kinds of genes involved in cancer. The results of this study showed that a total of 17 oncogenes and tumor suppressor gene in
pancreatic cancer occurrence and development of abnormal expression, it is there may be co-ordinated co-expression of
the gene cluster. Therefore, pancreatic cancer using cDNA microarray technology to study the genetic population level,
screening a number of areas in the tumor-related play an
important role in molecular and play a key role in regulating genes, in order to reveal the pancreatic cancer at the gene level, the nature of the of pancreatic cancer early diagnosis
and early treatment provide a theoretical basis.
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