GAS7基因於肺癌之分子變異及臨床相關性研究

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(1)Fig. 1 Frequent chromosomal deletion regions in tumors from lung cancer patients. Chromosomal location of 20 markers (squared) showing the high percentage of LOH (>48%) along with the markers analyzed at the respective chromosome. The frequency of LOH is noted on the side of the marker and the novel sites of frequent LOH is indicated by a symbol *. GAS7 gene is located at 17p13.1 region, which is between two markers named D17S938 and D17S974. Figure was taken from ref (14).. 50.

(2) Fig. 2 The functions of GAS gene family in controlling cell cycle. The mechanism controlling the maintenance and departure from the cellular growth-arrested state, which could be regulated by the genes predominantly expressed during the growth-arrest or growth arrest-specific genes (GAS). These GAS genes mediate a variety of biological functions including cell survival, proliferation and growth suppression, cell differentiation and apoptosis (Taken from web-site information of Dr. Sue Lin-Chao at institute of Molecular Biology: http://www.imb.sinica.edu.tw/~ mbsue/index_c.html in Academic Sinica).. 51.

(3) Alternative splicing. Fig. 3 Representative figure of GAS7 gene DNA and mRNA composition. DNA composition of GAS7 gene is shown by upper panel. There are 14 exons in this gene. Through alternative splicing, GAS7 gene can be transcribed into three different isofrom: GAS7C, 7B, and 7A, and their full length cDNA sizes are shown in bp. Figure was taken from ref (15). Exons are not drawn to real scale.. 52.

(4) [www.ncbi.nlm.nih.gov]. Fig. 4 Schematic representation of functional domain of GAS7C, 7B, and 7A. GAS7 protein has three different functional domain (from N-terminal to C terminal): Src homology 3 domain (SH3), WW domain, and Fes/CIP4 homology domain (FCH). FCH domain which can promote actin assembly and the crosslinking of actin filament. WW domain near the N-terminal region in GAS7 plays a role in signal transduction. SH3 domain acts as a receptor which binds to proline-rich ligand and cause the signal transduction event.. 53.

(5) Fig. 5 Schematic representation of primer design of GAS7 gene. In order to distinguish the three different isoforms of GAS7 gene, we designed their primers at the very 5’ end of GAS7 mRNA, which is the unique region for each isoform. Each primer pair for each GAS7 isoform is shown by red opposite arrow, and their PCR product size was indicated (in bp).. 54.

(6) Fig. 6 GAS7 mRNA and protein expressions in normal lung cells (IMR90 and Bes2B) and lung cancer cells (A549). (A) The GAS7A, 7B and 7C mRNA expressions are shown. The molecular weight of GAS7A, 7B and 7C are 555bp, 321bp and 282bp. β-actin is used as an internal control. (B) The GAS7B and 7C protein expressions in the cell lines are shown. The location and molecular weight of GAS7B, and GAS7C are as indicated.. 55.

(7) Fig. 7 GAS7C protein identified in IMR90 normal lung cell by Peptide MASS spectrum analysis. (A) We evaluated the GAS7C in the IMR90 cells which express predominantly the GAS7C isoform. (B) Trypsin-digested peptides of the eluted band on the Western blot corresponding to the GAS7C were purified and sent for the mess spectrum analysis. (C) Protein data base search indicated that the band was indeed GAS7C protein.. 56.

(8) Fig. 8 The localization of GAS7 protein in A549 and IMR90 cell line. Cells were stained by PI conjugated anti-GAS7 antibody (shown in red, left panel) and DAPI nuclear stain (shown in blue, meddle panel). The GAS7 and DAPI staining were merged in right panel. The green arrows indicate hollow nuclear in A549, which expresses GAS7B predominantly. For IMR90, which mainly expresses GAS7C, both cytoplasmic and nuclear staining were observed.. 57.

(9) Fig. 9 Representative figures of immunohistochemistry analysis of GAS7, in paraffin sections of lung tumors. GAS7 protein cytoplasmic and nucleus immunoreactivities were found in an AD (A) and an SQ (C) patients; and negative stainings were found in another AD (B) and SQ patients (D). (Original magnification: 200X). 58.

(10) Fig. 10 Representative figures of Western blot analysis of GAS7 protein in lung tumor tissues. (A) Two different protein bands can be observed on gel, and they represent for two different GAS7 isoforms, i.e., GAS7C and GAS7B. T: the tumor lung tissue. Patient 92 was negative for GAS7C protein expression. Patients 85 had GAS7B protein low expression. β-actin was used as the internal control for the analysis. The protein expression level of GAS7A is too low to be analyzed in patients.. (B). The. correlation. between. GAS7. protein. analyzed. by. immunohistochemistry (IHC) and Western blot (WB) assay. IHC-P or IHC-N means the immunohistochemistry stains are positive or negative expression, respectively. WB-P or WB-N means the western blots are positive or negative expression, respectively. This figure shows that IHC and Western blot is highly concordant.. 59.

(11) Fig. 11 Representative figure of GAS7B (A) and GAS7C (B) mRNA expression analysis in lung cancer. N: the normal lung tissue; T: the tumor lung tissue. The patients whose tumor tissues expressed the GAS7 mRNA at the low level are indicated by red. β-actin was used as the internal control for the analysis. MW, molecular weight marker.. 60.

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(13) Fig.12 Representative figures of BSP assay for the GAS7B and GAS7C methylation status in lung cancer patients. (A) Methylation status of the CpG sites at promoter region of the GAS7B after sodium bisulfite treatment of genomic DNA of normal lung cell lines IMR90 and several tumor lung tissues. Their mRNA expression statuses are indicated on the side. (B) In the paired lung cancer sample, CpG sites of the CpG island region of GAS7C promoter were completely methylated, indicating by underlined CpG regions, whereas in normal samples, most CpG sites were unmethylated, representing by TG regions. Horizontal red line indicates the genomic DNA region. Numbers below the horizontal line are the positions (in bp) of the genomic DNA. Bars under the sequence highlight the CpG sites at promoter region of each GAS7 isoform. The region between blue arrows (primer pair) is the CpG island region we detected. Transcriptional start sites are indicated by dashed arrows. Methylated CpG sites at different levels are indicated as 100% z, 75% 50%. , 25 %. ,. , and 0% {, calculated by the ratio between C peak (blue). and T peak (red).. 62.

(14) Fig.13 Representative figures for the detection of LOH at the di-nucleotide repeats microsatellite marker (AFMA070). The number on the X-axis indicates the length of the fragment, and the number on the Y-axis shows the fluorescence intensity of peak height (the amount of PCR product). The LOH status of three patients are shown as (A) NO LOH; (B) LOH.. 63.

(15) Fig. 14 Concordance analysis with (A) protein expression and mRNA expression, and (B) mRNA expression and promoter methylation of the GAS7 genes. The percentage of cases is indicated in the X-axis, whereas the type of comparison is plotted as the Y-axis. “+” indicates positive protein expression, positive mRNA expression, and hypermethylation of promoter, as opposed to “−”. The percentage of concordant group (gray bars) and non-concordant group (white bars) is indicated above. P values for association between protein expression, mRNA expression, and promoter methylation are also showed at the bottom.. 64.

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