Background
Methods
Tissues
Clinicopathologic features | IHC staining | Real-time PCR | PR (%) | Total |
---|---|---|---|---|
Case no. | 102 | 93 | 195 | |
Age(y) | ||||
< 60 | 61 | 48 | 55.9 | 109 |
≥60 | 41 | 45 | 44.1 | 86 |
Gender | ||||
Man | 76 | 70 | 74.9 | 146 |
Female | 26 | 23 | 25.1 | 49 |
Tumor size(cm) | ||||
< 5 | 57 | 58 | 59.0 | 115 |
≥5 | 45 | 35 | 41.0 | 80 |
Tissue differentiation | ||||
Well | 5 | 1 | 3.1 | 6 |
Moderately | 56 | 56 | 57.4 | 112 |
Poorly | 41 | 36 | 39.5 | 77 |
Depth of invasion | ||||
T1 + T2 | 27 | 22 | 25.1 | 49 |
T3 + T4 | 75 | 71 | 74.9 | 146 |
Lymph node metastasis | ||||
No | 32 | 22 | 27.7 | 54 |
Yes | 70 | 71 | 72.3 | 141 |
TNM staging | ||||
I + II | 43 | 29 | 36.9 | 72 |
III + IV | 59 | 64 | 63.1 | 123 |
Distant metastasis | ||||
Yes | 13 | 8 | 10.8 | 21 |
No | 89 | 85 | 89.2 | 174 |
Immunohistochemistry
Total RNA extraction and real-time PCR
Gene | Forward primer (5′-3′) | Reverse primer |
---|---|---|
Reg IV | CAGATCCTGGTCTGGCAAGT | ATTCGTTGCTGCTCCAAGTT |
SOX9 | GGAGATGAAATCTGTTCTGGGAATG | TTGAAGGTTAACTGCTGGTGTTCTG |
GAPDH | CAATGACCCCTTCATTGACC | GACAAGCTTCCCGTTCTCAG |
Overexpression of Reg IV gene in gastric carcinoma cells
-
Reg IV-KpnI-F: 5’-CGGGGTACCATGGCTTCCAGAAGCATG-3’
-
Reg IV-BamHI-R: 5’-CGCGGATCCCTATGGTCGGTACTTGCA-3’
Silencing of the Reg IV gene and SOX9 gene by siRNAs in gastric carcinoma cells
siRNA | Sequence 5′ → 3’ | |
---|---|---|
SOX9 | 1 | Sense: GCAGCGACGUCAUCUCCAATT |
Antisense: UUGGAGAUGACGUCGCUGCTT | ||
2 | Sense: GAACAAGCCGCACGUCAAGTT | |
Antisense: CUUGACGUGCGGCUUGUUCTT | ||
3 | Sense: GACCUUCGAUGUCAACGAGTT | |
Antisense: CUCGUUGACAUCGAAGGUCTT | ||
Reg IV | 1 | Sense: CAUGCUUCUGGAAGCCAUCTT |
Antisense: GAUGGCUUCCAGAAGCAUGTT | ||
2 | Sense: GCUCAUCUCAGCACAGUGCTT | |
Antisense: GCACUGUGCUGAGAUGAGCTT | ||
3 | Sense: CUUCAGGAAGCUGAGGAACTT | |
Antisense: GUUCCUCAGCUUCCUGAAGTT | ||
Negative control | Sense: UUCUCCGAACGUGUCACGUTT | |
Antisense: ACGUGACACGUUCGGAGAATT |
Western blotting
Invasion and migration assay of tumor cells transfected with PEGFP-Reg IV, siR-Reg IV, siR-SOX9 in vitro
Wound healing assay of tumor cells transfected with PEGFP-Reg IV, siR-Reg IV, siR-SOX9 in vitro
Statistical analysis
Results
Reg IV and SOX9 protein expressed in human gastric cancer
Parts of Fig. 1 | Score (Reg IV) | Parts of Fig. 1 | Score (SOX9) |
---|---|---|---|
b | 12 | e | 8.4 |
c | 12 | f | 10.8 |
Variable | Reg IV | P-Value | SOX9 | P-Value | ||
---|---|---|---|---|---|---|
+ | – | + | – | |||
Tumor tissues | 55 | 47 | 0.022* | 28 | 74 | 0.003* |
Adjacent tissues | 13 | 27 | 2 | 38 | ||
Age (year) | ||||||
< 60 | 30 | 31 | 0.241 | 17 | 44 | 0.908 |
≥ 60 | 25 | 16 | 11 | 30 | ||
Gender | ||||||
Man | 40 | 36 | 0.655 | 20 | 56 | 0.66 |
Female | 15 | 11 | 8 | 18 | ||
Tumor size (cm) | ||||||
< 5 | 32 | 25 | 0.613 | 14 | 43 | 0.462 |
≥ 5 | 23 | 22 | 14 | 31 | ||
Tissue differentiation | ||||||
Moderately-well | 33 | 28 | 0.965 | 16 | 45 | 0.736 |
Poorly | 22 | 19 | 12 | 29 | ||
Depth of invasion | ||||||
T1 + T2 | 14 | 30 | 0 | 9 | 18 | 0.424 |
T3 + T4 | 41 | 17 | 19 | 56 | ||
Lymph node metastasis | ||||||
No | 18 | 14 | 0.75 | 11 | 21 | 0.289 |
Yes | 37 | 33 | 17 | 53 | ||
TNM staging | ||||||
I + II | 23 | 20 | 0.94 | 12 | 31 | 0.93 |
III + IV | 32 | 27 | 16 | 43 | ||
Distant metastasis | ||||||
Yes | 5 | 8 | 0.251 | 4 | 9 | 0.93 |
No | 50 | 39 | 24 | 65 |
Reg IV and SOX9 transcript expression in human gastric cancer
Variable | Reg IV | P Value | SOX9 | P Value | ||||
---|---|---|---|---|---|---|---|---|
T/N > 2 | T/N 2–0.5 | T/N < 0.5 | T/N > 2 | T/N 2–0.5 | T/N < 0.5 | |||
Case no. | 40 | 17 | 36 | 29 | 37 | 27 | ||
Age (year) | ||||||||
< 60 | 22 | 9 | 17 | 0.789 | 14 | 20 | 14 | 0.897 |
≥ 60 | 18 | 8 | 19 | 15 | 17 | 13 | ||
Gender | ||||||||
Man | 28 | 15 | 27 | 0.344 | 21 | 27 | 22 | 0.673 |
Female | 12 | 2 | 9 | 8 | 10 | 5 | ||
Tumor size (cm) | ||||||||
< 5 | 28 | 8 | 22 | 0.257 | 13 | 25 | 20 | 0.055 |
≥ 5 | 12 | 9 | 14 | 16 | 12 | 7 | ||
Tissue differentiation | ||||||||
Moderately-well | 26 | 7 | 24 | 0.168 | 20 | 17 | 20 | 0.044 |
Poorly | 14 | 10 | 12 | 9 | 20 | 7 | ||
Depth of invasion | ||||||||
T1 + T2 | 8 | 8 | 20 | 0.005 | 10 | 21 | 5 | 0.007 |
T3 + T4 | 32 | 9 | 16 | 19 | 16 | 22 | ||
Lymph node metastasis | ||||||||
No | 7 | 5 | 25 | 0.000 | 13 | 18 | 6 | 0.082 |
Yes | 33 | 12 | 11 | 16 | 19 | 21 | ||
TNM staging | ||||||||
I + II | 11 | 7 | 11 | 0.591 | 8 | 12 | 9 | 0.878 |
III + IV | 29 | 10 | 25 | 21 | 25 | 18 |
Association of Reg IV protein expression and SOX9 protein expression
SOX9 | Reg IV | r | P-value | ||
---|---|---|---|---|---|
Positive | Negative | Total no. | |||
Positive | 24 | 4 | 28 | 0.392 | 0.000 |
Negative | 31 | 43 | 74 | ||
Total no. | 55 | 47 | 102 |
Association of Reg IV transcript expression and SOX9 transcript expression
SOX9 | Reg IV | |||||
---|---|---|---|---|---|---|
Up-regulation | No difference | Down-regulation | Total no. | r | P-value | |
Up-regulation | 16 | 5 | 8 | 29 | ||
No difference | 17 | 9 | 11 | 37 | 0.273 | 0.008 |
Down-regulation | 7 | 3 | 17 | 27 | ||
Total no. | 40 | 17 | 36 | 93 |