Background
Materials and methods
Clinical samples
Characteristics | Number of case | RP11-757G1.5 | expression | p value |
---|---|---|---|---|
High(n = 56) | Low(n = 56) | |||
Gender | p = 0.568 | |||
Male | 63 (56.3%) | 30 | 33 | |
Famale | 49 (43.7%) | 26 | 23 | |
Age at diagnosis | p = 0.676 | |||
< 60 | 32 (28.6%) | 15 | 17 | |
≥ 60 | 80 (71.4%) | 41 | 39 | |
Differentiation | p = 0.404 | |||
poor | 48 (42.9%) | 23 | 25 | |
Moderately | 28 (25.0%) | 17 | 11 | |
well | 36 (32.1%) | 16 | 20 | |
Tumor size (cm) | p = 0.003** | |||
< 5 | 54 (48.2%) | 19 | 35 | |
≥ 5 | 58 (51.8%) | 37 | 21 | |
Depth of invasion | p = 0.693 | |||
T1,T2 | 40 (35.7%) | 19 | 21 | |
T3,T4 | 72 (64.3%) | 37 | 35 | |
Location | p = 0.779 | |||
Transverse colon | 30 (26.8%) | 13 | 17 | |
Ascending colon | 34 (30.4%) | 19 | 15 | |
Descending colon | 19 (17.0%) | 9 | 10 | |
Sigmoid colon | 29 (25.8%) | 15 | 14 | |
Lymph node status | p = 0.008** | |||
N0 | 52 (46.4%) | 33 | 19 | |
N1 + N2 | 60 (53.6%) | 23 | 37 | |
TNM stage | p < 0.001*** | |||
I + II | 61 (54.5%) | 20 | 41 | |
III + IV | 51 (45.5%) | 36 | 15 |
Reagents
Cell culture
RNA extraction and RT-qPCR assay
sh-NC: 5′-GCGGATGCCCGATTGGGCCA-3’ | |
sh-RP11-757G1.5#1: 5′-ATCCGGCGGTAGCTAGCTAAGCAA-3’ | |
sh-RP11-757G1.5#2: 5′-GAAATTGCGTACAAGGAGAGTCA-3’ | |
pcDNA3.1-RP11-757G1.5 | |
F: 5′-AGGGCCTCCGCTGGAATTTTAAAGGGTCCCTGAAG-3′ (BamHI) | |
R: 5′-CAAGTCGAGGGTCGGGTGCAAAACAACCGATAC-3′ (XhoI) | |
miRNA-139-5p mimic: 5′-ACCGGTTGAAAAGTGCGATGCTACCCGGTAAGGCCGCCG-3’ | |
miRNA-139-5p inhibitor: | |
5′-AAGCTTGGTGGGTGGCACGCGCAAAGTGTGCAGTCATGAAGC-3’ | |
si-NC: 5′-AATTGCGGAACCGGTTGCATTAA-3’ | |
si-YAP1: 5′-GAAGGCAGTTTGAAGTGACGCGGG-3’ |
GAPDH | |
F: 5′-TGTGGGCATCAATGGATTTGG-3’ | |
R: 5′-ACACCATGTATTCCGGGTCAAT-3’ | |
RP11-757G1.5 | |
F: 5′-CGTAGGCAAAAAGCGTACGGAT-3’ | |
R: 5′-AAAAGCGGGAAAGCGAAAGT-3’ | |
miR-139-5p | |
F: 5′-ACACTCCAGCTGCACGTGTC-3′ | |
R: 5′-TGGTGTCGTGGAGTCGGTTGA-3’ | |
YAP1 | |
F: 5′-CGCTGACGGAGTACAAGTG-3’ | |
R: 5′-GTAGGAGCCGACCTCGTTG-3’ |
Isolation of cytoplasmic and nuclear RNA
Plasmid construction and transfection
Cell proliferation assay
In vitro cell migration and invasion assay
Luciferase reporter assay
RNA pull-down assay
Ago2 immunoprecipitation assay
Western blotting analysis
Immunohistochemistry analysis
In vivo studies
Statistical analyses
Results
Overexpression of RP11-757G1.5 in CRC associates with poor prognosis
Variable | Subset | Univariateanalysis | Multivariate analysis | ||
---|---|---|---|---|---|
p-value | HR (95% CI) | p-value | HR (95% CI) | ||
Gender | Male/female | 0.771 | 0.734 (0.415–1.859) | – | – |
Age at diagnosis (years) | < 60/≥60 | 0.474 | 0.626 (0.537–2.552) | – | – |
Differentiation | Well + moderately/poorly | 0.331 | 1.422 (0.614–2.683) | – | – |
Tumor size (cm) | < 5/≥5 | 0.002** | 2.742 (0.326–4.663) | 0.017* | 3.471 (0.527–6.028) |
Depth of invasion | T1 + T2/T3 + T4 | 0.095 | 1.116 (0.549–4.241) | – | – |
Location | Colon/rectum | 0.852 | 0.632 (0.412–1.743) | – | – |
Lymph node status | N0/N1 + N2 | 0.153 | 2.571 (1.306–4.663) | 0.216 | 2.154 (1.146–6.915) |
TNM stage | I + II/III + IV | 0.003** | 13.145 (5.044–34.378) | 0.004** | 7.603 (2.521–33.109) |
RP11-757G.15 | High/low | 0.008** | 3.441 (1.471–8.005) | 0.019* | 2.015 (1.018–5.856) |