Acquisition of clinical specimens. Colorectal cancer(CRC) were obtained from
archived tissue samples derived from patients with CRC who underwent surgical
treatment at The First Affiliated Hospital of Zhengzhou University, China from
January, 2003 through December, 2012. CRC was diagnosed according to the 2007
WHO Classification of Tumors of the colorectal cancer. All tumors were staged on the
basis of the pathologic tumor-node-metastasis classification of the International Union
Against Cancer [22]. The selection criteria were as follows: 1) the subject presented
with a diagnosis of colorectal cancer and no history of other tumors; 2) the subject
had complete demographic and clinical data, such as age, gender, clinical
manifestations, tumor size, extent of resection, adjuvant therapy, and date of relapse
and/or death; 3) the subject underwent evaluation by enhanced head MRI scanning for
tumor relapse or progression after surgery at least once every six months. Written
informed consent of the patients was provided by their legal surrogates to permit
surgical procedures and use of resected tissues. This study was approved by the
Specialty Committee on Ethics of Biomedicine Research, Zhengzhou University of
China. Human tissue acquisition and use in this study complied with the National
Regulations on the Use of Clinical Samples in China.
Collection of clinical information and follow up. Data was collected by review of
the clinical history. Information was recorded including the patient’s characteristics
(e.g., gender, age), relevant symptoms or history, tumor characteristics (e.g., size,
boundary, whether or not associated with a cystic change or evidence of necrosis),
extent of resection, post-surgical treatment protocol (e.g., whether the patient took
assistant radiotherapy or chemotherapy), overall survival time and progression-free
survival time, or miR-145 and RAD18 expression status (e.g., high levels or low
expression levels). For analysis, a patient's age was stratified into ≥60 or less than
60 years. The extent of resection was classified as gross total resection, subtotal
resection (i.e. greater than 95% of the enhancing tumor was resected), and partial
resection. The tumor size was described by mean tumor diameter (MTD, defined as
the geometric mean of 3 diameters on MRI scan), and sorted into ≥4 cm and <4 cm.
The follow-up was conducted by telephone or direct correspondence. Postsurgical
treatment, including adjuvant radiotherapy and chemotherapy, was fully discussed
with the patient or their relatives. The time of tumor relapse or death was verified by
the patient or their relatives, by medical recording, or by thesocial security record.
Overall survival (OS) was calculated in months from the date of diagnosis to the time
of death, regardless of cause. Progression free survival (PFS) was defined as the
period from the initial date of diagnosis to the time of tumor progression by MRI, or
to the time of death of the patient from CRC.
Apoptosis assay. Apoptosis was measured using the Apoptosis Detection Kit I (BD
Biosciences, Mountain View, CA). As a standard, 1 x 106/ml cells per treatment
condition were fixed and stained with 5ul Annexin-V-FITC (BD PharMingen) and 5µl
propidium iodide (Sigma). Flow-cytometric analysis was performed for 1 x 104 cells
and analyzed by FACScan (Becton Dickinson, NJ) using a single laser emitting
excitation light at 488 nm. Data was analyzed by CellQuest software (Becton Dickson,
San Jose, CA).
Immunocytochemistry and in situ hybridization. Immunohistochemical staining
was performed using anti-RAD18 antibody (Santa Cruz Biotechnology, 1:50 dilution).
In situ hybridization was performed using a miRNA-145 probe
from Exiqon (miRCURY LNA detection probe 5ʹ-DIG (digoxigenin)-labelled).
Detection of the probe was carried out using digoxigenin antibody (21H8; 1:200,
ab420, Abcam), LSAB2 System-HRP (K0672, Dako) and liquid DAB+ Substrate
Chromogen System (K3468, Dako) according to the manufacturer’s instructions.