CRC patients and follow-up
This study was approved by People’s Hospital of Baoan District Ethics Committee. From June 2013 to May 2015, a total of 62 CRC patients (36 males and 26 females) were enrolled. Age of the patients ranged from 46 years to 68 years (57.1 ± 5.6 year). CRC patients were diagnosed by histopathological exam. No initiated therapy was observed and recurrent CRC patients were excluded. Patients were grouped into American Joint Committee on Cancer (AJCC) stage I or II (n = 26) and III or IV (n = 36). Based on AJCC stages, the 62 patients were treated with different therapies. A follow-up (5 years) was performed monthly to record their survival. Informed consent was signed by all patients.
Crc Tissues And Cells
Prior to treatments, CRC and paired non-tumor tissues were obtained from all patients. Following confirmation by histopathological exam, the tissue samples were stored in liquid nitrogen before use.
WiDr (ATCC ® CCL-218™) and HT-29 (ATCC ® HTB-38™) human CRC cell lines from ATCC (USA) were included for cell experiments and were cultivated following the instructions from ATCC. Cell culture medium was ATCC-formulated McCoy's 5a Medium Modified (Catalog No. 30-2007) with 10% FBS. Cells were cultivated at 37°C.
Vector, Mirna And Cell Transfections
LINC 01436 expression vector (pcDNA3.1, Invitrogen) and miR-466 mimic (Sigma-Aldrich) were transfected into cells through transfections mediated by Lipofectamine 2000 (Invitrogen), which was used to transfect 40 nM miRNA or 1µg expression vector into 108 cells.
Total RNA isolation was done by using Ribozol reagent (Invitrogen). DNA removal from RNA was done by incubating with DNase I (Invitrogen) for 80 min at 37°C. Electrophoresis was performed using Urea-PAGE gels (5%) to determine RNA integrity. RNA purity was determined by calculating OD260/280 ratios.
RNA samples with an OD 260/280 ratio close to 2.0 were used to synthesize cDNA samples, followed by qPCRs with internal control 18S rRNA to study the expression of LINC 01436.
To determine the expression of miR-466 precursor, a sequence-specific reverse primer was used to perform reverse transcriptions (RTs), and sequence-specific forward and reverse primers were used to perform qPCRs. To determine the expression of mature miR-466, miRNAs were first added with poly (A), followed by RTs performed using poly (T) as reverse primer. After that, poly (T) and sequence-specific forward primer were used to perform qPCRs. All steps were completed using All-in-One™ miRNA qRT-PCR reagent kit (GeneCopoeia).
Ct values were subjected to 2−ΔΔCT method normalization. Primers were listed in supplemental Table.1.
At 48h post-transfection, WiDr and HT-29 cells were harvested and were subjected to cell apoptosis analysis through CCK-8 assay using a CCK8 kit (ab228554, Abcam). A 96 well plate was used to cultivate cells (3000 cells per well) at 37°C. At 2h after the addition of CCK-8 (10%) the measurement of OD values (450 nm) was performed.
Average values of gene expression in three technical replicates of paired tissues were subjected o paired t test. Mean ± SD values in three biological replicates were subjected to ANOVA Tukey’s test. Correlation analysis was performed by linear regression. With the median level of LINC 01436 in CRC tissues as a cutoff value, patients were classified into high and low level group (n = 31). Log-rank test was used for comparison. P < 0.05 was deemed statistically significant.