Patients, treatment and follow-up
A total of 105 cases of SCLC were admitted to Yongchuan Hospital of Chongqing Medical University between April 2010 and April 2014. Form these patients the present study enrolled 60 cases (42 males and 18 females; 40 to 69 years, 54.1 ± 6.6 years). This study was approved by Yongchuan Hospital of Chongqing Medical University committee before patient admission, and that was conducted in accordance with the Declaration of Helsinki. The written informed consent was obtained from all patients. Inclusion criteria: 1) new SCLC cases; 2) no therapies were initiated; 3) confirmed by histopathological exam. Exclusion criteria: 1) patients transferred from other hospital; 2) patients with recurrent SCLC; 3) therapies were initiated; 4) multiple clinical disorders were diagnosed.
The 60 patients included 15, 20 and 25 cases at AJCC clinical stage II-IV, respectively. According to patients’ conditions, surgery only, or surgery in combination with chemotherapy or radiotherapy, or chemotherapy or radiotherapy only, were performed. A 5-year follow-up was performed since admission in a monthly manner through telephone or outpatient visit. Patients died of causes unrelated to SCLC were excluded.
Specimen collection and SCLC cell model
MRI-guided biopsy was performed to collect both SCLC (tumor) and adjacent non-tumor lung tissues from each patient. Histopathological exams were performed to confirm all tissue samples.
Human SCLC cell line SHP-77 (ATCC, USA) was used. 90% RPMI-1640 medium was mixed with 10% FBS to cultivate cells at 37 °C with 5% CO2.
Transient transfections
Negative control (NC) miRNA and miR-148a mimic were from Sigma-Aldrich (USA). Vectors expressing MCM3AP-AS1 and ROCK1 were constructed using pcDNA3 vector by Sangon (Shanghai, China). SHP-77 cells collated at 80% confluence were transfected with 45 nM miRNAor 10 nM vector using lipofectamine 2000 (Sigma-Aldrich). Control (C) cells were untransfected cells. Empty vector- or NC miRNA-transfected cells were NC cells. At 4h after transfections, cells were harvested and used in following experiments.
RNA extractions and digestion
To detect gene expression, total RNAs in tissue sample (0.03g per sample) or SHP-77 cells (3×105 cells per transfection group) were extracted using RNAzol (Sigma-Aldrich). All steps were completed following the instructions from Sigma-Aldrich. It is worth noting that RNA precipitation was performed using 75% ethanol. The purpose is to harvest miRNA. DNase I digestion was performed on all RNA samples.
QPCR
Tetro Reverse Transcriptase (Bioline) was used to perform all reverse transcriptions and qPCR reactions were carried out using QuantiTect SYBR Green PCR Kit (Qiagen). With GAPDH as endougenous control, MCM3AP-AS1 and ROCK1 mRNA expression levels were normalized.
All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) was used to measure the expression levels of mature miR-148a. After the addition of poly (A), miRNA reverse transcription and qPCR assays were sequentially performed. Expression of miR-148a was normalized to U6.
Primer sequences were MCM3AP-AS1: Forward-CTGCTAATGGCAACACTGA and Reverse-AGGTGCTGTCTGGTGGAGA; GAPDH : Forward-CAGGAGGCATTGCTGATGAT and Reverse-GAAGGCTGGGGCTCATTT; ROCK1: Forward-CCTGTAACCCAAGGAGATGT and Reverse-CACAATTGGCAGGAAAGTG; miR-148a: Forward-AAAGTTCTGAGACACTCCG. U6 primers and miR-148a reverse primer were included in the kit. All Ct values were processed using 2-ΔΔCT method and PCR reactions were repeated 3 times.
Cell invasion and migration analysis
SHP-77 cells were harvested at 48 h post-transfection and cell invasion as well as migration abilities were analyzed by performing Transwell assays. To prepare single cell suspensions, non-serum RPMI-1640 Medium was mixed with SHP-77 cells in a ratio of 4×104 cells per 1 ml cell culture medium. After that, cell suspensions were injected into the upper Transwell chamber (0.1ml per well). In contrast, the lower chamber was filled with 80% RPMI-1640 medium and 20% FBS. Before invasion assays, Matrigel (200ug/ml, Millipore, USA) was used to coat the membranes for 8h at 37 °C to mimic cell invasion. The chambers were cultivated at 37 °C for 15 h. After that, membranes were collected at stained for 15min at room temperature with 0.1% crystal violet (Sigma-Aldrich, USA). Invasion and migrating cells were observed under a light microscope.
Western blot
SHP-77 cells were harvested at 48h post-transfection and the expression of ROCK1 was detected by performing western blot. RIPA solution (Sigma-Aldrich) was mixed with SHP-77 cells in a ratio of 105 cells per 1 ml to extract total proteins. BCA assay (Sigma-Aldrich) was then performed to quantify protein samples. Electrophoresis (12 % SDS-PAGE gel) was performed after the protein samples were denatured in boiling water for 15 min, followed by transferring the proteins to PVDF membranes. Blocking was performed in 5% fat-free milk at 24°C for 1h, following by incubation with primary antibodies including rabbit anti-GAPDH (1: 1600, ab22555, Abcam) and anti-ROCK1 (1: 1600, ab97592, Abcam), and the incubation was performed at 4°C for 12h. Following that, the membranes were further incubated with secondary antibody of goat HRP (IgG) (1:2000; ab6721; Abcam) at 24°C for 1h. Following that, membranes were incubated with ECL Western Blotting Substrate (ab65623, Abcam) to develop signals, which were processed using Image J v1.46 software.
Statistical analysis
Mean values of 3 replicates were calculated and used to perform following statistical analyses. Pearson’s correlation coefficient was for correlation analysis. The 60 patients were grouped into low and high MCM3AP-AS1 level groups with the median expression level of MCM3AP-AS1 in SCLC as cutoff value. Survival curves were plotted and compared by log-rank test. To explore differences between tissue types (non-tumor and SCLC) and among multiple cell groups, paired t test and ANOVA (on-way) and Tukey test were used. p<0.05 was statistically significant.