Overexpression of LARP1 predicts poor prognosis of colorectal cancer and is expected to be a potential therapeutic target

Tissue samples were obtained from patients who had undergone radical resection at the General Surgery Department of Shanghai General Hospital. No chemotherapy or radiation therapy was applied to these patients before radical resection. We collected a total of 40 cases of colorectal cancer specimens between December 2014 and September 2015. The diagnosis was confirmed by two pathologists, and the cancer staging was determined based on pathological findings according to the American Joint Committee on Cancer (AJCC). All the CRC and corresponding adjacent normal mucosa specimens were collected and preserved after the approval of Institutional Research Ethics Committee of Shanghai Jiao Tong University Affiliated Shanghai General Hospital. All of the 40 patients in the study signed the informed consent. The CRC tissues and adjacent normal mucosae (at least 10 cm away from the primary site) were obtained after surgical resection and immediately frozen in liquid nitrogen and stored at −80 °C to extract RNA and protein later.

The total RNA was extracted from cultured cells, tumor tissues, and paired normal mucosa from 40 colorectal cancer patients according to protocols of the manufacturer (TRIzol, TaKaRa, Japan). The first-strand cDNA was synthesized using 1 μg of total RNA by a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Lithuania). Reverse transcription-polymerase chain reaction (RT-PCR) was performed using a ViiA 7 Real-Time PCR System (Life Technology, New York, USA), according to the manufacturer’s protocol. The primer sequences used were as follows: LARP1 sense 5′-GCAACCTAAAGACACTAC-3′ and antisense 5′-CCTCTTCTTCACTTCAATC-3′, PCNA sense 5′-GTACCTGAACTTCTTTACAAA-3′ and antisense 5′-TGCCTAAGATCCTTCTTC-3′, cyclinD1 sense 5′-GCTGCGAAGTGGAAACCA-3′ and antisense 5′-CCTCCTTCTGCACACATTTG-3′, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense 5′-GGAAGCTTGTCATCAATG-3′ and antisense 5′-CCCCACTTGATTTTGGAG-3′. The amplification conditions were as follows: 1 cycle of 2 min at 95 °C followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. All experiments were performed in triplicate. The relative quantities of the expression of LARP1 were calculated by 2^−ΔΔCt (cycle threshold) values, with GAPDH as an internal control.

The CRC cells HCT8 and RKO were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All the cells were cultured at 37 °C under a moist atmosphere with 5 % CO₂ and maintained in Dulbecco’s modified Eagle medium supplemented with 10 % fetal bovine serum (Gibco, USA), 1 % streptomycin, and penicillin. The shRNA plasmid for LARP1 and the control-shRNA plasmid were purchased from Gene Pharma Company (Shanghai, China). For plasmid transfection, 4 × 10⁴ cells/well in six-well plates were cultured overnight and then transfected with plasmids using Lipofectamine 2000 (Invitrogen, CA, USA), following the manufacturer’s protocols. Stable clones of HCT8 and RKO cells expressing LARP1 shRNA or control shRNA were obtained by puromycin selection. The shRNA target sequence for LARP1 was 5′-GCCAGTCTCAGGAGATGAACA-3′. The control-shRNA target sequence was 5′-TGTTCATCTCCTGAGACTGGC-3′.

Total protein was isolated using a radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, China) according to the manufacturer’s instruction. Then, the concentration of extracted protein was measured using the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology). The same amount of protein (30 μg) was dropped into 10 % sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5 % fat-free milk in Tris-buffered saline and 0.1 % Tween 20 solution at room temperature, and then incubated with primary antibody against LARP1 (1:2000 dilution; Abcam, Cambridge, UK) and GAPDH (1:1000 dilution; Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, the membranes were incubated with secondary antibody (1:5000 dilution; Abgent, San Diego, CA, USA), which has a role of horseradish peroxide (HRP)-tagged detection at room temperature for 2 h. Images of proteins were displayed using an enhanced chemiluminescence reagent (Applygen Technologies Inc., Beijing, China). The Quantity One software (Bio-Rad, California, USA) was used to quantitate the gray scale of each protein strips.

The TMA containing 117 paired CRC specimens was constructed in cooperation with Xin Chao Company (Shanghai, China). Tumors were resected from patients between May 2005 and March 2007. The follow-up lasted until December 2014. The median follow-up time in survivors was 63.5 months (range, 4–88 months). The TMA data include 55 men and 62 women with a mean age of 66.32 years (range, 26–89 years). After being dewaxed and rehydrated in xylene and a graded series of ethanol, antigen retrieval for tissue sections were implemented using 0.01 M sodium citrate buffer (pH 6.0). Then, the tissue sections were incubated in the dark with the primary antibody against LARP1 (1:500 dilution; Abcam, Cambridge, UK) or PCNA (1:10,000 dilution; Abcam, Cambridge, UK) for 16 h at 4 °C. The primary antibody was detected using an HRP-conjugated secondary antibody (Gene Tech, Shanghai, China) for 30 min at room temperature. The immunoreactivity was evaluated independently by two pathologists who were blind to the prognosis of patients. The evaluation included both the intensity and the area of staining. Staining intensity for LARP1 scores were as follows: 0 for negative, 1 for weak, 2 for moderate, and 3 for strong. The extent of staining was calculated according to the percentage of the cells stained positively; scores were as follows: 0 (0 %), 1 (1–25 %), 2 (26–50 %), 3 (51–75 %), and 4 (76–100 %). After multiplying the staining intensity score by the staining extent score, the specimens were divided into two groups based on the final score: low (0–6) and high (7–12). The PCNA index was based on the percentage points of the positive nuclear staining cells, and the specimens were divided into two groups based on this: low (less than 10 % of cells with positive nuclei) and high (more than 10 % of cells with positive nuclei).

The cell proliferation ability was measured using Cell Counting Kit-8 (CCK8) (Dojindo, Japan) according to the instructions of the manufacturer. In simple terms, cells were seeded into 96-well plates (2 × 10³ cells/well) in triplicate. At the appropriate time (12, 24, 36, 48, 60, 72, 96 h), the cells were incubated with 10-μL CCK-8 solution for 2 h at 37 °C under a moist atmosphere with 5 % CO₂. The absorbance of 450 nm was measured using the Gen5 microplate reader (BioTek, Vermont, USA).

In the plate colony formation assays, 1000 log-phase cells per well were seeded in six-well plates and then cultured at 37 °C under a moist atmosphere with 5 % CO₂ for 2 weeks. After fixing with methyl alcohol for 15 min, the cells were stained with Giemsa solution for 20 min. Then, counting and photographing were carried out for colonies of each well. All assays were repeated independently in triplicate.

The SPSS statistical software program version 22 (SPSS, IL, USA) was used for statistical analysis. The two-tailed Student’s t test was used for continuous variables. The χ ² test or Fisher’s exact test for categorical data was used to analyze the relationship between LARP1 and clinicopathological features. The disease-free survival (DFS) and overall survival (OS) rates were defined as the interval from the initial surgery to clinically or radiologically proven recurrence/metastasis and death, respectively. The Kaplan–Meier method was used to calculate the survival rates, and the differences between the survival curves were examined by the log-rank tests. Cox proportional hazards models were applied to estimate the effects of the expression of LARP1 and CRC clinicopathological variables on survival in univariate and multivariate analyses. P < 0.05 was considered statistically significant.