LncRNA CASC15 upregulates cyclin D1 by downregulating miR-365 in laryngeal squamous cell carcinoma to promote cell proliferation

Research patients and follow-up

This study included 58 LSCC patients (36 males and 22 females, 42 to 67 years old, mean age 54.1 ± 7.3 years old) who were selected from the 134 LSCC patients admitted at Changhai Hospital, Navy Military Medical University from January 2011 to January 2014. Inclusion criteria: (1) LSCC patients who were newly diagnosed; (2) major organs were still in good condition (based on electrocardiogram, urine test, blood test, scatoscopy, X-ray and B-mode ultrasonography); (3) no previous anti-tumor therapies were initiated; (4) willing to participate in a 5-year follow-up. Exclusion criteria: (1) recurrent cases; (2) clinical disorders other than LSCC were observed; (3) history of malignancies. All patients were followed up for 5 years and patients who were lost during follow-up or died of other causes were excluded from this study. According to the stage standard established by AJCC, 10, 12, 18 and 18 cases were classified into stages I–IV, respectively. Patients’ specific clinical features were shown in Table 1.

Table 1 Clinical Characteristics of patients includedEthical considerations

All the 58 LSCC patients were informed with the experimental protocols, and they all singed the informed consent. Before patient admission, this study was approved by the Ethics Committee of the Changhai Hospital, Navy Military Medical University.

Tissues and cells

All LSCC patients were diagnosed through histopathological biopsies. Both tumor (LSCC) and adjacent (within 3 cm around tumors) non-tumor tissues from 3 different sites of each patient were collected. The weight of each tissue sample ranged from 0.1 to 0.14 g. All tissues were confirmed by 3 experienced pathologists. Human LSCC cell line UM-SCC-17A (MilliporeSigma, USA) was used. DMEM containing 10% FBS was used to cultivate UM-SCC-17A cells at 37 °C with 5% CO2.

Vectors and miRNA mimic

CASC15 and cyclin D1 expression vectors were constructed by Songon (Shanghai, China) using pcDNA3.1 vectors. Negative control miRNA and miR-365 mimic were purchased from Sigma-Aldrich (USA).

Transient cell transfections

UM-SCC-17A cells were harvested at the confluence of 70–90%. In a 6-well plate, 105 cells were transfected with 10 nM CASC15 expression vector, cyclin D1 expression vector, 10 nM pcDNA3.1 vector (negative control, NC), 50 nM miR-365 mimic, or 50 nM negative control miRNA (negative control, NC) using lipofectamine 2000 transfection reagent (Sigma-Aldrich, USA). Cells without transfections were control (C) cells. Cells were harvested at 24 h after transfections to be used in the following experiments.

RT-qPCR

UM-SCC-17A cells were harvested at 24 h after transfections and mixed with Ribozol reagent (Invitrogen, USA, 105 cells with 1 ml solution) to extract total RNAs. Tissues were ground in liquid nitrogen, followed by the addition of Ribozol reagent (0.5 g tissue with 1 ml solution) to extract total RNAs. RNA samples were treated with DNase I to remove genomic DNA. RNA samples were then used as template to synthesize cDNA through reverse transcription (25 °C for 10 min, 55 °C for 20 min and 85 °C for 10 min) using AMV Reverse Transcriptase (Canvax Biotech, USA). SYBR Green Master Mix (Bio-Rad, USA) was used to prepare RT-qPCR mixtures. The expression of CASC15 and cyclin D1 were detected using 18S rRNA and GAPDH as endogenous control, respectively. Using the same amount of cells and tissues (0.5 ng tissues with 1 * 10–6 ml solution), miRNA extractions were performed using miRNA Isolation Kit (Geneaid, USA). After that, reverse transcriptions and RT-qPCRs were performed using All-in-One™ miRNA RT-qPCR Detection Kit* (Genecopoeia, Guangzhou, Shanghai). The expression of miR-365 was determined with U6 as the endogenous control. Three replicates were included for each experiment and 2−ΔΔCT method was used to process data.

Cell proliferation assay

DMEM containing 10% FBS (totally 10 ml) was mixed with 3 × 104 UM-SCC-17A cells to prepare single cell suspensions. UM-SCC-17A cells were collected at 24 h post-transfections. The mycoplasma test of the cell line was performed, and cells were confirmed by STR analysis. A 96-well cell culture plate was used to cultivate cells at 37 °C with 5% CO2 in 0.1 ml cell suspension per well. Three replicate wells were set for each experiment. To detect cell proliferation, 10 μl cell counting kit-8 solution (Sigma-Aldrich, USA) was added into each well every 24 h for 4 times. After that, cells were cultivated for additional 2 h and 10 μl DMSO was added. Finally, OD values at 450 nm were measured to detect cell proliferation.

Western blot analysis

UM-SCC-17A cells from 3 biological replicates of each experiment were collected at 24 h post-transfection, and 105 cells were mixed with 1 ml RIPA solution (Beyotime, Jiangsu, China) to extract total proteins. Proteins were denatured and subjected to 12% SDS-PAGE gel electrophoresis. Following gel transfer (PVDF membrane) and blocking (in 5% non-fat milk at 25 °C for 2 h), cyclin D1 (1:1,500, ab31392, Abcam) or GAPDH (1:1,500, ab9845, Abcam) rabbit polyclonal primary antibody was added to incubate with the membranes at 4 °C overnight, followed by incubation with the secondary antibody of IgG-HRP goat anti rabbit (1:1,000, MBS435036, MyBioSource) at 25 °C for 2 h. ECL (Sigma-Aldrich, USA) was used to develop signals. The X-ray film (Sangon, Shanghai, China) was exposed for 10–60 s and Image J v1.46 software was used to process the signals.

Statistical analyses

Mean values were calculated using the data from 3 biological replicates. Differences between 2 types of tissue were analyzed using paired t test. One-way ANOVA and Tukey test were used for the analysis of differences among multiple groups. Linear regression was used for correlation analysis. The 58 LSCC patients were divided into high (n = 28) and low (n = 30) CASC15 groups (Youden’s index) and survival curves were plotted using K-M method and were compared by log-rank test. P < 0.05 was statistically significant.

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