LncRNA AB209371 up-regulated Survivin gene by down-regulating miR-203 in ovarian carcinoma

Sixty one OC patients (epithelial, high-grade serous tumor) were selected from the 105 OC patients who were admitted to School of Life Science, College of Health Sciences, Jiangsu Normal University from May 2016 to January 2019. This study was approved by the review board of Ethics Committee of School of Life Science, College of Health Sciences, Jiangsu Normal University. The inclusion criteria are: 1) newly diagnosed OC cases; 2) no other clinical disorders were diagnosed; 3) no therapies were initiated. And the exclusion criteria are: 1) recurrent OC patients; 2) history of malignancies; 3) therapies were performed within three months before admission. After admission, the patients were informed of the principle of the present study. All of them signed informed consent. According to the system established by AJCC, there were 12, 23, and 26 cases at clinical stage II-IV, respectively.

Before the initiation of any therapies, an ovarian biopsy was performed on each patient to obtain both non-tumor ovarian (within 2 cm around the tumor and contained less than 2% cancer cells) and OC tissues (contained more than 95% cancer cells). All tissues were directly dropped into a liquid nitrogen tank for storage. UWB1.289 (ATCC, USA) human OC cell line was used. A mixture of 50% MEGM medium and 50% RPMI-1640 Medium (3% FBS) was used to cultivate cells. Cell culture conditions were: 37 °C, 95% humidity, and 5% CO₂.

AB209371 and Survivin expression vectors were constructed using by inserting AB209371 and Survivin cDNAs into the pcDNA3.1 vector (RIBOBIO, Guangzhou, China). Negative control (NC) miRNA (5′-GGUUCUCGGAAUCGUACGAGCUAGC-3′) and miR-203 mimic (5′-AGUGGUUCUUAACAGUUCAACAGUU-3′) were synthesized by RIBOBIO. UWB1.289 cells were harvested at the confluence of 70–80%, followed by lipofectamine 2000 (RIBOBIO) to transfect 35 nM miRNA (NC miRNA as NC group) or 10 nM vector (empty vector as NC group) into 5 × 10⁵ cells in each well of a 6-well plate. Cells were all harvested at 24 h post-transfection to perform subsequent experiments. Control (C) cells in all groups were un-transfected cells.

UWB1.289 cells were harvested at 24 h after all transfections and counted. OC and non-tumor tissues were ground in liquid nitrogen. Total RNAs in these cells and tissue samples were extracted using RNAzol (Sigma-Aldrich, USA). It is noted that 85% ethanol was used to precipitate RNAs to harvest miRNAs.

After RNA extractions, all RNA samples were digested for 1 h at 37 °C with DNase I to remove genomic DNA. Following that, reverse transcriptions were performed using AMV Reverse Transcriptase (Promega Corporation) with total RNAs as a template. To measure the expression levels of AB209371 and Survivin mRNA, QuantiTect SYBR Green PCR Kit (Qiagen) was used to prepare all qPCR reaction mixtures with GAPDH as an endogenous control.

To measure the expression levels of miR-203, 3′-polyadenylation of mature miRNAs, reverse transcriptions, and qPCR assays were all performed using All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia). All steps were carried out according to the instructions from Genecopoeia. U6 was used as the endogenous control. All experiments were performed with three replicates and analyzed by the 2^-ΔΔCT method.

CCK-8 kit (Sigma-Aldrich, USA) was used to analyze the effects of different transfections on the proliferation of UWB1.289 cells. Briefly, UWB1.289 cells were collected at 24 h post-transfection, and the numbers were counted. Following that, 3 × 10⁴ UWB1.289 cells were mixture with 1 ml cell above culture medium (3% FBS) to make cell suspensions. The cell was cultured using a 96-well plate (100 μl in each well) at 37 °C. 10 μl CCK-8 solution was added into each well to monitor cell proliferation at 2 h before the termination of cell culture. After the end of cell culture, each well was supplemented with 10 μl DMSO, followed by the measurement of OD values (450 nm).

UWB1.289 cells were collected at 24 h post-transfection, and numbers were counted. Following that, total proteins in 5 × 10⁵ UWB1.289 cells were extracted using RIPA solution (Sangon, USA). After denaturing for 5 min in boiling water, electrophoresis was performed to separate different proteins using 10% SDS-PAGE gel. After that, proteins were transferred to PVDF membranes. Then the membrane was blocked for 2 h in PBS containing 5% non-fat milk at room temperature. Rabbit Survivin (1: 1200, ab469, Abcam) and GAPDH (1: 1000, ab37168, Abcam) primary antibodies were used to incubate the membranes for 15 h at 4 °C, followed by incubating by anti-goat IgG- HRP (1:1000; ab6721; Abcam) for 2 h at 24 °C. ECL substrate (ab65623, Abcam) was utilized to develop signals. Image J v1.48 software was used to process all signals.

Expression levels of AB209371 and Survivin mRNA in two types of tissues were measured by qPCR and compared by paired t-test. Comparing to the expression levels in non-tumor tissues, expression levels of AB209371 (Fig. 1a) and Survivin mRNA (Fig. 1b) were significantly higher in non-tumor tissues (p < 0.05). According to the system established by AJCC, 61 OC patients had 12, 23, and 26 cases diagnosed with clinical stage II-IV, respectively. Expression levels of AB209371 and Survivin mRNA in OC tissues were compared among three clinical stages by ANOVA (one-way) and Tukey test. It can be observed that mRNA expression levels of AB209371 (Fig. 1c) and Survivin (Fig. 1d) increased significantly by the elevation of clinical stages (p < 0.05).

Linear regression was used to analyze the correlations between AB209371 and Survivin mRNA. It can be observed that the expression level of AB209371 was significantly and positively correlated with the expression level of Survivin mRNA in OC tissues (Fig. 2a). In contrast, expression levels of AB209371 and Survivin mRNA were not significantly correlated in non-tumor tissues (Fig. 2b).

UWB1.289 cells were transfected with AB209371 and Survivin vectors as well as a miR-203 mimic. At 24 h post-transfection, expression levels of AB209371, Survivin mRNA, and miR-203 were measured. Comparing to C and NC groups, expression levels of AB209371, Survivin mRNA, and miR-203 were significantly increased (Fig. 3a, p < 0.05). Moreover, AB209371 over-expression led to up-regulated Survivin mRNA, while miR-203 over-expression led to down-regulated Survivin mRNA in OC cells (Fig. 3b, p < 0.05). Western blot was performed to evalulate the effects of AB209371 and miR-203 over-expression on the expression of Survivin at protein level, and the representative bands were shown in Fig. 3c. Analysis of the quantification data of western blot showed that, AB209371 over-expression led to up-regulated Survivin protein, while miR-203 over-expression led to down-regulated Survivin protein in OC cells (Fig. 3d, p < 0.05). Moreover, miR-203 over-expression attenuated the effects of AB209371 on Survivin expression (Fig. 3e, p < 0.05). In addition, AB209371 over-expression led to down-regulated miR-203, while miR-203 over-expression failed to affect AB209371 but down-regulated Survivin (Fig. 3c, p < 0.05).

The effects of AB209371, Survivin mRNA, and miR-203 over-expression on cell proliferation were analyzed by CCK-8 assay. Comparing to NC and C groups, AB209371 and Survivin over-expression resulted in the increased proliferation rate of OC cells. MiR-203 over-expression played the opposite role and attenuated the effects of AB209371 over-expression (Fig. 4, p < 0.05).