SOX2 Overlapping Transcript (SOX2-OT) Enhances the Lung Cancer Malignancy Through Interaction with miR-194-5p/ SOX5 Axis

Abstract

Background: Lung cancer is one of the most common types of cancer and a leading cause of cancer-related deaths worldwide. Therefore, it is useful to know the biomarkers involved in the malignancy of lung cancer.

Objectives: This study aimed to show that SOX2-OT as a long non-coding RNA (IncRNA) regulates gene expression via the SOX2-OT/miR-194-5p/SOX5 axis molecular pathway in lung cancer.

Materials and methods: A549 cells transfected with siRNA-SOX2-OT and the expression of SOX2-OT and miR-194-5p genes were analyzed by real-time PCR before and after transfection. In addition, the expression of the B-catenin, MMP9, phosphorylated and activated STAT3 (p-STAT3), SOX5, and VEGF proteins before and after transfection was investigated by Western blotting.

Results: After using siRNA-SOX2-OT, an increase in the expression of miR-194-5p and a decrease in the expression of B-catenin, SOX5, p-STAT3 activated STAT3, VEGF, and MMP9 proteins was observed.

Conclusions: According to the results of the present study, an increase in SOX2-OT in lung cancer seems to stimulate the expression of beta-catenin, SOX5, MMP9, and VEGF thus support the malignancy of lung cancer cells.

Keywords: MMP9; SOX5; VEGF; p-STAT3; β-catenin; SOX2-OT; miRNA-194-5P.

Figure 1

The A549 cells before and after si-SOX2-OT transfection. magnification, 20X.

Figure 2

The Expression assessment of SOX2-OT in A549 cells before and after si-SOX2-OT transfection. The control sample is the untreated A549 cells; siRNA 1 50nM are A549 cells treated with siRNA 1, 50 nM only, and the third group are A549 cells transfected with siRNA 2, 50 nM. The final column shows A549 cells transfected with 100 nM of siRNA2. The A549 siRNA 1 (50nM) group was not significantly different from the A549 control group (P > 0.05). SOX2-OT expression exhibited a dramatic decrease (to about 0.196) in the A549 siRNA 2 (50nM) group (* P < 0.05). The cells with siRNA2 (100nM) transfection suppressed SOX2-OT expression significantly (to about 0.073) 2 days after transfection (** P < 0.01). The error bar indicates the mean±SE. B-actin gene expression was used as the housekeeping gene control to normalize Cq values.

Figure 3

The Expression assessment of SOX2 in A549 cells before and after si-SOX2-OT transfection. The expression of the SOX2 gene is reduced with the decrease in the SOX2-OT gene expression compared to the control group. SOX2 expression is 0.097 (** P < 0.01), 0.050 (** P < 0.01), and 0.025 (*** P < 0.001) for A549 treated with siRNA1 50 nM, siRNA2 50 nM, and siRNA2 100 nM, respectively. The error bar indicates the mean±SE. -actin gene expression was used as the housekeeping gene control to normalize Cq values.

Figure 4

The Expression assessment of miR-194-5p in A549 cells before and after si-SOX2-OT transfection. The miR-194-5p gene expression significantly increased (4.17 fold) (* P < 0.05) after the decrease in SOX2-OT gene expression (** P < 0.01) compared to the control groups. The error bar indicates the mean±SE. The RNU48 gene expression was used as the housekeeping gene control to normalize Cq values.

Figure 5

Western blot of β-actin, β-catenin, SOX5, p-STAT3, VEGF, and MMP9 proteins before and after si-SOX2-OT transfection. Western blotting showed a decrease in the intensity of the band and, as a result, a decrease in the expression of all the studied proteins after si-SOX2-OT transfection (50nM from each siRNAs). β-actin was used as a reference protein.

Figure 6

Proposed pathway for the SOX2-OT molecule in lung cancer. It seems that SOX2-OT increases the expression of SOX5 by inhibiting miR-194-5p, and SOX5 increases VEGF and MMP9 by activating STAT3. On the other hand, SOX2-OT increases the expression of beta-catenin.