LncRNA ANRIL Regulates Ovarian Cancer Progression and Tumor Stem Cell-Like Characteristics via miR-324-5p/Ran Axis

Abstract

Objective: Long non-coding RNA (lncRNA) ANRIL is emerging as a crucial role in ovarian cancer progression and prognosis. However, the precise molecular mechanism of ANRIL on ovarian cancer is not known. Thus, we aim to study the underlying mechanism of ANRIL on the action.

Methods: The MTT assay assessed cell viability. Cell migration and invasion were determined using the wound healing assay, Transwell migration, and invasion assay. The relationships of ANRIL, miR-324-5p, and RAN were evaluated using luciferase activity assay and RNA pull-down assay. Cancer stem cell was identified by flow cytometry. Sphere formation assay was conducted to determine the stem-like properties. Xenograft tumor was established to assess tumor growth in vivo. qRT-PCR and Western blot were used to detect gene expression.

Results: ANRIL was elevated while miR-324-5p was decreased in ovarian cancer tissues and cells. Besides, downregulated ANRIL enhanced miR-324-5p expression, and the luciferase reporting experiment and RNA pull-down assay showed the binding interaction between ANRIL and miR-324-5p. miR-324-5p directly targeted Ran and negatively modulated the expression of Ran. Besides, Ran was promoted by overexpressed ANRIL, which was reversed by overexpression of miR-324-5p. Furthermore, decreased ANRIL and increased miR-324-5p suppressed tumor growth, migration capacity, drug resistance, and alleviated stem-like characteristics in vitro and in vivo. Ran mediated the regulation of ANRIL on cell viability, stem-like properties, and drug resistance of ovarian cancer cells.

Conclusion: The ANRIL/miR-324-5p/Ran axis regulated ovarian cancer development, making the axis meaningful targets for ovarian cancer therapy.

Keywords: ANRIL; cancer stem cell; drug resistance; migration; ovarian cancer; tumorigenicity.

Figure 1

ANRIL is increased and miR-324-5p is decreased in ovarian cancer. (A) The levels of ANRIL and miR-324-5p were detected in human ovarian patient tissues by qRT-PCR. **P<0.01, versus the normal group. (B) ANRIL expression was detected in normal human ovarian surface epithelial cells (HOSEPiCs) and ovarian cancer cells SKOV3 and SKOV3/DDP by qRT-PCR. U6 was assigned as the control gene. **P<0.01, versus HOSEPiCs cells.

Figure 2

ANRIL directly binds to miR-324-5p in SKOV3 cells. (A) The ANRIL level was assessed in SKOV3 cells after si-ANRIL transfection by qRT-PCR. **P<0.01, versus si-NC group. (B) The miR-324-5p level was determined in SKOV3 cells transfected with si-ANRIL by qRT-PCR. U6 was assigned as the control gene. **P<0.01, versus si-NC group. (C) Predict targeting regions between ANRIL and miR-324-5p. The relations of ANRIL and miR-324-5p were verified using Luciferase reporter assay. SKOV3 cells were co-transfected with either 50 nM miR-324-5p mimics or NC oligos and 200 ng of pMIR -ANRIL-wt or pMIR -ANRIL-mut using Lipofectamine 3000. **P<0.01 versus miR-NC group. (D) RNA pull-down assay was performed to further demonstrate the binding relationship between ANRIL and miR-324-5p. **P<0.01 versus Bio-mt-miR-324-5p group. (E) The relationship between the expression of ANRIL and miR-324-5p was analyzed by Spearman correlation analysis.

Figure 3

miR-324-5p negatively regulates Ran expression in ovarian cancer cells. (A) The Ran mRNA level was measured in SKOV3 and SKOV3/DDP cells by qRT-PCR. **P<0.01, versus HOSEPiCs group. (B) The Ran protein level was determined in SKOV3 and SKOV3/DDP cells. **P<0.01, versus HOSEPiCs group. (C) The expression of miR-324-5p was detected in SKOV3 cells transfected with miR-324-5p mimics. U6 was assigned as the control gene. **P<0.01, versus miR-NC group. (D) qRT-PCR was used to detect the Ran mRNA level was detected in SKOV3 cells transfected with miR-324-5p mimics. **P<0.01, versus miR-NC group. (E and F) The protein expression of Ran in SKOV3 cells transfected with miR-324-5p mimics was evaluated by Western blot. **P<0.01, versus miR-NC group. (G) Predict targeting regions between RAN and miR-324-5p. The relations of Ran and miR-324-5p were verified using Luciferase reporter assay. **P<0.01, versus miR-NC group. (H) RNA pull-down assay was performed to further prove the target relationship between miR-324-5p and Ran. **P<0.01 versus Bio-mt-miR-324-5p group. (I) The relationship between the expression of miR-324-5p and Ran mRNA level was analyzed by Spearman correlation analysis. (J) The protein levels of Ran in SKOV3 cells with overexpression of ANRIL and miR-324-5p were determined by Western blot.

Figure 4

Downregulation of ANRIL and overexpression of miR-324-5p suppresses ovarian cancer cell growth, migration, and EMT. SKOV3 cells were divided into 4 groups: si-NC, si-ANRIL, miR-NC, and miR-324-5p mimics groups. (A) The knockout efficiency of 3 siRNAs against ANRIL was determined using the qRT-PCR. **P<0.01, versus si-NC group. (B) MTT assay was carried out to assess the cell viability of SKOV3 cells in different group. *P<0.05, **P<0.01 versus si-NC group or miR-NC group. (C) The SKOV3 cells migration was measured though conducting wound healing assays. **P<0.01, versus si-NC group or miR-NC group. (D) The SKOV3 cells invasion was assessed using Transwell invasion assays. **P<0.01, versus si-NC group or miR-NC group. (E) Wound healing assay, Transwell migration assay and Transwell invasion assay were performed to detect cell migration and invasion ability. (F) The protein levels of E-cadherin, Vimentin, N-cadherin, Snail and cyclinD1were determined in SKOV3 cells through performing Western blot. **P<0.01, versus si-NC group or miR-NC group. (G) MTT assay was performed to determine the cell viability of SKOV3 cells in different group with treatment of different concentration of cisplatin. **P<0.01 versus si-NC group or miR-NC group. (H) MTT assay was performed to determine the cell viability of SKOV3 cells in different group with treatment of different concentration of doxorubicin. **P<0.01 versus si-NC group or miR-NC group.

Figure 5

Decreased ANRIL and increased miR-324-5p alleviates stem-like properties in vitro and in vivo. Cells were divided into 4 groups: si-NC, si-ANRIL, miR-NC, and miR-324-5p mimics groups. (A) Sphere formation assay was conducted to assess the stem-like properties. (B) The number and size of spheres were counted. **P<0.01, versus si-NC group or miR-NC group. ns: P>0.05. (C) Flow cytometry for detection of cancer stem cell in SKOV3 cells was conducted. (D) The mRNA expression levels of cancer stem cell biomarkers were assessed by qRT-PCR. **P<0.01, versus si-NC group or miR-NC group. (E) Mice were divided into 4 groups: KD-NC, KD-ANRIL, OE-NC, and OE-miR-324-5p groups. The recombinant lentiviruses and the negative control (NC) lentivirus were used to mediate animal gene expression. (F) The volume of tumors in mice established using SKOV3 and A2780 cells was assessed. *P<0.05, **P<0.01 versus KD-ANRIL group or OE-NC group. (G) The mRNA expression levels of cancer stem cell biomarkers in tumor tissues of mice established using SKOV3 and A2780 cells were assessed by qRT-PCR. **P<0.01 versus KD-ANRIL group or OE-NC group.

Figure 6

Ran mediates the modulation process of ANRIL on stem-like characteristics in ovarian cancer cells. Cells were divided into 4 groups: si-NC, si-ANRIL, si-ANRIL + pcDNA, and si-ANRIL + pcDNA-Ran groups. (A) The mRNA expression levels of Ran and RhoA were detected. **P<0.01, compared with si-NC group or si-ANRIL + pcDNA group. (B) The protein levels of Ran and RhoA were determined. **P<0.01, versus si-NC group or si-ANRIL + pcDNA group. (C) MTT assay was conducted to assess the cell viability of SKOV3 cells in different group. *P<0.05, versus si-NC group or si-ANRIL + pcDNA group. (D and E) Sphere formation assay was conducted to assess the stem-like properties. **P<0.01, versus si-NC group or si-ANRIL + pcDNA group. ns: P>0.05. (F) The mRNA expression levels of cancer stem cell biomarkers were assessed by qRT-PCR. **P<0.01, versus si-NC group or si-ANRIL + pcDNA group. (G) MTT assay was conducted to assess the cell viability of SKOV3 cells in different group after treated by cisplatin and doxorubicin. *P<0.05, **P<0.01, versus si-NC group or si-ANRIL + pcDNA group.