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Long non-coding RNAs (lncRNAs) are important players in cancer development. LncRNA FGD5-AS1 has been reported as a potential oncogene in ovarian cancer (OC). The present paper focused on the action mechanism of FGD5-AS1 in OC. Clinical OC samples were collected for expression analyses of FGD5-AS1, RBBP6, and miR-107. The expression of FGD5-AS1, RBBP6, and miR-107 in OC cells was altered by transfection. OC cell proliferation was assessed by MTT and colony formation assays, and angiogenesis of human umbilical vein endothelial cells (HUVECs) cultured with OC cell supernatants by matrigel angiogenesis assay. The interactions among FGD5-AS1, miR-107, and RBBP6 were detected by luciferase reporter assay. FGD5-AS1 and RBBP6 were strongly expressed and miR-107 was poorly expressed in clinical OC samples and OC cell lines. FGD5-AS1 or RBBP6 overexpression in Hey and SKOV3 cells could potentiate OC cell proliferation and HUVEC angiogenesis, while FGD5-AS1 or RBBP6 knockdown in OC cells inhibited the above cellular processes. FGD5-AS1 targeted miR-107 to positively regulate RBBP6 expression. Additionally, miR-107 overexpression or RBBP6 knockdown in SKOV3 cells partially reversed the FGD5-AS1-dependent stimulation of OC cell proliferation and HUVEC angiogenesis. FGD5-AS1 may act as a promoter of OC via miR-107/RBBP6 axis.
Keywords: Angiogenesis, FGD5-AS1, miR-107, ovarian cancer, proliferation, RBBP6
How to cite this URL:
Zhang W, Shi J, Liu G. Abnormal expression of long non-coding RNA FGD5-AS1 affects the development of ovarian cancer through regulating miR-107/RBBP6 axis. Chin J Physiol [Epub ahead of print] [cited 2023 Mar 24]. Available from: https://www.cjphysiology.org/preprintarticle.asp?id=371727 |
| Introduction | |  |
Ovarian cancer (OC) ranks as the most frequent cause of cancer-related death in women worldwide,[1],[2],[3] and the 5-year survival rate of patients with OC is unsatisfactory.[4] Almost 300,000 new cases of OC were diagnosed worldwide in 2018, and the insufficiency of early diagnosis partially accounts for a high mortality (over 152,000 deaths).[5],[6] Moreover, the treatment of OC is complicated by its molecular and genetic heterogeneity. Thus, searching for potential biomarkers and mechanism-based research could be crucial for the advancement of OC therapy.
Long non-coding RNAs (lncRNAs) are RNAs with lengths of more than 200 nucleotides,[7],[8] playing vital roles in various human cancers. Dysregulation of lncRNAs has been demonstrated in OC. For instance, lncRNA HOST2 could regulate microRNA (miRNA) let-7b to exert its modulation on cell biological behaviors in epithelial OC.[9] FGD5-AS1, a novel lncRNA, has been reported as an oncogene in many cancers, including OC,[10],[11],[12] but the action mechanisms of FGD5-AS1 in OC have been poorly studied.
miRNAs are short non-coding RNAs that play important roles in gene regulation.[13] Recently, a series of studies have revealed a new mechanism by which lncRNAs function as “sponges” of miRNAs to regulate downstream mRNAs in OC and other malignant diseases.[14],[15],[16] FGD5-AS1 promotes non-small cell lung cancer cell proliferation by sponging hsa-miR-107,[11] but the FGD5-AS1/miR-107 axis has not been established in OC. Moreover, there are contradictory findings about the role of miR-107 in OC. The study by Liu et al. indicates that miR-107 is downregulated by lncRNA AFAP1-AS1 to facilitate OC cell development.[17] However, lncRNA DUBR reduces the malignant properties of OC cells by downregulating miR-107.[18] The aforementioned findings provoked us to dig into the interaction of FGD5-AS1 with miR-107 in OC.
The aims of our study were to detect the expression status of FGD5-AS1 and miR-107 in OC and explore their regulatory roles and mechanisms in OC progression in respect of proliferation and angiogenesis. This work may provide a new train of thought for illuminating the function of FGD5-AS1 in OC.
| Materials and Methods | | |
Clinical samples
Tumor tissues and adjacent normal tissues were obtained from 20 patients who were diagnosed with OC in the First Affiliated Hospital of Jinzhou Medical University from March 2021 to December 2021. The tumor tissues were the diseased tissues collected during surgical resection and confirmed by postoperative pathological examinations. All the patients did not receive other treatments before surgery. The samples were frozen at −80°C immediately after collection. The patients were informed of the use of the samples, which was also approved by the Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University (Approval No. 202044). All the procedures followed the Declaration of Helsinki.
Cell culture
Normal human ovarian surface epithelial cells (HOSEpiC), human embryonic kidney (HEK)-293T cells, human OC cell lines (Hey and SKOV3), and human umbilical vein endothelial cells (HUVECs) were from the American Type Culture Collection. HOSEpiC, Hey, and SKOV3 were cultured in RPMI-1640 in 5% CO2 at 37°C, while HEK-293T and HUVECs in DMEM; all media were from Thermo Fisher Scientific (MA, USA) and supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.
Cell transfection
OC cells (Hey and SKOV3) in the logarithmic phase of growth were transfected with 2 μg of pcDNA3.1, pcDNA3.1-FGD5-AS1, sh-NC, sh-FGD5-AS1, pcDNA3.1-RBBP6, sh-RBBP6, 100 nM of mimic NC, miR-107 mimic, inhibitor NC, and/or miR-107 inhibitor (RiboBio, Guangzhou, Guangdong, China) via Lipofectamine 2000 (Thermo Fisher Scientific) and correspondingly divided into the following groups: pcDNA3.1 group, pcDNA3.1-FGD5-AS1 group, sh-NC group, sh-FGD5-AS1 group, pcDNA3.1-RBBP6 group, sh-RBBP6 group, mimic NC group, miR-107 mimic group, inhibitor NC group, miR-107 inhibitor group, pcDNA3.1-FGD5-AS1 + mimic NC group, pcDNA3.1-FGD5-AS1 + miR-107 mimic group, pcDNA3.1-FGD5-AS1 + sh-NC group, and pcDNA3.1-FGD5-AS1 + sh-RBBP6 group.
MTT assay
Hey and SKOV3 cells were cultured in 96-well plates (100 μL/well, 104–105 cells), with 3 duplicates for each group, for 24, 48, 72, and 96 h and then incubated with MTT solution (20 μL/well, 5 mg/mL; Sigma, MO, USA) for 4 h. DMSO (150 μL/well) was then added to promote the dissolution of crystal for 10 min. Absorbance of each well was tested at 540 nm wavelength for 3 times in a microplate reader.
Colony formation assay
After being digested by 0.25% trypsin, monolayer Hey and SKOV3 cells in the logarithmic phase were made into single-cell suspensions with a 10% FBS medium and inoculated in culture dishes (containing 10 ml of culture medium prewarmed at 37°C) at densities of 50, 100, and 200 cells/dish for 2–3 weeks. When the colonies were visible to naked eyes, the cells were washed twice in PBS and fixed with 5 ml of acetic acid/methanol (1:3, by Vol.). After 15 min of fixation, the acetic acid/methanol was removed and the cells were stained with Giemsa for 10–30 min. The staining solution was washed away and the culture dishes were dried. The number of colonies with more than 10 cells was counted directly with naked eyes or under an optical microscope at a low magnification.
Matrigel angiogenesis assay
Matrigel (BD, NJ, USA) was thawed overnight at 4°C in a refrigerator. The supernatants of Hey and SKOV3 cells were centrifuged at 1500 r/min for 10 min and mixed with DMEM at a ratio of 1:1. Matrigel coating was carried out in an ice box. Matrigel was diluted with DMEM at a ratio of 1:1 and added to 24-well plates (80 μL/well). Next, the plates were placed in an incubator (37°C) for 1 h and each well was then plated with 2 × 104 HUVECs and added to the prepared medium. The tube formation was observed under a microscope.
Reverse transcription–polymerase chain reaction
Tissues, HOSEpiC, Hey, and SKOV3 cells were dissolved in TRIzol (16096020; Thermo Fisher Scientific, MA, USA) for RNA extraction. After quantification by NanoDrop 2000 (Waltham, MA, USA), RNA was reverse transcribed into cDNA. The PCR system was prepared using a fluorescence quantitative PCR kit (Takara, Dalian, China), and the assay was performed on a ABI7500 PCR instrument (Applied Biosystems Inc., Shanghai, China) with the conditions: predenaturation at 95°C (10 min) and 40 cycles of denaturation at 95°C (10 s), annealing at 60°C (20 s), and extension at 72°C (34 s). The expression levels of FGD5-AS1, miR-107, and RBBP6 were detected using corresponding primers (GENEWIZ Inc., Beijing, China) [Table 1], with GAPDH (for lncRNA and mRNA) and U6 (for miRNA) as internal references. The 2-ΔΔCt method was adopted for data analysis.[19] ΔΔCt = [Ct (target gene) – Ct (reference gene)]experimental group– [Ct (target gene)– Ct (reference gene)]control group. | Table 1: Primer sequence for reverse transcription–polymerase chain reaction to determine the expression levels of FGD5-AS1, miR-107, and RBBP6
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Western blotting
Tissues, HOSEpiC, Hey, and SKOV3 cells were lysed with a RIPA lysis buffer on ice for 30 min. The protein samples were collected by centrifugation and subpacked in 0.5 mL centrifuge tubes for storage at −20°C or quantification with BCA kits (Sigma). After denaturation in 6× SDS loading buffer, the proteins were separated by electrophoresis and transferred onto a membrane in a transfer buffer (precooled at 4°C) for 1.5 h. After 5% nonfat milk-TBST blocking (1 h), the membrane was probed with TBST-diluted primary antibodies of GAPDH (1:10,000, ab181602) or RBBP6 (1:1000, ab237514) (Abcam, MA, USA) at 4°C overnight and later re-probed with goat anti-rabbit immunoglobulin G (1:5000, Beijing ComWin Biotech Co., Ltd., China) at room temperature for 2 h. The color of the bands was developed for detecting the protein expression.
Dual-luciferase reporter assay
The binding sites for miR-107 in FGD5-AS1 and RBBP6 were predicted by the online software starBase. According to the prediction, the mutant sequence and wild sequence of the binding site in FGD5-AS1 and RBBP6 were designed. The mutant and wild sequence fragments were cloned and conjugated to luciferase reporter vectors (Promega, WI, USA), which were designated as mutant-type FGD5-AS1 (MT-FGD5-AS1), WT-FGD5-AS1, MT-RBBP6, and WT-RBBP6, respectively, and then separately co-transfected with miR-107 mimic or miR-107 inhibitor into HEK-293T cells. After 48 h, the luminescence intensity was measured using a luciferase reporter assay kit (Promega).
Statistical analysis
Data were analyzed utilizing SPSS 18.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6.0. Measurement data were expressed as mean ± standard deviation. Two groups were compared using t-test, otherwise utilizing one-way analysis of variance. P < 0.05 was regarded as statistically significant.
| Results | | |
Overexpression of FGD5-AS1 and RBBP6 in ovarian cancer
FGD5-AS1, a novel lncRNA, has been reported as an oncogene in many malignancies, including OC.[10],[11],[12] RBBP6 is also a potential promoter of OC,[20] but there is no report about the relationship between FGD5-AS1 and RBBP6. The lncRNA-miRNA-mRNA network has been established for many years as an important working mechanism of lncRNAs. Therefore, this study aimed to determine whether FGD5-AS1 regulates RBBP6 in OC through a miRNA. FGD5-AS1 promotes non-small cell lung cancer cell proliferation by sponging hsa-miR-107.[11] miR-107 is downregulated by lncRNA AFAP1-AS1 to facilitate OC cell development,[17] but the FGD5-AS1/miR-107 axis has not been established in OC. The starBase database was searched for downstream targets of miR-107 and predicted binding sites in RBBP6[21] mRNA for miR-107. Therefore, FGD5-AS1 was assumed to promote OC progression via the miR-107/RBBP6 axis. First, reverse transcription–polymerase chain reaction (RT-PCR) and Western blotting experiments were designed to determine the expression of FGD5-AS1 and RBBP6 in OC tissues and cells.
RT-PCR for detection of FGD5-AS1 expression revealed that FGD5-AS1 expression was higher in OC tissues than in adjacent normal tissues [[Figure 1]a, P < 0.001]. RT-PCR and Western blotting for detection of RBBP6 mRNA and protein showed that RBBP6 was also overexpressed in OC tissues [[Figure 1]b, [Figure 1]c, P < 0.001]. In addition, Pearson correlation analysis demonstrated a positive correlation between FGD5-AS1 expression and RBBP6 expression in OC tissues [[Figure 1]d, P < 0.05]. Compared to HOSEpiC, OC cell lines Hey and SKOV3 also showed higher expression of FGD5-AS1 [[Figure 1]e, P < 0.001] and RBBP6 mRNA and protein [[Figure 1]f, [Figure 1]g, P < 0.01]. These results suggest that the abnormal expression of FGD5-AS1 and RBBP6 may be implicated in the progression of OC. | Figure 1: Aberrant expression of FGD5-AS1 and RBBP6 in OC. RT-PCR was used to detect the expression of FGD5-AS1 in clinical OC samples and normal tissues (n = 20) (a). RT-PCR (b) and Western blotting (c) were used to detect the expression of RBBP6 mRNA and protein in clinical OC samples and normal tissues (n = 20). Pearson correlation analysis was used to determine the correlation between FGD5-AS1 expression and RBBP6 expression in OC tissues (d). RT-PCR was used to detect the expression of FGD5-AS1 in HOSEpiC, Hey, and SKOV3 cells (e). RT-PCR (f) and Western blotting (g) were used to detect the levels of RBBP6 mRNA and protein in HOSEpiC, Hey, and SKOV3 cells. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001, versus paracancer or HOSEpiC group. OC: Ovarian cancer, RT-PCR: Reverse transcription–polymerase chain reaction, HOSEpiC: Human ovarian surface epithelial cells, SD: Standard deviation.
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FGD5-AS1 drives ovarian cancer cell proliferation and angiogenesis
Upregulation of FGD5-AS1 was found in OC tissues and cells, suggesting that it might promote the progression of OC. To decipher the effect of FGD5-AS1 on OC cells, FGD5-AS1 was overexpressed or silenced in Hey and SKOV3 cells by transfection with pcDNA3.1-FGD5-AS1 or sh-FGD5-AS1, respectively. After transfection, the OC cells were subjected to MTT and colony formation assays for assessing cell proliferation. HUVECs were incubated with the supernatants of Hey and SKOV3 cells from pcDNA3.1-FGD5-AS1, pcDNA3.1, sh-FGD5-AS1, and sh-NC groups. A matrigel angiogenesis assay was performed to evaluate the angiogenic ability of these HUVECs.
RT-PCR to determine the transfection effectiveness revealed that FGD5-AS1 was upregulated in pcDNA3.1-FGD5-AS1 group (vs. pcDNA3.1 group) [[Figure 2]a, P < 0.001]; however, FGD5-AS1 exhibited very low expression in sh-FGD5-AS1 group, compared to that in sh-NC group [[Figure 2]a, P < 0.01]. pcDNA3.1-FGD5-AS1 group had stronger proliferative ability and increased cell colonies (vs. pcDNA3.1 group). Reversely, sh-FGD5-AS1 group had weaker proliferative ability and decreased cell colonies as compared with sh-NC group [[Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, P < 0.01]. The in vitro vascularization was increased in pcDNA3.1-FGD5-AS1 group (vs. pcDNA3.1 group) and decreased in sh-FGD5-AS1 group (vs. sh-NC group) [[Figure 2]f, [Figure 2]g, P < 0.01]. Altogether, FGD5-AS1 could facilitate OC cell proliferation and HUVEC angiogenesis. | Figure 2: FGD5-AS1 promotes OC cell proliferation and angiogenesis. After pcDNA3.1, pcDNA3.1-FGD5-AS1, sh-NC, or sh-FGD5-AS1 plasmid was transfected into Hey and SKOV3 cells, RT-PCR was used to detect FGD5-AS1 expression (a). The proliferation ability of Hey and SKOV3 cells was detected by MTT assay (b-c). The number of Hey and SKOV3 cell colonies was counted (d-e). Vascularization was monitored by matrigel angiogenesis assay (f-g). Data were presented as mean ± SD. **P < 0.01, ***P < 0.001; OC, Ovarian cancer, RT-PCR: Reverse transcription–polymerase chain reaction, SD: Standard deviation.
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RBBP6 stimulates ovarian cancer cell proliferation and angiogenesis
Upregulation of RBBP6 was found in OC tissues and cells, suggesting that it might promote the progression of OC. Over- or underexpression of RBBP6 was induced by transfection with pcDNA3.1-RBBP6 or sh-RBBP6 to evaluate the effects of RBBP6 on OC cells. After transfection, the OC cells were subjected to MTT and colony formation assays for assessing cell proliferation. HUVECs were incubated with the supernatants of Hey and SKOV3 cells from pcDNA3.1-RBBP6, pcDNA3.1, sh-RBBP6, and sh-NC groups. A matrigel angiogenesis assay was performed to evaluate the angiogenic ability of these HUVECs.
RT-PCR and Western blot for determining the transfection effectiveness revealed that pcDNA3.1-RBBP6 group had increased levels of RBBP6 mRNA and protein in contrast to pcDNA3.1 group. Opposite expression patterns of RBBP6 were detected in sh-RBBP6 group (vs. sh-NC group) [[Figure 3]a, [Figure 3]b, P < 0.05]. The aforementioned findings showed satisfactory transfection efficiency of RBBP6 overexpression and silencing plasmids in both Hey and SKOV3 cells. The proliferation of OC cells and the number of cell colonies were elevated in pcDNA3.1-RBBP6 group (vs. pcDNA3.1 group) and decreased in sh-RBBP6 group (vs. sh-NC group) [[Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 3]f, P < 0.01], indicating that RBBP6 could stimulate Hey and SKOV3 cell proliferation. Additionally, pcDNA3.1-RBBP6 group had more vessel tubes than pcDNA3.1 group, while sh-RBBP6 group had less vessel tubes than sh-NC group [[Figure 3]g, [Figure 3]h, P < 0.01]. These results suggest that RBBP6 is conducive to OC cell proliferation and angiogenesis. | Figure 3: RBBP6 augments OC cell proliferation and angiogenesis. pcDNA3.1, pcDNA3.1-RBBP6, sh-NC, or sh-RBBP6 plasmid was transfected into Hey and SKOV3 cells, and the levels of RBBP6 were evaluated by RT-PCR and Western blot (a-b). MTT assay was conducted to determine the proliferation ability of Hey and SKOV3 cells (c-d). Colony formation of Hey and SKOV3 cells were assessed (e-f). Vascularization was tested by matrigel angiogenesis assay (g-h). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; OC, Ovarian cancer, RT-PCR: Reverse transcription–polymerase chain reaction, SD: Standard deviation.
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FGD5-AS1 targets miR-107 to positively regulate RBBP6
FGD5-AS1 and RBBP6 were both upregulated in OC tissues and cells. As a functional lncRNA, FGD5-AS1 may regulate the expression of RBBP6 based on a competitive endogenous RNA network, in which lncRNAs compete with protein-coding mRNAs for binding to miRNAs.[22] We found that there were binding sites for miR-107 in FGD5-AS1 and RBBP6 by searching DIANA and starBase databases. Hence, we speculated that FGD5-AS1 could target miR-107 to regulate RBBP6 expression, thereby modulating OC cell proliferation and angiogenesis. The expression of FGD5-AS1 or miR-107 was altered in OC cells to evaluate its effect on the expression of miR-107 or RBBP6, respectively. Dual-luciferase reporter assays were performed to determine the relationship between FGD5-AS1 and miR-107 or between miR-107 and RBBP6.
First, the expression of miR-107 in clinical OC samples and normal tissues was detected. RT-PCR showed that miR-107 was expressed at a low level in OC tissues [[Figure 4]a, P < 0.001]. Moreover, Pearson correlation analysis demonstrated a negative correlation between miR-107 expression and FGD5-AS1 expression in OC tissues [[Figure 4]b, P < 0.05]. Downregulation of miR-107 was also evidenced in Hey and SKOV3 cells [[Figure 4]c, P < 0.01]. Next, FGD5-AS1 was overexpressed or underexpressed in Hey and SKOV3 cells. RT-PCR assay for detecting miR-107 showed that miR-107 expression was inhibited in pcDNA3.1-FGD5-AS1 group (vs. pcDNA3.1 group) and stimulated in sh-FGD5-AS1 group (vs. sh-NC group) [[Figure 4]d, P < 0.001]. The predicted binding site and mutant binding site in FGD5-AS1 for miR-107 are shown in [Figure 4]e. To verify their targeting relationship, luciferase reporter plasmids containing wild-type FGD5-AS1 (WT-FGD5-AS1) and MT-FGD5-AS1 were generated. Transfection of WT-FGD5-AS1 plasmid and miR-107 mimic significantly restricted the luciferase activity, while transfection of WT-FGD5-AS1 plasmid and miR-107 inhibitor obviously elevated the luciferase activity (vs. mimic NC or inhibitor NC) [[Figure 4]f, P < 0.01]. However, the luciferase activity remained unaffected after co-transfection of MT-FGD5-AS1 with miR-107 mimic or miR-107 inhibitor [Figure 4]f. These results indicate that FGD5-AS1 interacts directly with miR-107. | Figure 4: FGD5-AS1 targets miR-107 to positively regulate RBBP6. RT-PCR was used to detect the expression of miR-107 in clinical OC samples and normal tissues (n = 20) (a). Pearson correlation analysis was used to determine the correlation between FGD5-AS1 expression and miR-107 expression in OC tissues (b). RT-PCR was used to detect the expression of miR-107 in HOSEpiC, Hey, and SKOV3 cells (c). After pcDNA3.1, pcDNA3.1-FGD5-AS1, sh-NC, or sh-FGD5-AS1 plasmid was transfected into Hey and SKOV3 cells, RT-PCR was used to detect the content of miR-107 (d). The miR-107 binding site in FGD5-AS1 was predicted by starBase and a mutant binding site was designed (e). After mimic NC, miR-107 mimic, inhibitor NC, or miR-107 inhibitor was co-transfected with WT-FGD5-AS1 or MT-FGD5-AS1 into HEK-293T cells, the luciferase activity was determined by dual-luciferase reporter assay (f). After mimic NC, miR-107 mimic, inhibitor NC, or miR-107 inhibitor was transfected into Hey and SKOV3 cells, RT-PCR was utilized for analysis of miR-107 expression (g). The levels of RBBP6 were estimated using RT-PCR and Western blot (h-i). Pearson correlation analysis was used to determine the correlation between RBBP6 expression and miR-107 expression in OC tissues (j). Online software starBase was used to predict the binding site for miR-107 in RBBP6 mRNA, and mutations in the binding site were designed (k). After miR-107 mimic, miR-107 inhibitor, mimic NC, or inhibitor NC was transfected with WT-RBBP6 or MT-RBBP6 into HEK-293T cells, a luciferase reporter assay kit was applied to the measurement of luciferase activity (l). Data were presented as mean ± SD. **P < 0.01, ***P < 0.001. RT-PCR: Reverse transcription–polymerase chain reaction, HOSEpiC: Human ovarian surface epithelial cells, SD: Standard deviation, MT-FGD5-AS1: Mutant-type FGD5-AS1.
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Overexpression or inhibition of miR-107 was induced in Hey and SKOV3 cells by transfection with miR-107 mimic or miR-107 inhibitor, respectively [[Figure 4]g, P < 0.01]. Noteworthy, as reflected by RT-PCR and Western blotting, overexpression of miR-107 inhibited the expression of RBBP6 mRNA and protein, whereas inhibition of miR-107 promoted that [[Figure 4]h, [Figure 4]i, P < 0.01]. These data indicate that miR-107 inversely regulates RBBP6 expression. Additionally, Pearson correlation analysis revealed a negative correlation between miR-107 expression and RBBP6 expression in OC tissues [[Figure 4]j, P < 0.05]. Next, the online software starBase predicted that there was a specific binding site in RBBP6 3'-UTR for miR-107 [Figure 4]k. To verify this, we constructed luciferase reporter plasmids containing wild-type RBBP6 3'-UTR (WT-RBBP6) and mutant-type RBBP6 3'-UTR (MT-RBBP6). Transfection of miR-107 mimic and WT-RBBP6 plasmid effectively lowered the luciferase activity, while transfection of miR-107 inhibitor and WT-RBBP6 promoted the luciferase activity (P < 0.01). However, there was no significant change in luciferase activity when MT-RBBP6 was co-transfected with miR-107 mimic or inhibitor [Figure 4]l. Hence, RBBP6 mRNA interacts directly with miR-107.
FGD5-AS1 facilitates ovarian cancer cell proliferation and angiogenesis via miR-107/RBBP6 axis
To further investigate whether FGD5-AS1 promoted OC cell proliferation and angiogenesis by regulating miR-107/RBBP6 axis, pcDNA3.1-FGD5-AS1, miR-107 mimic, and sh-RBBP6 were transfected/co-transfected into SKOV3 cells. MTT and colony formation assays were conducted to assess the proliferation of SKOV3 cells. HUVECs were incubated with the supernatants of the transfected SKOV3 cells. A matrigel angiogenesis assay was performed to evaluate the angiogenic ability of HUVECs.
The proliferation and colony formation of SKOV3 cells were promoted in pcDNA3.1-FGD5-AS1 group (vs. pcDNA3.1 group) but suppressed dramatically in pcDNA3.1-FGD5-AS1 + miR-107 mimic group (vs. pcDNA3.1-FGD5-AS1 + mimic NC group) and pcDNA3.1-FGD5-AS1 + sh-RBBP6 group (vs. pcDNA3.1-FGD5-AS1 + sh-NC group) [[Figure 5]a, [Figure 5]b, P < 0.05]. These results indicate that miR-107 mimic and sh-RBBP6 partially abolish the pro-proliferative effect of pcDNA3.1-FGD5-AS1 on OC cells. Western blotting revealed that pcDNA3.1-FGD5-AS1 group had an increased protein level of RBBP6 (vs. pcDNA3.1 group), whereas pcDNA3.1-FGD5-AS1 + miR-107 mimic and pcDNA3.1-FGD5-AS1 + sh-RBBP6 groups had decreased protein levels of RBBP6 in comparison with their control groups [[Figure 5]c, P < 0.01]. Matrigel angiogenesis assay revealed that the in vitro endothelial tube formation was stimulated in pcDNA3.1-FGD5-AS1 group (vs. pcDNA3.1 group) but suppressed in pcDNA3.1-FGD5-AS1 + miR-107 mimic and pcDNA3.1-FGD5-AS1 + sh-RBBP6 groups in contrast to their negative controls [[Figure 5]d, P < 0.01], indicating that miR-107 mimic and RBBP6 depletion could partially impede the pro-angiogenic action of FGD5-AS1. Altogether, FGD5-AS1 modulates the miR-107/RBBP6 axis to promote OC cell proliferation and angiogenesis. | Figure 5: FGD5-AS1 potentiates OC cell proliferation and angiogenesis via miR-107/RBBP6 axis. pcDNA3.1-FGD5-AS1, miR-107 mimic, sh-RBBP6, and their negative control plasmids were transfected/co-transfected into SKOV3 cells. The proliferation ability of SKOV3 cells was evaluated by MTT assay (a). The number of colonies formed by SKOV3 cells was counted (b). The protein levels of RBBP6 were monitored by Western blotting (c). Matrigel angiogenesis assay was employed to detect vascularization (d). Data were presented as mean ± SD. *P < 0.05, **P < 0.01; OC, Ovarian cancer, SD: Standard deviation.
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| Discussion | | |
Here, in this study, upregulation of FGD5-AS1 was demonstrated to have associations with OC progression. Knockdown of FGD5-AS1 efficiently inhibited OC cell proliferation, accompanied by inhibition of tumor angiogenesis in vitro. Mechanistic investigation indicates that the miR-107/RBBP6 axis plays a crucial role in FGD5-AS1-mediated tumor proliferation and angiogenesis in OC.
Abnormally expressed lncRNAs can act as oncogenes or tumor suppressor genes in the tumorigenesis of multiple cancers, including pancreatic cancer, gastric cancer, colorectal carcinoma, and OC.[23],[24],[25],[26],[27] FGD5-AS1 is a relatively new lncRNA and has been reported to participate in clear cell kidney carcinoma and periodontitis.[28],[29] Nevertheless, the expression and role of FGD5-AS1 in the tumorigenesis of OC have not been fully determined. Herein, RT-PCR analysis demonstrated that FGD5-AS1 was upregulated in OC tissues and cells. Furthermore, gain or loss of FGD5-AS1 function was performed to evaluate the effect of FGD5-AS1 over the proliferation ability of OC cells. Transfection of FGD5-AS1 overexpression plasmid raised the number of cell colonies; inhibition of FGD5-AS1 brought about inverse results. In non-small cell lung cancer, upregulated FGD5-AS1 acted as a tumor promoter through accelerating cancer cell proliferation, invasion, and autophagy.[30] These data imply that FGD5-AS1 could potentiate tumor cell proliferation. Moreover, in the present study, silencing FGD5-AS1 significantly suppressed tumor angiogenesis in vitro. However, the inner molecular mechanism of the effect of FGD5-AS1 in OC cells is still unclear.
RBBP6 is a 250kD protein shown to be upregulated in numerous cancers,[31] which was consistent with our detection of RBBP6 in OC. Then, both over- and underexpressions of RBBP6 were conducted to measure its effects on OC cells and HUVECs. MTT, colony formation, and matrigel angiogenesis assays demonstrated a pro-proliferative and pro-angiogenic role of RBBP6 in OC. Previously, the mechanism of RBBP6 in other types of cancers has been widely studied. For instance, RBBP6 promotes epithelial-mesenchymal transition and metastasis in colorectal cancer mainly through regulating ubiquitination and degradation of IκBα to enhance p65 nuclear translocation and activate NF-κB pathway.[32] In cervical cancer, RBBP6 drives the biological processes of cancer cells through activating JNK signaling pathway, indicating the potential of RBBP6 inhibition for cancer treatment.[33] Our results provide solid evidence and broaden our understanding concerning the role of RBBP6 in OC progression.
Mechanistically, lncRNAs can regulate tumor progression by sequestering miRNAs to upregulate downstream target mRNAs.[34],[35],[36] In the present research, miR-107 expression in OC cells declined following transfection of FGD5-AS1 overexpression plasmid, whereas miR-107 expression was elevated when OC cells were transfected with FGD5-AS1 silencing vector. The luciferase reporter assay showed that FGD5-AS1 interacted directly with miR-107. Meanwhile, transfection of miR-107 mimic could inhibit the mRNA and protein levels of RBBP6, implying that miR-107 inversely regulates RBBP6 expression. Furthermore, the luciferase reporter assay indicated that miR-107 interacted directly with RBBP6 at the 3'-UTR. The regulation of FGD5-AS1 on miR-107/RBBP6 axis in promoting OC cell proliferation and angiogenesis was further verified in vitro.
| Conclusion | | |
This study reports the positive role of lncRNA FGD5-AS1 in tumor proliferation and angiogenesis. Mechanistic investigation indicates that FGD5-AS1 boosts tumor cell proliferation and endothelial angiogenesis by sponging miR-107 to upregulate RBBP6 expression in OC. This study offers a novel understanding of the pathogenesis of OC and also supports that lncRNA FGD5-AS1 can be a therapeutic target for OC.
Data availability statement
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Guoyan Liu,
Department of Obstetrics and Gynecology, Tianjin Medical University Cancer Hospital, No. 1 Binshui Ave., Hexi District, Tianjin 300060
China
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/cjop.CJOP-D-22-00084
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1] |
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