Up-regulation of circ_LARP4 suppresses cell proliferation and migration in ovarian cancer by regulating miR-513b-5p/LARP4 axis

Abstract

Background: Ovarian cancer (OC) is a common fatal malignant tumor of female reproductive system worldwide. Growing studies have proofed that circular RNAs (circRNAs) engage in the regulation of various types of cancers. However, the underlying biological functions and effect mechanism of circular RNA_LARP4 (circ_LARP4) in OC have not been explored.

Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to detect the expression of circ_LARP4 in OC cells. The function of circ_LARP4 was measured by cell counting kit-8 (CCK-8), colony formation assay and transwell assay. RNA immunoprecipitation (RIP) assay and luciferase reporter assays assessed the binding correlation between miR-513b-5p and circ_LARP4 (or LARP4).

Results: The expression of circ_LARP4 in OC cells was much lower than that in human normal ovarian epithelial cells. Overexpressing circ_LARP4 impaired cell proliferation, invasion and migration abilities. Circ_LARP4 worked as a competing endogenous RNA (ceRNA) to sponge miR-513b-5p. Furthermore, LARP4 was indirectly modulated by circ_LARP4 as the downstream target of miR-513b-5p, as well as the host gene of circ_LARP4.

Conclusion: Circ_LARP4 could hamper cell proliferation and migration by sponging miR-513b-5p to regulate the expression of LARP4. This research may provide some referential value to OC treatment.

Keywords: LARP4; OC; ceRNA; circ_LARP4; miR-513b-5p.

Fig. 1

Circ_LARP4 expression is markedly down-regulated in OC cell lines. a Utilizing quantitative real-time polymerase chain reaction (qRT-PCR) to assess the expression of OC cell lines (SKOV3, A2780, SW626, OVCAR3, OVCAR4) and normal ovarian epithelial cells (HOSEpiC). b RNA expression of circ_LARP4 and LARP4 in two cells (SKOV3 and A2780) treated with actinomycin D was tested by qRT-PCR analysis. c The qRT-PCR analysis was used to the expression of circ_LARP4 and linear LARP4 with adding RNase R. d Circ_LARP4 expressions in cDNA and gDNA was measured by qRT-PCR analysis. All results were displayed as the mean ± SD. *P < 0.05, **P < 0.01

Fig. 2

Up-regulating circ_LARP4 hampers cell proliferation and migration of OC cell lines. a The qRT-PCR analysis was employed to determine the expression of circ_LARP4 with increasing circ_LARP4. b Cell counting kit-8 (CCK-8) assay was applied to detect cell viability under circ_LARP4 overexpression (OE-circ_LARP4). c Colony formation assay was carried out to estimate cell proliferation ability with OE-circ_LARP4. d Implementing western blot assay to detect apoptosis related protein level (Bax, Bcl-2, Cleaved caspase-3, Total caspase-3, Cleaved caspase-9 and Total caspase-9) under the condition of OE-circ_LARP4. e, f Transwell assay was conducted to test cell invasion and migration capacities. g Invasion and migration related protein levels (MMP2, MMP7 and MMP9) were measure by Western blot assay. All data were performed as the mean ± SD. **P < 0.01

Fig. 3

Circ_LARP4 up-regulates its host gene LARP4 after transcription. a, b Subcellular fractionation assay and fluorescence in situ hybridization (FISH) were performed to determine that circ_LARP4 was located in cytoplasm. c The expression and protein level of LARP4 after increasing circ_LARP4 were respectively determined by qRT-PCR analysis and western blot assays. d Luciferase reporter assay was used to test the luciferase activity of LARP4 promoter region. e qRT-PCR analysis and western blot assays were applied to analyze the mRNA and protein expression of LARP4 in different groups. f The proliferation ability of transfected cells was measured via colony formation. g Cell invasion capability in different groups was evaluated via transwell. h Western blot analysis of migration-related proteins was conducted. Results were revealed as the mean ± SD. **P < 0.01

Fig. 4

Circ_LARP4 works as a sponge for miR-513b-5p to modulate LARP4. a Three websites (PITA, microT and miRmap) were utilized to predict the potential miRNAs which could bind to LARP4. b Venn diagram was used to screen out the target miRNA which could bind to circ_LARP4 and LARP4 simultaneously. RNA pull down assay and qRT-PCR tested the expression of candidate RNAs. c RNA immunoprecipitation (RIP) assay was utilized to verify that circ_LARP4, miR-513b-5p and LARP4 co-existed in RNA-induced-silencing-complex (RISC). d The potential binding sites between circ_LARP4 and miR-513b-5p were depicted by starBase website. e The binding relationship between circ_LARP4 and miR-513b-5p was attested by luciferase reporter assay. f The binding sites between LARP4 and miR-513b-5p were acquired from starBase website. g Luciferase reporter assay was performed to detect the binding correlation between LARP4 and miR-513b-5p. All data were showed as the mean ± SD. **P < 0.01

Fig. 5

Circ_LARP4 inhibits the development of OC by regulating miR-513b-5p/LARP4 axis. a The qRT-PCR analysis and western blot assay were carried out to separately determine the expression and protein level of LARP4 in cells transfected with OE-circ_LARP4 and miR-513b-5p mimics. b, c CCK-8 assay and colony formation assay were performed to measure cell proliferation capacities. d Cell apoptosis-related protein levels were tested by western blot assay. e, f Transwell assay was carried out to estimate cell invasion and migration abilities. g The expression of proteins which were correlated to cell invasion and migration was measured by western blot analysis. All data were presented as the mean ± SD. **P < 0.01