CircDDX17 targets miR-223-3p / RIP3 to regulate the proliferation and apoptosis of non-small cell lung cancer cells

Abstract

Objective: To explore the molecular mechanism of circDDX17 regulating the proliferation and apoptosis of non-small cell lung cancer cells by targeting the miR-223-3p/RIP3 molecular axis. Methods: The expression levels of circDDX17, miR-223-3p, and RIP3 in human normal lung epithelial cell lines BEAS-2B and non-small cell lung cancer cells H1299, A549, and H446 were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The plasmids of pcDNA, pcDNA-circDDX17, anti-miR-con, anti-miR-223-3p, pcDNA-circDDX17 and miR-con, pcDNA-circDDX17 and miR-223-3p mimics were transfected into H1299 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Flow cytometry was used to detect the cell cycle and cell apoptosis. Plate cloning experiment was used to detect cell proliferation ability. The dual luciferase report experiment was applied to verify the targeting relationship between miR-223-3p with circDDX17 and RIP3. Western blot was used to detect the protein expression of cyclinD1, CDK2, cleaved caspase-3 and Bax. Results: The expression levels of circDDX17 and RIP3 mRNA in H1299, A549, and H446 cells were significantly reduced (P<0.05), the expression level of miR-223-3p mRNA was significantly increased (P<0.05) compared with BEAS-2B. The cell viability [(69.46±4.68)%], the number of cell clones (83.49±7.86), the proportion of cells in S phase [(22.52±1.41) %], the protein expression levels of cyclinD1 and CDK2 in PCDNa-CircDDX17 group were lower than those in pcDNA group [(97.54±7.72)%, 205.03±13.37, (28.69±1.49)%, respectively, P<0.05], while the percentage of G0/G1 phase cells [(64.45±3.56)%], apoptosis rate [(18.36±1.63)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17 group were higher than those of pcDNA group [(51.33±2.76) % and (5.21±0.54) %, respectively, P<0.05]. The viability [(72.64±5.44)%], the number of cell clones (78.16±8.23), the proportion of S-stage cells [(21.34±1.59) %], the protein expression levels of CyclinD1 and CDK2 in anti-miR-223-3p group were lower than those in anti-miR-con group [(103.47±6.25)%, 169.32±14.53, (28.43±1.26)%, respectively, P<0.05]. Percentage of G0/G1 phase cells [(62.86±3.28)%], apoptosis rate [(14.64±1.67)%], the protein expression levels of cleaved caspase-3 and Bax in the anti-miR-223-3p group were higher than those of anti-miR-con group [(51.33±2.71)% and (4.83±0.39)%, respectively, P<0.05]. MiR-223-3p has complementary sites with circDDX17 or RIP3. The viability [(135.45±9.28)%], the number of cell clones (174.64±10.68), the proportion of S-phase cells [(26.39±2.25)%], the protein expression levels of cyclinD1 and CDK2 in pcDNA-circDDX17+miR-223-3p group were higher than those in pcDNA-circDDX17+miR-con group [(101.56±6.68)%, 107.65±7.62, (21.64±1.72)%, P<0.05]. Percentage of G0/G1 phase cells [(56.64±2.76)%], apoptosis rate [(8.34±0.76)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17+miR-223-3p group were lower than those of pcDNA-circDDX17+miR-con group [(64.03±3.48)% and (15.21±1.18)%, respectively, P<0.05]. Conclusion: circDDX17 could inhibit the proliferation and induce apoptosis of non-small cell lung cancer cells via targeting the miR-223-3p / RIP3 molecular axis.