MALAT1 was significantly up-regulated and miR-655-3p was remarkably down-regulated in RB
Firstly, we measured the levels of MALAT1 and miR-655-3p in thirty RB tissues and eighteen normal globe tissues. As presented in Fig. 1A, the level of MALAT1 was apparently elevated in RB tissues compared to that in normal tissues. Also, the level of miR-655-3p was distinctly decreased in RB tissues (Fig. 1B). Besides, the scatter diagram exhibited that the level of miR-655-3p in RB was negatively linear correlated with the level of MALAT1 (Fig. 1C). These data indicated that MALAT1 was highly expressed, while miR-655-3p was lowly expressed in RB.
MALAT1 silencing constrained cell proliferation, metastasis, and EMT but promoted apoptosis in Y79 and WERI-Rb-1 cells
In order to investigate the functions of MALAT1 in RB, we firstly measured the level of MALAT1 in RB cells. The results displayed that MALAT1 was obviously increased in RB cell lines (Y79 and WERI-Rb-1) in contrast to that in retinal pigment epithelial cell line ARPE19 (Fig. 2A). The qRT-PCR results confirmed the knockdown efficiency, indicated by the dramatical reduction of MALAT1 in Y79 and WERI-Rb-1 cells transfected with si-MALAT1 (Fig. 2B). Moreover, the transfection of si-MALAT1 resulted in the marked decline of cell viability in Y79 and WERI-Rb-1 cells in comparison with that in si-con group (Fig. 2C-D). While the apoptotic rate was notably facilitated in Y79 and WERI-Rb-1 cells transfected with si-MALAT1 (Fig. 2E). Since p21, CyclinD1 as proliferation-related factors and Bcl-2, cleaved-casp-3 as apoptosis-associated markers, the protein levels of them were tested in Y79 and WERI-Rb-1 cells. The Western blotassay exhibited that the protein levels of p21 and cleaved-casp-3 were strikingly accelerated, but the protein levels of CyclinD1, Bcl-2 were conspicuously decreased in si-MALAT1-transfected siY79 and WERI-Rb-1 cells (Fig. 2F-G). Besides, the introduction of si-MALAT1 notably reduced the migrated and invaded abilities of Y79 and WERI-Rb-1 cells compared with that in si-con group (Fig. 2H-I). Given that E-cadherin, N-cadherin, and Vimentin as epithelial-mesenchymal transition (EMT) protein markers, the levels of these protein were detected. As exhibited in Fig. 2J-K, the protein level of E-cadherin was apparently elevated, while N-cadherin and Vimentin were strikingly decreased in si-MALAT1 group in comparison with that in si-con group. Taken together, the depletion of MALAT1 inhibited cell proliferation, migration, invasion, and EMT but facilitated apoptosis in Y79 and WERI-Rb-1 cells.
MiR-655-3p overexpression suppressed cell proliferation, migration, invasion, and EMT while boosted apoptosis in Y79 and WERI-Rb-1 cells
Meanwhile, the level of miR-655-3p was evidently down-regulated in Y79 and WERI-Rb-1 cells (Fig. 3A). The level of miR-655-3p was extremely enhanced in Y79 and WERI-Rb-1 cells transfected with miR-655-3p mimics, affirming the transfection efficiency (Fig. 3B). Furthermore, CCK8 assay indicated that cell viability, migrated and invaded abilitieswere significantly declined in Y79 and WERI-Rb-1 cells transfected with miR-655-3p related to that in miR-con group (Fig. 3C-D and H-I). The transfection of miR-655-3p greatly augmented apoptotic rate compared to that in miR-con group (Fig .3E). As shown in Fig. 3F-G, the introduction of miR-655-3p apparently expedited the protein levels of p21, cleaved-casp-3 but drastically reduced the protein levels of CyclinD1, Bcl-2 in Y79 and WERI-Rb-1 cells. Besides, the transfection of miR-655-3p contributed to the distinctly down-regulation of the level of E-cadherin protein, as well as the remarkably decrease of the protein levels of N-cadherin and Vimentin in miR-655-3p group related to that in miR-con group (Fig. 3J-K). To sum up, these results disclosed that the overexpression of miR-655-3p retarded RB progression.
MALAT1 negatively interacted with miR-655-3p in Y79 and WERI-Rb-1 cells
To explore the mechanism of MALAT1 in RB, starBase online database (http://starbase.sysu.edu.cn/starbase2/) was used to predict the putative targets of MALAT1. The results showed that miR-655-3p was a candidate of MALAT1 (Fig. 4A). Following dual-luciferase reporter assay demonstrated that the transfection of miR-655-3p contributed to the remarkable reduction of luciferase activity of WT-MALAT1 reporter related to that in miR-con group, while the luciferase activity of MUT-MALAT1 reporter had no change (Fig. 4B-C). In addition, the level of miR-655-3p was enhanced in si-MALAT1-transfected Y79 and WERI-Rb-1 cells, while decreased in pcDNA-MALAT1 group (Fig. 4D-E). These data implicated that MALAT1 sponged miR-655-3p in Y79 and WERI-Rb-1 cells.
MALAT1 knockdown inhibited cell proliferation but facilitated apoptosis in RB by sponging miR-655-3p
To explore the mechanism and functions of MALAT1 and miR-655-3p in RB, si-MALAT1 and anti-miR-con were transfected into Y79 and WERI-Rb-1 cells. . As exhibited in Fig. 5A, the level of miR-655-3p was distinctly increased in si-MALAT1-transfected Y79 and WERI-Rb-1 cells, while obviously declined by the introduction of anti-miR-655-3p. Moreover, the emergence of anti-miR-655-3p relieved the inhibitory effects on cell viability and the abilities of migrated and invaded of Y79 and WERI-Rb-1 cells inhibited by si-MALAT1 (Fig. 5B-C and G-H). MiR-655-3p inhibitor attenuated the promoted impact on apoptotic rate in Y79 and WERI-Rb-1 cells induced by MALAT1 silencing (Fig.5D). In addition, the transfection of miR-655-3p inhibitor counteracted the facilitated effect on the protein levels of p21, cleaved-casp-3, and E-cadherin, as well as the restraint effect on the protein levels of CyclinD1, Bcl-2, N-cadherin, and Vimentin in Y79 and WERI-Rb-1 cells caused by si-MALAT1 (Fig. 5E-F and I-J).These data implicated that the silencing of MALAT1 suppressed RB progression by sponging miR-655-3p.
ATAD was a candidate target of miR-655-3p in Y79 and WERI-Rb-1 cells
In order to explore the mechanism of miR-655-3p in RB, starBase online database was used to search the putative target of miR-655-3p in RB. As shown in Fig. 6A, ATAD2 3’UTR had complementary binding sites with miR-655-3p. The luciferase activity of WT-ATAD2 reporter was markedly declined in Y79 and WERI-Rb-1 cells transfected with miR-655-3p, while MUT-ATAD2 reporter’s activity had no apparent fluctuation in any group (Fig. 6B-C). Besides, the mRNA and protein levels of ATAD2 were highly expressed in RB tissues and cells (Fig. 6D-G). Also, the level of ATAD2 was negatively correlated with miR-655-3p (Fig. 6H). The qRT-PCR and Western blot assay showed that the mRNA and protein levels of ATAD2 were distinctly reduced in miR-655-3p-transfected Y79 and WERI-Rb-1 cells, while dramatically enhanced in Y79 and WERI-Rb-1 cells transfected with anti-miR-65-3p (Fig. 6I-L). These data demonstrated that ATAD21 was negatively interacted with miR-655-3p in Y79 and WERI-Rb-1 cells.
MiR-655-3p negatively regulated ATAD2 expression to impede cell proliferation and impel apoptosis in Y79 and WERI-Rb-1 cells
To investigate whether the effects of miR-655-3p on RB progression were mediated by ATAD2, the restoration experiments of ATAD2 was conducted. As shown in Fig. 7A-C, the mRNA and protein levels of ATAD2 were remarkably down-regulated in Y79 and WERI-Rb-1 cells transfected with miR-655-3p, while relieved in miR-655-3p and pcDNA-ATAD2 group Furthermore, the transfection of pcDNA-ATAD2 receded the repressive impacts on cell viability and migrated and invaded abilities retarded by miR-655-3p in Y79 and WERI-Rb-1 cells (Fig. 7D-E). The apoptotic rate was distinctly enhanced in Y79 and WERI-Rb-1 cells transfected with miR-655-3p, while partly attenuated in Y79 and WERI-Rb-1 cells co-transfected with miR-655-3p and pcDNA-ATAD2 (Fig. 7F). Finally, the transfection of pcDNA-ATAD2 mitigated the accelerated effect on the protein levels of p21, cleaved-casp-3, E-cadherin as well as the restraint effect on the protein levels of CyclinD1, Bcl-2, N-cadherin, and Vimentin in Y79 and WERI-Rb-1 cells caused by miR-655-3p mimics (Fig. 7G-H and K-L). These data implicated that miR-655-3p hampered cell proliferation, metastasis, and EMT while impelled apoptosis in Y79 and WERI-Rb-1 cells modulating ATAD2.
MALAT1 positively regulated ATAD2 expression to accelerate cell proliferation but block apoptosis in RB by sponging miR-655-3p
To further explore the relationship among MALAT1, miR-655-3p and ATAD2 in RB, we detected the protein level of ATAD2 in Y79 and WERI-Rb-1 cells transfected with si-con, si-MALAT1, si-MALAT1 + anti-miR-con, or si-MALAT1 + anti-miR-655-3p. As presented in Fig. 8A-C, the level of ATAD2 mRNA and protein were apparently down-regulated in Y79 and WERI-Rb-1 cells transfected with si-MALAT1, while regained by the introduction of anti-miR-655-3p. Also, the scatter plot showed that the level of MALAT1 was positively linear correlated with ATAD2 (Fig. 8D). Meanwhile, the introduction of si-MALAT1 effectively down-regulated the levels of mRNA and protein in Y79 and WERI-Rb-1 cells, while reversed by the emergence of pcDNA-ATAD2 (Fig. 8E-G). Moreover, the transfection of pcDNA-ATAD2 weakened the suppressive impacts on cell viability, migrated and invaded abilitiesin Y79 and WERI-Rb-1 cells inhibited by si-MALAT1 (Fig.8H-I and M-N). Whereas, the apoptotic rate showed the opposite trend. Briefly, the apoptotic rate was obviously increased in Y79 and WERI-Rb-1 cells transfected with si-MALAT1, while partly attenuated in Y79 and WERI-Rb-1 cells co-transfected with si-MALAT1 and pcDNA-ATAD2 (Fig. 8J). The Western blot assay indicated that the emergence of pcDNA-ATAD2 neutralized the promoted effects on the protein levels of p21, cleaved-casp-3, and E-cadherin, as well as the inhibitory effects on the protein levels of CyclinD1, Bcl-2, N-cadherin, and Vimentin in Y79 and WERI-Rb-1 cells caused by MALAT1 silencing (Fig.8K-L and O-P). These data demonstrated that MALAT1 positively modulated ATAD2 expression to regulate cell behaviors in Y79 and WERI-Rb-1 cells by sponging miR-655-3p.
MALAT1 depletion impeded xenograft tumor growth in vivo
To further verify the effects of MALAT1 in vivo, the mice model experiments were constructed. Firstly, we affirmed sh-MALAT1 or sh-con transfection efficiency, indicated by the striking down-regulation of MALAT1 in Y79 cells (Fig. 9A). The measurement results presented that the weight and volume were both substantially declined in sh-MALAT1 group related to that in sh-NC group (Fig. 9B-C). In addition, the level of miR-655-3p was significantly elevated in sh-MALAT1 group, while the mRNA and protein levels of ATAD2 were notably reduced in sh-MALAT1 group (Fig. 9D-E). These results suggested that the silencing of MALAT1 blocked xenograft tumor growth in vivo.