Circular RNA hsa_circ_0000511 Improves Epithelial Mesenchymal Transition of Cervical Cancer by Regulating hsa-mir-296-5p/HMGA1

Abstract

As the second largest gynecological cancer, cervical cancer has been widely reported in recent years in which circular RNA is involved in the disease process. We earlier found that the expression of hsa_circ_0000511 in cervical cancer cells increased significantly, but its role in the process of cervical cancer is not clear. The purpose of this study is to explore its possible mechanisms in cervical cancer. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), cell counting kit-8 assay, Transwell test, cell transfection, RNA pull-down assay and dual-luciferase reporter assay, and Western blot analysis were used to detect the expression and distribution of hsa_circ_0000511 in SiHa and HeLa cells, the ability of invasion and proliferation, and the modulated relationships between hsa_circ_0000511 and hsa-mir-296-5p, hsa-mir-296-5p, and HMGA1. hsa_circ_0000511 had the highest expression in SiHa and HeLa cells, and the expression in the cytoplasm was significantly higher than that in the nucleus, and its expression was not affected by RNase R. When hsa_circ_0000511 was silenced, its expression in SiHa and HeLa cells was significantly decreased; the proliferation, invasion, and migration abilities of the two kinds of cells were significantly enhanced; and the protein expression of E-cadherin was significantly upregulated, while the protein expression of N-cadherin was significantly downregulated. The expression of hsa-mir-296-5p was lower in SiHa and HeLa cells; however, its expression was increased when hsa_circ_0000511 was inhibited and decreased when hsa_circ_0000511 was overexpressed, so did the ability of proliferation, invasion, and migration and the protein expression of E-cadherin. Interestingly, the protein expression of HMGA1 also changed in these two cells when hsa-mir-296-5p was inhibited or overexpressed. Our results indicate that the upregulated hsa_circ_0000511 can inhibit the proliferation, invasion, and migration of SiHa and HeLa cells by regulating hsa-mir-296-5p/HMGA1, suggesting that the hsa_circ_0000511/hsa-mir-296-5p/HMGA1 pathway may be a potential target for the treatment of cervical cancer.

Figure 1

hsa_circ_0000511 was highly expressed in SiHa and HeLa cells and mainly located in the cytoplasm. The expression levels of hsa_circ_0000511 in SiHa and HeLa cells were significantly higher than those in H8 and C-33A cells (a) (mean ± SD; the data were analyzed by one-way analysis of variance; vs. H8, ^∗∗p < 0.01; vs. C-33A, ^##p < 0.01). hsa_circ_0000511 (b) and its gene symbol (RPPH1) (c) were mainly expressed in the cytoplasm in SiHa and HeLa cells. hsa_circ_0000511 can be resistant to RNase R⁺ (d), while RPPH1 is not (e) (mean ± SD; the data were analyzed by an unpaired two-sided t-test; ^∗∗∗p < 0.001).

Figure 2

Inhibition of hsa_circ_0000511 significantly promoted the invasion and migration of SiHa and HeLa cells. The proliferation of SiHa (a) and HeLa (b) cells was increased when hsa_circ_0000511 was silenced; naturally, the expression of hsa_circ_0000511 was significantly decreased in the silenced cells (c). Moreover, when hsa_circ_0000511 was silenced, the invasion and migration abilities of SiHa (d) and HeLa (f) cells were also significantly enhanced; that is, the number of cells in lower Transwell chamber was significantly increased (e, g), respectively. In addition, the results of Western blot analysis (h) showed that the protein expression of E-cadherin was significantly increased (i) but that of N-cadherin was significantly decreased (j) in the hsa_circ_0000511 silenced group. Mean ± SD; the data were analyzed by an unpaired two-sided t-test; vs. the control, ^∗∗p < 0.01 and ^∗∗∗p < 0.001; vs. si-circ-NC, ^##p < 0.01 and ^###p < 0.001.

Figure 3

hsa_circ_0000511 promoted the invasion and migration of SiHa and HeLa cells by regulating hsa-miR-296-5p. Contrary to hsa_circ_0000511, the expression of hsa-miR-296-5p in SiHa and HeLa cells was significantly lower than that in H8 and C-33A cells (a) (mean ± SD; the data were analyzed by one-way analysis of variance, vs. H8, ^∗∗p <0.01; vs. C-33A, ^##p <0.01), and that was increased when hsa_circ_0000511 was silenced (b) (mean ± SD, the data were analyzed by an unpaired two-sided t-test, vs. the control, ^∗∗p < 0.01; vs. si-circ-NC, ^##p < 0.01). The result of dual-luciferase reporter assay (c) shows that hsa-miR-296-5p is a direct target of hsa_circ_0000511 in SiHa (d) and HeLa (e) cells (mean ± SD; the data were analyzed by one-way analysis of variance; vs. circRNA-wt+miRNA NC, ^∗∗p < 0.01). The proliferation (f, g), invasion (h), and migration (i) of SiHa and HeLa cells were increased because hsa-miR-296-5p was regulated by hsa_circ_0000511; that is to say, the number of cells in a lower Transwell chamber was increased significantly (j, k). In addition, the expression of hsa-miR-296-5p (l) changed when whether hsa_circ_0000511 (m) was silenced or not. (f, g, j–m) Mean ± SD; the data were analyzed by one-way analysis of variance; vs. si-circ+miRNA mimics, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; vs. miRNA mimics, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001; vs. mimics NC, ^{^}p < 0.05, ^{^^}p < 0.01, and ^{^^^}p < 0.001; vs. si-circ+miRNA inhibitor, ^&&p < 0.01 and ^&&&p < 0.001.

Figure 4

HMGA1 is a direct target of hsa-miR-296-5p, and overexpression of HMGA1 can promote proliferation of SiHa and HeLa cells. Like hsa_circ_0000511, HMGA1 was highly expressed in SiHa and HeLa cells than in H8 and C-33A cells (a) (mean ± SD; the data were analyzed by one-way analysis of variance; vs. H8, ^∗∗p < 0.01; vs. C-33A, ^##p < 0.01). The results of Western blot (b) showed that the protein expressions of HMGA1 were changed when hsa-miR-296-5p was inhibited or not in SiHa (c) and HeLa (d) cells, and a dual-luciferase reporter assay (e) indicated that HMGA1 is a direct target of hsa-miR-296-5p ((c, d) mean ± SD, the data were analyzed by one-way analysis of variance; vs. mimics-NC, ^∗∗p < 0.01 and ^∗∗∗p < 0.001; vs. si-circ+miRNA inhibitor, ^###p < 0.001; (f, g), vs. HMGA1 3′-UTR (wt)+miRNA NC, ^∗∗p < 0.01). Moreover, CCK8 detection shown that overexpression of HMGA1 could promote proliferation of SiHa (h) and HeLa (i) cells. Mean ± SD; the data were analyzed by one-way analysis of variance; vs. the control, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; vs. si-circRNA, ^###p < 0.001; vs. OV-HMGA1, ^{^^^}p < 0.001; vs. si-circRNA+OV-HMGA1+miRNA mimics, ^&&p < 0.01.

Figure 5

hsa_circ_0000511 promoted EMT by regulating hsa-miR-296-5p/HMGA1 signal. Overexpression of HMGA1 increased the ability of invasion (a) and migration (b) in SiHa and HeLa cells, and the number of cells in the lower Transwell chamber was increased significantly (c, d). Further, the results of Western blotting (e, f) indicated that the expression of E-cadherin and N-cadherin (h) was consistent with that of HMGA1 (g). Overexpressed HMGA1 inhibited the protein expression of E-cadherin and promoted that of N-cadherin, and vice versa. Mean ± SD; the data were analyzed by one-way analysis of variance; vs. control, ^∗∗p < 0.01 and ^∗∗∗p < 0.001; vs. si-circRNA+OV-HMGA1+miRNA mimics, ^###p < 0.001.

Figure 6

hsa_circ_0000511 promotes EMT of cervical cancer cells in vivo by downregulating hsa-miR-296-5p/HMGA1. Inhibition of hsa_circ_0000511 significantly reduced the tumor volume in vivo (a, b), and Western blot analysis (c) showed that the protein expression of HGMA1 was decreased in turn when hsa_circ_0000511 was silenced or hsa-miR-296-5p was activated (d), while the protein expression ration between E-cadherin and N-cadherin was opposite to that of HGMA1 (e). Meanwhile, the results of qRT-PCR showed that the expression of hsa_circ_0000511 was consistent with the protein expression of HGMA1 (f) but the expression of hsa-miR-296-5p (g) was opposite to that of hsa_circ_0000511. Besides, changes of average optical density of PCNA (h, i) were consistent with the expression of hsa_circ_0000511. Mean ± SD; the data were analyzed by one-way analysis of variance; vs. the control, ^∗∗p < 0.01 and ^∗∗∗p < 0.001; vs. si-circRNA, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001.