CircRNA hsa_circRNA_0000069 promotes the proliferation, migration and invasion of cervical cancer through miR-873-5p/TUSC3 axis

Abstract

Background: Cervical cancer (CC) is the second leading cause of cancer deaths in women worldwide, still lacking effective biomarkers and therapies for diagnosis and treatment. CircRNAs are a class of endogenous RNAs that regulate gene expression through interacting with miRNAs, implicating in the progression of cancers. Yet the roles of circRNAs in CC are not fully characterized.

Methods: Fifty pairs of tumor and adjacent normal tissues from CC patients, as well as four CC cell lines and a normal human cervical epithelial cell line were subjected to qRT-PCR assay to assess the mRNA levels of hsa_circ_0000069. CCK-8 and colony formation assays were conducted to detect the proliferation of CC cells. Transwell assay was used to evaluate the migration and invasion capabilities of CC cells. RNA pull-down and luciferase assays were used to determine the interaction between hsa_circ_0000069 and miR-873-5p. A xenograft model of CC was established to verify the in vivo function of hsa_circ_0000069 in CC progression.

Results: We firstly demonstrated that hsa_circ_0000069 was significantly upregulated and closely related to the lymph node metastasis, and poor prognosis of CC patients. Besides, hsa_circ_0000069 promoted CC cell proliferation, migration, and invasion. The knockdown of hsa_circ_0000069 also inhibited CC tumor growth in vivo. Mechanically, we revealed that hsa_circ_0000069 functioned as an oncogene in CC, which is the sponge of miR-873-5p to facilitate the TUSC3 expression, consequently promoting CC progression.

Conclusion: We demonstrated a critical hsa_circ_0000069-miR-873-5p-TUSC3 function network involved in the CC progression, which provides mechanistic insights into the roles of CircRNAs in CC progression and a promising therapeutic target for CC treatment.

Keywords: Cervical cancer; CircRNA; Invasion; Migration; Proliferation; hsa_circ_0000069; miRNA.

Fig. 1

CircRNA hsa_circ_0000069 is upregulated in CC and associated with CC progression. a Heatmap of differentially expressed circular RNAs in CC tissues and normal tissues according to the online data set (GSE102686), (absolute log2 fold change > 1, P < 0.05). b Relative mRNA expression levels of hsa_circ_0000069 in 50 pairs of CC tissues and adjacent normal tissues were measured using qRT-PCR. c Relative mRNA expression levels of hsa_circ_0000069 in CC cell lines (SiHa, C-4I, HeLa and C-33A) and normal human cervical epithelial cell line, End1/E6E7. d Relative mRNA expression levels of hsa_circ_0000069 in lymphatic metastatic and non-metastatic CC tissues were measured using qRT-PCR. e Higher expression of hsa_circ_0000069 was linked to the lower survival rate of CC patients as determined by Kaplan–Meier analysis (n = 50 patients, n = 25 in each arm of low expression or high expression). All data are representative of three independent experiments and expressed as mean ± SD. P values were determined by a two-tailed Student’s unpaired t-test, **P < 0.01

Fig. 2

hsa_circ_0000069 promotes proliferation, migration and invasion in CC cell lines. a qRT-PCR analysis of hsa_circ_0000069 expression in SiHa and HeLa cells transfected with NC or si-hsa_circ_0000069 (si-1# and 2#). b, c Cell counting kit-8 and colony formation assays were used to measure the proliferation ability of siHa and HeLa cells transfected with NC or si-hsa_circ_0000069 (si-1# and 2#). d, e Transwell assays were used to determine the effects of hsa_circ_0000069 on CC cell migration and invasion. All data is representative of three independent experiments and expressed as mean ± SD. P value in Fig. 2b was determined by a one-way ANOVA, and the others were determined by two-tailed Student’s unpaired t-test, **P < 0.01. NC: negative control; si: small interfering. f Representative western blots of the protein expression levels for cell proliferation and migration biomarkers in siHa and HeLa cells transfected with NC or si-hsa_circ_0000069 (si-1# and 2#). GAPDH was used as an internal reference

Fig. 3

hsa_circ_0000069 promotes CC tumor growth in vivo. a The tumor growth curves of nude mice with si‐hsa_circ_0000069 (si-1# and 2#) or NC SiHa and HeLa cells. P values were determined by a one-way ANOVA, **P < 0.01. b Tumor weights of each group were analyzed at the endpoint of the experiment. Data are representative of three independent experiments and expressed as mean ± SD. c Relative expression levels of hsa_circ_0000069 in xenografts of each group were assessed using qRT-PCR. P values were determined by two-tailed Student’s unpaired t-test, **P < 0.01

Fig. 4

hsa_circ_0000069 directly interacts with miR-873-5p. a The mRNA levels of nuclear control (U6), cytoplasmic control (GAPDH) and hsa_circ_0000069 were analyzed using qRT‐PCR in nuclear and cytoplasmic fractions. b A predicted binding site of miR-873-5p within hsa_circ_0000069 by bioinformatic analysis using the CircInteractome database (https://circinteractome.nia.nih.gov/). And the binding sequences “AGUUCCUGA” in hsa_circ_0000069 were mutated to “CAGGAAGUC” for generating Mut‐hsa_circ_0000069. c, d Luciferase reporter and RNA pull-down assays were used to determine the interaction between hsa_circ_0000069 and miR-873-5p. e Relative mRNA expression of miR-873-5p in hsa_circ_0000069-knockdown SiHa and Hela cells. f Relative mRNA expression of miR-873-5p in CC tissues and adjacent normal tissues was examined using qRT‐PCR. g Spearman correlation analysis between miR-873-5p and hsa_circ_0000069 expressions in 50 pairs of CC tissues. All data are representative of three independent experiments and shown as mean ± SD. P values were determined by two-tailed Student’s unpaired t-test, **P < 0.01; ns, not significant

Fig. 5

miR-873-5p represses the proliferation, migration and invasion. a Relative mRNA expression of miR-873-5p in siHa and HeLa cells transfected with miR‐NC or miR-873-5p mimics. b, c. Cell counting kit‐8 and colony formation assays for evaluation of cell proliferation of SiHa and HeLa cells transfected with miR‐NC or miR-873-5p mimics. d, e Cell migration and invasion of SiHa and HeLa cells were measured using transwell assays. Data are representative of three independent experiments and shown as mean ± SD. P value in Fig. 5b was determined by a one-way ANOVA, and the others were determined by two-tailed Student’s unpaired t-test, **P < 0.01

Fig. 6

hsa_circ_0000069 promotes TUSC3 expression by sponging miR-873-5p. a A predicted binding site of miR-873-5p within the TUSC3 3′-UTR region using Targetscan. And the binding sequences “GUUCCUG” in hsa_circ_0000069 were mutated to “UCCAAGU” for generating Mut-TUSC3. Luciferase reporter assays were used to evaluate the interaction between TUSC3 3′-UTR and miR-873-5p. b Relative protein levels of TUSC3 in miR-873-5p-overexpressed SiHa and HeLa cells were measured using densitometric quantification by image J. Representative western blots of SiHa and Hela cells transiently transfected with miR-NC or miR-873-5p mimics. The data are normalized to GAPDH and presented as mean ± SD. of two experiments. c Relative protein expression levels of TUSC3 in hsa_circ_0000069-knockdown or miR-873-5p inhibitor-treated SiHa and HeLa cells were measured using densitometric quantification by image J. Representative western blots of SiHa and Hela cells transiently transfected with NC or si-hsa_circ_0000069 (si-1#) or both si-1# and miR-873-5p inhibitor. The data are normalized to GAPDH and presented as mean ± SD. of two experiments. d Spearman correlation analysis between miR-873-5p and TUSC3 expressions in CC tissues. Data are shown as mean ± SD. P values were determined by two-tailed Student’s unpaired t-test, **P < 0.01. inh: inhibitor

Fig. 7

Restoration of TUSC3 reversed the effects of hsa_circ_0000069 knockdown in CC cells. a Relative protein expression levels of TUSC3 in hsa_circ_0000069-knockdown, or both hsa_circ_0000069-knockdown and TUSC3-overexpressed SiHa and HeLa cells were measured using densitometric quantification by image J. Representative western blots of SiHa and Hela cells transiently transfected with NC or si-hsa_circ_0000069 (si-1#), or both si-1# and pcDNA3.1-TUSC3. The data are normalized to GAPDH and presented as mean ± SD. of two experiments. b, c Cell counting kit‐8 and colony formation assays were used to measure the proliferation of SiHa and HeLa cells. d, e Transwell assays were used to determine the migration and invasion of SiHa and HeLa cells. Data are representative of three independent experiments and shown as mean ± SD. P value in Fig. 7b was determined by a one-way ANOVA, and the others were determined by two-tailed Student’s unpaired t-test, **P < 0.01. NC negative control, si small interfering, OE Overexpression