Hsa_circ_0072008 regulates cell proliferation, migration, and invasion in cervical squamous cell carcinoma via miR-1305/helicase, lymphoid specific (HELLS) axis

Abstract

Cervical squamous cell carcinoma (CESC) is one of the most common cancers in women. Recent studies have proved that circular RNAs (circRNAs) could regulate the progress of CESC, but the mechanism is still indistinct. In this work, we explored the roles of circ_0072008 in CESC. The expression levels of circ_0072008, microRNA-1305 (miR-1305) and mRNA of HELLS (helicase, lymphoid specific) were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) in CESC tissues. Meanwhile, the level of HELLS was quantified by western blot analysis. Besides, the cell functions were examined by colony formation assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay, wound healing assay, flow cytometry assay and western blot. Furthermore, the interaction between miR-1305 and circ_0072008 or HELLS was detected by dual-luciferase reporter assay. The function of circ_0072008 in CESC has also been further verified in vivo by xenograft model experiments. The levels of circ_0072008 and HELLS were upregulated, and the miR-1305 level was decreased in CESC tissues in contrast to that in normal tissues. For functional analysis, silencing circ_0072008 inhibited cell proliferation and cell migration, whereas enhanced cell apoptosis in CESC cells. In mechanism, circ_0072008 acted as a miR-1305 sponge to regulate the level of HELLS. Moreover, miR-1305 was confirmed to repress the progression of CESC cells by suppressing HELLS. Meanwhile, knockdown of circ_0072008 inhibited CESC cells growth in vivo. In conclusion, circ_0072008 facilitated CESC cell proliferation, migration, and invasion through increasing HELLS expression by regulating miR-1305, which also offered an underlying targeted therapy for CESC treatment.

Keywords: Cervical squamous cell carcinoma; HELLS; circ_0072008; miR-1305.

Graphical abstract

Figure 1.

Circ_0072008 expression was enhanced in CESC tissues. (a) The expression of circ_0072008 in CESC tissues was detected by qRT-PCR. (b) The relative levels of circ_0072008 in CESC cells were detected by qRT-PCR. (c and d) The levels of circ_0072008 and MYO10 mRNA after RNase R treatment were detected by qRT-PCR. (e and f) The levels of circ_0072008 and MYO10 mRNA after Act D treatment were detected by qRT-PCR. ***P < 0.001.

Figure 2.

Circ_0072008 knockdown inhibited CESC progression. (a) The silencing efficiency of si-circ_0072008 was measured by qRT-PCR. (b) The colony formation assay revealed the cell proliferation. (c) The EdU assay unfolded the cell proliferation. (d) The wound healing assay measured the cell migration. (e) The flow cytometry assay showed the cell apoptosis. (f and g) The protein level of PCNA and Bax were examined by Western blot. ***P < 0.001.

Figure 3.

Circ_0072008 acted as a sponge for miR-1305. (a) The targeted miRNAs of circ_0072008 were forecast by starbase. (b and c) The expression of miR-1305 in CESC tissues and cells was measured by qRT-PCR. (d and e) Dual-luciferase reporter assay was used to verify the relationship between circ_0072008 and miR-1305. (f) RNA pull-down assay was used to verify the relationship between circ_0072008 and miR-1305. ***P < 0.001.

Figure 4.

Circ_0072008 facilitated the progression of CESC by sponging miR-1305. (a) The expression of miR-1305 was detected by qRT-PCR. (b and c) The cell proliferation, (d) the cell migration, (e) the cell apoptosis, (f and g) the protein levels of PCNA and Bax were detected by colony formation assay, EdU assay, wound healing assay, flow cytometry assay, and western blot. **P < 0.01, ***P < 0.001.

Figure 5.

MiR-1305 targeted HELLS in CESC cells. (a) The binding site between miR-1305 and HELLS was analyzed by starbase. (b–e) The expression of HELLS was detected by qRT-PCR and western blot. (f and g) Dual-luciferase reporter assay was used to confirm the relationship between miR-1305 and HELLS. (h–k) The expression of HELLS was detected by western blot. **P < 0.01, ***P < 0.001.

Figure 6.

Circ_0072008 regulated the progression of CESC by targeting HELLS. (a) The expression of HELLS was detected by western blot. (b and c) The cell proliferation, (d) the cell migration, (e) the cell apoptosis, (f and g) the protein level of PCNA and Bax were detected by colony formation assay, EdU assay, wound healing assay, flow cytometry assay, and western blot. **P < 0.01, ***P < 0.001.

Figure 7.

Circ_0072008 restricted tumor growth in vivo. (a) The tumor volume was measured every week. (b) Tumor weight was measured after 35 days when the mice were killed. (c) IHC analysis was implemented to examine the expression of Ki-67 in tumor tissues from different groups. (d–f) The expression of circ_0072008, miR-1305, and HELLS in these excised tumor tissues was detected by qRT-PCR. ***P < 0.001.