circKRT7-miR-29a-3p-COL1A1 Axis Promotes Ovarian Cancer Cell Progression

Abstract

Background: Circular RNA (circRNA) has emerged as an important regulator in the progression of human diseases. However, the role of circRNAs in ovarian cancer remains largely unknown.

Materials and methods: DNA sequencing and PCR were used to identify the existence and expression of circKRT7. The targeting relationship between circKRT7/miR-29a-3p and miR-29a-3p/COL1A1 was verified by fluorescence reporter assay. In vitro, colony formation, transwell and wound healing assay were used to detect the effects of circKRT7 and miR-29a-3p on the proliferation, migration and invasion ability of ovarian cancer cells. In vivo, xenograft tumor model was performed to validate the role of circKRT7 and miR-29a-3p in tumor growth.

Results: We found that circKRT7 can promote the proliferation and metastasis of ovarian cancer cells by absorbing miR-29a-3p, which leads to the up-regulation of COL1A1. In vitro, knock-down of circKRT7 can inhibit the migration and invasion of ovarian cancer cells. This effect of circKRT7 is achieved by adsorbing miR-29a-3p and subsequently COL1A1 release. In vivo experiments, the reduction of circKRT7 expression can also slow tumor growth, and this inhibition was partly counteracted after miR-29a-3p repression.

Conclusion: Overall, circKRT7 promotes EMT-related cell progression by absorbing miR-29a-3p in ovarian cancer. This suggests the crucial role of circular RNA in the malignant evolution in cancer.

Keywords: EMT; KRT7; circular RNA; ovarian cancer.

Figure 1

circKRT7 was highly expressed in ovarian cancer. (A) Expression of circKRT7 in 10 pair ovarian cancer and adjacent normal tissues. (B) Biogenesis of circKRT7 and predicted miR-29a-3p binding sites. (C and D) Tumor genesis (C) and tumor volume (D) in nude mice after circKRT7 knocked down. (E) circKRT7 levels in these solid tumors. (F) Relative miR-29a-3p expression in these tumors. (G) qRT-PCR analysis of circKRT7 and KRT7 mRNA after treatment with RNase R. Experiments were performed in triplicat. Statistical significance was considered at P < 0.05 and labeled with *.

Figure 2

Knock-down of circKRT7 inhibited ovarian cancer cell progression. ES-2 and SKOV3 cells were treated with sh-circKRT7. (A) The expression of circKRT7 in 5 ovarian cancer cells. (B) Expression of circKRT7 in ES-2 and SKOV3 cells after circKRT7 knock-down. (C) Scanning electron microscopy of cell morphology after circKRT7 knock-down. (D) Invasion analysis by transwell assay after circKRT7 knock-down. (E) Wound healing to evaluate cell migration ability after circKRT7 knock-down. (F) Colony formation assay to detect cell proliferation after circKRT7 knock-down. (G) EMT markers, E-cadherin and Vimentin, were detected by Western blot. Experiments were performed in triplicate. Statistical significance was considered at P < 0.05 and labeled with *.

Figure 3

circKRT7 adsorbed miR-29a-3p to release COL1A1. (A) Predicted binding sites of miR-29a-3p in circKRT7 and COL1A1 3ʹUTR. (B) Localization of circKRT7 and miR-29a-3p in ES-2 cells. (C) Luciferase activity assay analyzes the binding ability of miR-29a-3p and circKRT7. (D) Luciferase activity assay analyzes the binding ability of miR-29a-3p and COL1A1 3ʹUTR. (E) miR-29a-3p and circKRT7 expression after over-expression of wild type or mutated circKRT7. (F) Western blot analysis COL1A1 protein level after treated with circKRT7 shRNA or along with miR-29a-3p ASO. Experiments were performed in triplicate. Statistical significance was considered at P < 0.05 and labeled with *.

Figure 4

miR-29a-3p inhibited ovarian cancer cell migration and invasion. ES-2 and SKOV3 cells were transfected with miR-29a-3p mimics or control mimics. (A) Western analysis was used to detect the expression of COL1A1, E-cadherin and Vimentin. (B) Scanning electron microscopy of cell morphology. (C) Cell invasive ability was analyzed by transwell assay. (D) Wound healing assay was performed to assess cell migration. (E) Colony formation was performed to detect cell proliferation. Experiments were performed in triplicate. Statistical significance was considered at P < 0.05 and labeled with *.

Figure 5

COL1A1 counteracted the role of miR-29a-3p in ovarian cancer cells. ES-2 and SKOV3 cells were transfected with miR-29a-3p mimics alone or along with COL1A1. (A) Western blot analyzed the protein levels of COL1A1, E-cadherin and Vimentin in ES-2 cells. (B) Wound healing assay was used to detected cell migration ability. (C) Cell invasion was analyzed by transwell assay. (D) Colony formation was performed to detect cell proliferation. Experiments were performed in triplicate. Statistical significance was considered at P < 0.05 and labeled with *.

Figure 6

Role of circKRT7 in ovarian cancer was mediated by miR-29a-3p. ES-2 and SKOV3 cells were treated with sh-circKRT7 alone or along with miR-29a-3p inhibitor. (A) Expression of circKRT7 and miR-29a-3p in ES-2 cells. (B) Western blot analyzed the protein levels of COL1A1, E-cadherin and Vimentin in ES-2 cells. (C) Cell invasion was analyzed by transwell assay. (D) Wound healing assay was used to detected cell migration ability. (E) Colony formation was performed to detect cell proliferation. Experiments were performed in triplicate. Statistical significance was considered at P < 0.05 and labeled with *.

Figure 7

Knock-down of circKRT7 inhibited tumor growth of ovarian cancer in vivo. (A) Pictures of tumor-bearing animals after euthanasia. (B) Tumor volumes of each group. (C) IHC was performed to detect the expression of COL1A1, E-cadherin and Vimentin. (D) Expression of circKRT7 and miR-29a-3p in solid tumors. Experiments were performed in triplicate. Statistical significance was considered at P < 0.05 and labeled with *.