CRNDE Contributes Cervical Cancer Progression by Regulating miR-4262/ZEB1 Axis

Abstract

Background: Cervical cancer is a lethal gynecologic cancer in women. Long non-coding RNA colorectal neoplasia differentially expressed (LncRNA CRNDE) was recognized as a significant oncogene in multiple cancers. However, the functional role of CRNDE in cervical cancer remains poorly explored.

Methods: The expression of CRNDE, microRNA-4262 (miR-4262) and zinc-finger E-box binding homeobox 1 (ZEB1) in cervical cancer tumors and cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Colony formation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were performed to detect cell viability. Flow cytometry and caspase-3 activity assay were conducted to evaluate cell apoptosis. The interaction between miR-4262 and CRNDE or ZEB1 was verified by dual-luciferase reporter system. Transwell assay was employed to evaluate cell migration and invasion. The relative protein expression was assessed by Western blot.

Results: CRNDE and ZEB1 were up-regulated, while miR-4262 was down-regulated in cervical cancer tissues and cells. We found that CRNDE sponged miR-4262 and ZEB1 was a target of miR-4262. In addition, miR-4262 inhibitor abolished CRNDE silencing-induced repression on cell proliferation, EMT, migration, invasion and promotion on cell apoptosis. Furthermore, ZEB1 rescued the effects of miR-4262 overexpression or CRNDE deletion on cervical cancer progression. Our data showed that CRNDE targeted miR-4262 to regulate ZEB1 expression in cervical cancer cells. Besides, CRNDE expedited cervical cancer progression through wnt/β-catenin pathway via sponging miR-4262 and altering ZEB1 expression.

Conclusion: Our findings demonstrated that CRNDE facilitated the progression of cervical cancer through activation of wnt/β-catenin pathway by regulating miR-4262/ZEB1 axis, representing a prospective targeted therapy for cervical cancer.

Keywords: CRNDE; ZEB1; cervical cancer; miR-4262; wnt/β-catenin pathway.

Figure 1

CRNDE directly interacted with miR-4262. (A and B) CRNDE expression was detected by qRT-PCR in cervical cancer tumors and cells (C-33A, Hela, SiHa, CasKi) as well as the normal tissues and cells (ECT1/E6E7). (C) CRNDE expression was detected using qRT-PCR in Hela and SiHa cells transfected with sh-NC, pcDNA, sh-CRNDE and CRNDE. (D) Prediction of the putative binding sites between CRNDE and miR-4262 using StarBase. (E and F) Luciferase activity of Hela and SiHa cells co-transfected with CRNDE-WT or CRNDE-MUT and miR-4262 or miR-NC was determined by dual-luciferase reporter assay. (G and H) The expression of miR-4262 in cervical cancer tumors and cells as well as the normal counterparts was detected by qRT-PCR. (I) Analysis of the correlation between CRNDE and miR-4262 by Person’s correlation coefficient (R=−0.61, P<0.0001). (J) Detection of miR-4262 expression in Hela and SiHa cells transfected with anti-miR-NC, miR-NC, anti-miR-4262 and miR-4262 by qRT-PCR. (K) Measurement of miR-4262 expression in Hela and SiHa cells transfected with sh-NC, pcDNA, sh-CRNDE and CRNDE by qRT-PCR. *P<0.05.

Figure 2

MiR-4262 inhibitor restored CRNDE silencing-induced suppressive effects on cervical cancer progression. Hela and SiHa cells were transfected with sh-NC, sh-CRNDE, sh-CRNDE+anti-miR-NC and sh-CRNDE+anti-miR-4262. (A) Examination of miR-4262 expression in transfected cells by qRT-PCR. (B) Analysis of colonies of transfected cells by colony formation assay. (C and D) Cell viability was detected by MTT assay. (E and F) The protein expression of Ki-67 and PCNA was detected by Western blot. (G) Cell apoptosis was determined by flow cytometry. (H and I) Detection of apoptosis-associated proteins including Bcl-2 and Bax using Western blot. (J) Identification of caspase-3 activity of transfected cells by caspase-3 activity kit. *P<0.05.

Figure 3

MiR-4262 inhibitor abrogated CRNDE silencing-mediated inhibition on cervical cancer cell migration, invasion and EMT. Hela and SiHa cells were transfected with sh-NC, sh-CRNDE, sh-CRNDE+anti-miR-NC and sh-CRNDE+anti-miR-4262. (A and B) Cell migration and invasion were assessed by transwell assay. (C and D) The protein expression of N-cadherin, E-cadherin, Vimentin and Snail was analyzed by Western blot. *P<0.05.

Figure 4

ZEB1 was a target of miR-4262. (A) The putative binding sites between ZEB1 and miR-4262 were predicted by StarBase. (B and C) Dual-luciferase reporter assay was conducted to evaluate the luciferase activity of Hela and SiHa cells co-transfected with ZEB1 3ʹUTR-WT or ZEB1 3ʹUTR-MUT and miR-4262 or miR-NC. (D and E) ZEB1 mRNA and protein expression in cervical cancer tumors compared with normal tissues were examined by qRT-PCR and Western blot. (F) Analysis of the correlation between ZEB1 and miR-4262 by Person’s correlation coefficient (R=−0.60, P<0.0001). (G) Protein expression of ZEB1 in cervical cancer cells and normal cells was measured by Western blot. (H–K) Detection of ZEB1 mRNA and protein expression in Hela and SiHa cells transfected with anti-miR-NC, anti-miR-4262, miR-NC and miR-4262 using qRT-PCR and Western blot. *P<0.05.

Figure 5

ZEB1 attenuated miR-4262-mediated inhibition on cell proliferation and promotion on cell apoptosis in cervical cancer. Hela and SiHa cells were transfected with miR-NC, miR-4262, miR-4262+Vector and miR-4262+ZEB1. (A–C) Detection of ZEB1 mRNA and protein expression in transfected cells using qRT-PCR and Western blot. (D) Measurement of colonies of transfected cells by colony formation assay. (E and F) Cell viability was detected by MTT assay. (G and H) Western blot was performed to examine the expression of Ki-67 and PCNA. (I) Cell apoptosis was measured by flow cytometry. (J and K) Detection of apoptosis-associated proteins including Bcl-2 and Bax using Western blot. (L) Examination of caspase-3 activity of transfected cells by caspase-3 activity kit. *P<0.05.

Figure 6

ZEB1 abolished miR-4262-induced suppression on the migration, invasion and EMT of cervical cancer cells. Hela and SiHa cells were transfected with miR-NC, miR-4262, miR-4262+Vector and miR-4262+ZEB1. (A and B) Transwell assay was applied to evaluate cell migration and invasion. (C and D) Protein expression of MMP9, Vimentin, N-cadherin and E-cadherin was analyzed by Western blot. *P<0.05.

Figure 7

CRNDE regulated ZEB1 expression by sponging miR-4262. Hela and SiHa cells were transfected with miR-NC, miR-4262, pcDNA and CRNDE. (A and B) Evaluation of ZEB1 mRNA expression in transfected cells using qRT-PCR. (C and D) Analysis of ZEB1 protein expression in transfected cells by Western blot. *P<0.05.

Figure 8

CRNDE activated the wnt/β-catenin pathway by targeting miR-4262/ZEB1 axis in cervical cancer. Hela and SiHa cells were transfected with sh-NC, sh-CRNDE, sh-CRNDE+anti-miR-NC, sh-CRNDE+anti-miR-4262, sh-CRNDE+Vector and sh-CRNDE+ZEB10. (A and B) Detection of the protein expression of β-catenin, GSK-3β, c-myc and cyclin D1 in transfected cells by Western blot. *P<0.05.