Knockdown of LncRNA DLEU2 Inhibits Cervical Cancer Progression via Targeting miR-128-3p

Abstract

Objective: Cervical cancer is one of the most common female malignancies worldwide and represents a major global health challenge. The fast growth of tumor and high rates of metastasis still lead to a poor prognosis of cervical cancer patients. It is urgent to clarify the mechanism and identify predictive biomarkers for the treatment of cervical cancer. Long non-coding RNAs (LncRNAs) have been identified in cervical cancer and are related to malignant phenotypes of cervical cancer cells. However, the roles and mechanism of LncRNA deleted in lymphocytic leukemia (DLEU2) in the tumorigenesis and progression of cervical cancer remain unknown.

Materials and methods: qPCR was performed to analyze the expression of DLEU2, Cyclin D1, CDK4, Bax, Bcl2 and mi-128-3p. Western blot was performed to detect the cell cycle hallmarks expression. CCK8 was used to examine cell proliferation. Cellular apoptosis was analyzed by Hoechst 33,258 staining and AV/PI staining with flow cytometry. Cell cycle was analyzed by flow cytometry. The xenograft model in nude mice was used to elucidate the function of DLEU2 in vivo. Bioinformatics analysis and luciferase reporter assay were proceeded to clarify whether miR-128-3p directly binds with lncRNA DLEU2. Pull‑down assay and RNA-binding protein immunoprecipitation assay were used for exploring the relationship between DLEU2 and miR-128-3p.

Results: We demonstrated that DLEU2 was upregulated in cervical cancer tumor tissues. Downregulation of DLEU2 inhibited cell proliferation, induced apoptosis and cell cycle arrest at G2/M phase of cervical cancer cells in vitro, and suppressed tumor growth in vivo. Further, LncRNA DLEU2 is one of the targets of miR-128-3p. miR-128-3p inhibitor abrogated the cell proliferation suppressed by knockdown of DLEU2, apoptosis induced by knockdown of DLEU2 and reversed the expression of cell cycle hallmarks regulated by knockdown of DLEU2.

Conclusion: Taken together, these results suggested knockdown of DLEU2 inhibited cervical cancer progression via targeting miR-128-3p.

Keywords: DLEU2; LncRNAs; cervical cancer; miR-128-3p; tumor growth.

Figure 1

LncRNA DLEU2 is upregulated in cervical cancer tissues and suppresses cell proliferation of cervical cancer cells. (A) The expression of DLEU2 was analyzed by qPCR in cervical cancer tissues and their paired normal tissues. (B) The expression of DLEU2 in HaCaT, HeLa, SiHa, C-33A and CaSKi cells was analyzed by qPCR. (C) The knockdown efficiency of si-DLEU2-1^# and si-DLEU2-2^# was analyzed in HeLa and SiHa cells by qPCR, respectively. (D) The knockdown efficiency of si-DLEU2 pool was analyzed in HeLa and SiHa cells by qPCR. (E) CCK8 assay in HeLa cells. (F) CCK8 assay in SiHa cells. GAPDH was used as an internal standard for DLEU2. **p<0.01.

Figure 2

Silencing DLEU2 induces apoptosis and cell arrest at G2/M phase of cervical cancer cells. (A and B) Cells were stained by Hoechst 33,258 for observation of apoptosis in Hela and SiHa cells. (C and D) FITC-Annexin V/PI-staining in DLEU2 silenced Hela and SiHa cells. (E and F) The cell cycle was analyzed 48 h after knockdown of DLEU2 in Hela and SiHa cells by flow cytometry. The expression of cell cycle hallmarks Cyclin D1 and CDK4 was analyzed by qPCR and Western blot in Hela (G) and SiHa (H) cells after knockdown of DLEU2. GAPDH was used as an internal standard. **p<0.01.

Figure 3

Silencing DLEU2 suppresses cervical cancer growth in vivo. (A) Representative morphologies of the tumors derived from the si-NC Hela cells and si-DLEU2 pool Hela cells (left) and si-NC SiHa cells and si-DLEU2 pool SiHa cells (right). The tumors were then dissected and photographed. The tumor volume curve (B) and tumor weight (C) of the DLEU2 silenced group mice versus the control group mice are expressed as the means±SD (n=6). (D) The expression of cell cycle hallmarks in the mice tumors was detected by qPCR and Western blot in Hela cells. (E) The expression of cell cycle hallmarks in the mice tumors was detected by qPCR and Western blot in SiHa cells. GAPDH was used as an internal standard. *p<0.05.**p<0.01.

Figure 4

DLEU2 is one of targets of miR-128-3p. (A) A predicted binding site of miR-128-2p within the DLEU2 3ʹ-UTR region using Starbase. The binding sequences “CACUGUG” in DLEU2 were mutated to “UGAGACU” for generating DLEU2 MUT. (B) Luciferase reporter assays were used to evaluate the interaction between DLEU2 and miR-128-3p. (C) RNA pull-down assays were used to determine the interaction between DLEU2 and miR-128-3p in SiHa and HeLa cells. (D) RIP assay in SiHa and HeLa cells. The extracted RNA was subjected to miR-128-3p (left) and DLEU2 (right) at the mRNA level using RT-qPCR. (E) The expression of miR-128-3p was analyzed with cervical cancer tissues and their paired normal tissues of 50 cervical cancer patients by qPCR. U6 was used as an internal standard for miR-128-3p and GAPDH was used as an internal standard for DLEU2. (F) Spearman correlation analysis between DLEU2 and miR-128-3p expressions in cervical cancer tissues. **p<0.01.

Figure 5

DLEU2 knockdown-inhibited cervical cancer is dependent on targeting miR-128-3p. MiR-128-3p inhibitor (inh) is utilized in the si-DLEU2 pool cells. (A) CCK8 assay in HeLa cells. (B) CCK8 assay in SiHa cells. (C) Apoptosis of DUEU2-knocked down Hela cells after treatment with miR-128-3p inhibitor. (D) Apoptosis of DUEU2-knocked down SiHa cells after treatment with miR-128-3p inhibitor. (E) The expression of cell cycle hallmarks was analyzed by qPCR and Western blot in Hela cells after treatment with miR-128-3p inhibitor. (F) The expression of cell cycle hallmarks was analyzed by qPCR and Western blot in SiHa cells after treatment with miR-128-3p inhibitor. GAPDH was used as an internal standard. **p<0.01 (siNC vs si-DLEU2 pool), ^##p<0.01 (si-DLEU2 pool vs si-DLEU2 pool+miR-128-3p inh).