MIR155HG promotes metastasis and cisplatin resistance of cervical cancer cells by regulating the miR-409-3p and ZEB1 axis

Abstract

Long non-coding RNA (lncRNA) exhibits a crucial role in multiple human malignancies. The expression of lncRNA MIR155HG, reportedly, is aberrantly up-regulated in several types of tumors. In this research, we studied the role and mechanism of MIR155HG in the progression of cervical cancer (CC). We analyzed the expression pattern of MIR155HG in CC patients using public database TCGA, and overexpressed its expression in HeLa cells to explore its cellular functions. Cellular phenotype experiments were conducted to assess the influence of MIR155HG on HeLa cells, including proliferation, cell cycle, migration, invasion, and cisplatin sensitivity. Then we evaluated the impact on epithelial-mesenchymal transition (EMT) of MIR155HG and identified the underlying regulatory mechanism. We found that MIR155HG was highly expressed and positively correlated with overall survival (OS) prognosis results in CC patients. Overexpression of MIR155HG (MIR155HG-OE) demonstrated higher proliferation and migration levels in HeLa cells, while MIR155HG inhibited the apoptosis rate of HeLa cells. Meanwhile, we found MIR155HG can reduce the effect of cisplatin sensitivity on HeLa cells. Besides this, MIR155HG-OE significantly changed the expression pattern of EMT biomarkers, including ZEB1, TWIST1, Vimentin, N-cadherin, and E-cadherin. To explore the underlying mechanism, we found MIR155HG can promote ZEB1 expression by sponging miR-409-3p, forming a competitive endogenous RNA system to inhibit cisplatin sensitivity. In this study, we deeply explored the molecular and cellular functions of MIR155HG in CC cells. Our results highlight the important role and potential targeting value of MIR155HG in CC pathogenesis and development.

Keywords: Cervical cancer; Cisplatin; MIR155HG; ZEB1; miR-409-3p.

Fig. 1

MIR155HG was dysregulated in CESC patients and associated with the prognosis results of CESC patients. (A) Box plot showing the expression level of MIR155HG in normal and primary tumor samples of CESC patients. Student’s t-test, P-value < 0.01. (B) Box plot showing the methylation level at the promoter region of MIR155HG in normal and primary tumor samples of CESC patients. Student’s t-test, P-value < 0.01. (C) Line plot showing the different OS probability of two CESC patient groups divided by MIR155HG expression levels. (D) The same as (C) but for the OS analysis result by GEPIA2 software. (E) Bar plot showing the cellular location of MIR155HG in HeLa cells by RT-qPCR experiment. N = 5; Student’s t-test, *** p-value < 0.001.

Fig. 2

The effects of MIR155HG on proliferation,migration and invasion of CC cells. (A) Bar plot showing the successful overexpression of MIR155HG in CC cells by RT-qPCR experiment; N = 6. (B) Cell flow cytometry and bar plot showing the decreased apoptosis level of CC cells by MIR155HG-OE; N = 3. (C) Bar plot showing the increased proliferation level of CC cells by MIR155HG-OE; N = 3. (D) Cell flow cytometry and bar plot showing the percentage of cells at different cell cycle states; N = 3. Two-way ANOVA test, * P < 0.05. (E) Cell culture and bar plot showing the increased migration and invasion levels of CC cells by MIR155HG-OE; N = 9. Two-way ANOVA test, * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001.

Fig. 3

MIR155HG significantly regulated expression levels of EMT biomarkers. (A) Bar plot showing the quantitative results for RT-qPCR results. N = 5; Student’s t-test, *** p-value < 0.001, **** p-value < 0.0001. (B) Western blot result showing the expression levels of EMT biomarkers. (C) Quantitative results for the WB experiment in (B). N = 3; Student’s t-test, ** p-value < 0.01.

Fig. 4

Effect of MIR155HG on cisplatin sensitivity of cervical cancer cells. (A) Growth inhibition of Hela cells was observed at different concentrations of cisplatin by CCK-8 method (mean ± STD, n = 5). (B) Growth inhibition of Hela cells was reduced after overexpression of MIR155HG at different concentrations of cisplatin. (C) Plate clonality assays was used to measure the impact of cisplatin to cell clonality in Hela cells after overexpression of MIR155HG. (D) Flow cytometry was adopted to examine apoptotic rate in Hela cells with cisplatin after overexpression of MIR155HG. (E). Effect of cisplatin on proliferation of Hela cells after overexpression of MIR155HG at different time. N = 3 for C-D, and N = 5 for A-B and E; One-way ANOVA test, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001; ns, non-significant.

Fig. 5

MIR155HG interacted with miR-409-3p and repressed its expression in HeLa cells. (A) Base pair presentation for MIR155HG and has-miR-409-3p that was predicted by LncTar. (B) Bar plot showing the quantitative results for dual luciferase reporter assay. N = 3; Student’s t-test, ** p-value < 0.01, **** p-value < 0.0001. (C) Bar plot showing the expression level for miR-409-3p in three groups. N = 3; Student’s t-test, **** p-value < 0.0001.

Fig. 6

MIR155HG regulated the expression of ZEB1 in HeLa cells. (A) Bar plot showing the expression level changes of ZEB1 in three groups. (B) Western blot showing the expression pattern of ZEB1 in HeLa cells. Left panel was the gel result, and right panel was the quantitative result. (C) Base pair presentation for ZEB1 and has-miR-409-3p that was predicted by TargetScan. (D) Bar plot showing the expression levels of MIR155HG and its target genes after cisplatin intervention. N = 5; Student’s t-test, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.