lncRNA PART1 and MIR17HG as ΔNp63α direct targets regulate tumor progression of cervical squamous cell carcinoma

Abstract

Cervical cancer (CC) remains one of the leading causes of mortality of female cancers worldwide, with more than 90% being cervical squamous cell carcinoma (CSCC). ΔNp63α is the predominant isoform expressed in cervical epithelial tissues and exerts its antitumor function in CSCC. In this study, we have identified 39 long noncoding RNAs as ΔNp63α targets in CSCC through RNA sequencing and chromatin immunoprecipitation sequencing, in which we further confirmed and focused on the two tumor-related long noncoding RNAs, PART1 (lncPART1) and MIR17HG (lncMIR17HG). Experiments from stable overexpression/knockdown cell lines revealed that lncPART1 and lncMIR17HG regulated cell proliferation, migration, and invasion. In vivo experiments further showed that lncPART1 suppresses tumor growth in CSCC-derived tumors. Examinations of clinical tissues indicated that the expression of lncPART1 was positively correlated with ΔNp63α expression, while lncMIR17HG was negatively correlated with ΔNp63α expression, suggesting that ΔNp63α plays a central role via regulating its direct targets in the progression of CSCC. These findings provide novel insights in targeted therapy of cervical cancers.

Keywords: CSCC; cervical cancer; lncMIR17HG; lncPART1; ΔNp63α.

FIGURE 1

Identification of target long noncoding RNAs (lncRNAs) regulated by ΔNp63α in cervical squamous cancer cells. A, Analyses of RNA sequencing (RNA‐seq) and chromatin immunoprecipitation sequencing (ChIP‐seq); overlapped lncRNAs are shown. B, All the 39 candidate lncRNAs were analyzed by qRT‐PCR in ME‐180/Con and ME‐18 0/shp63 cells. C, qRT‐PCR analysis of the expression of PART1 in the ME180/shp63 transfected with ME180/Con cells group. D, qRT‐PCR analysis of the expression of lncMIR17HG in the ME180/shp63 transfected with ME180/Con cells group. E, The expression level of lncPART1 in normal tissues and cervical cancer tissues. F, The relationship between lncPART1 and P63 in cervical cancer tissues. G, The expression level of lncMIR17HG in normal tissues and cervical cancer tissues. H, The relationship between lncMIR17HG and P63 in cervical cancer tissues. I, ChIP‐seq peaks for both lncRNAs and ChIP efficiency. All the experiments were performed in triplicates. Error bars show SD; data are means ± SEM. n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, based on the Student's t‐test

FIGURE 2

LncPART1 inhibits proliferation of cervical squamous cells in vitro. A, Expression levels of lncPART1 in SiHa/lncPART1 cells (SiHa cells with stable overexpression of lncPART1). B, Cell proliferation curves (as detected by RTCA assay) of SiHa cells in the two groups. C, Colony formation assays of SiHa/Con and SiHa/lncPART1 cells. Quantification of colony formation number is also shown. D, Cell cycle distribution analyses of SiHa/Con and SiHa/lncPART1 cells. Quantification of colony formation number is also shown. E, Representative images of wound healing in SiHa/Con and SiHa/lncPART1 cells. Quantification of healing rate is also shown. F, Representative images of transwell migration (up) and Matrigel invasion assays (down) of SiHa/Con and SiHa/lncPART1 cells. Quantification of migrating cells is also shown. All the experiments were performed in triplicates. Error bars show SD; data are means ± SEM. n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, based on the Student's t‐test

FIGURE 3

LncPART1 inhibits proliferation of cervical squamous cells in vitro. A, Expression levels of lncPART1 in ME180/shlncPART1‐1 and shlncPART1‐2 cells (ME‐180 cells with stable knockdown of lncPART1). B, Cell proliferation curves (as detected by RTCA assay) of SiHa cells in the three groups. C, Colony formation assays of ME‐180/Con, ME180/shlncPART1‐1, and shlncPART1‐2 cells. Quantification of colony formation number is also shown. D, Cell cycle distribution analyses of ME‐180/Con, ME180/shlncPART1‐1, and shlncPART1‐2 cells. Quantification of colony formation number is also shown. E, Representative images of wound healing in ME‐180/Con, ME180/shlncPART1‐1, and shlncPART1‐2 cells. Quantification of healing rate is also shown. F, Representative images of transwell migration (up) and Matrigel invasion assays (down) of ME‐180/Con, ME180/shlncPART1‐1. and shlncPART1‐2 cells. Quantification of migrating cells is also shown. All the experiments were performed in triplicates. Error bars show SD; data are means ± SEM. n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, based on the Student's t‐test

FIGURE 4

LncPART1 inhibits proliferation of cervical squamous cells in vivo. A, B, Images of the xenograft tumors in the SiHa/Con and SiHa/lncPART1 cells groups 30 days after injection. C, The tumor weight distribution in the SiHa/Con and SiHa/lncPART1 cells groups. D, E, Images of the xenograft tumors in the ME‐180/Con, ME180/shlncPART1‐1, and shlncPART1‐2 cells groups 30 days after injection. F, The tumor weight distribution in the ME‐180/Con, ME180/shlncPART1‐1, and shlncPART1‐2 cells groups. All the experiments were performed in triplicates. Error bars show SD; data are means ± SEM. n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, based on the Student's t‐test

FIGURE 5

LncMIR17HG promotes proliferation of cervical squamous cells in vitro. A, Expression levels of lncMIR17HG in SiHa/shlncMIR17HG cells (SiHa cells with knockdown of lncMIR17HG). B, Proliferation curves (as detected by RTCA assay) of SiHa/shlncMIR17HG cells compared with the corresponding controls. C, Colony formation assays of SiHa/Con and SiHa/shlncMIR17HG cells. Quantification of colony formation number is also shown. D, Representative images of wound healing in SiHa/Con and SiHa/shlncMIR17HG cells. Quantification of healing rate is also shown. E, Representative images of transwell migration (up) and Matrigel invasion assays (down) of SiHa/Con and SiHa/shlncMIR17HG cells. Quantification of migrating cells is also shown. All the experiments were performed in triplicates. Error bars who SD; data are means ± SEM. n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, based on the Student's t‐test