LncRNA BBOX1-AS1 upregulates HOXC6 expression through miR-361-3p and HuR to drive cervical cancer progression

Abstract

Objectives: Over the past years, growing attention has been paid to deciphering the pivotal role of long non-coding RNAs (lncRNAs) in regulating the occurrence and development of human malignancies, cervical cancer (CC) included. Nonetheless, the regulatory role of lncRNA BBOX1 antisense RNA 1 (BBOX1-AS1) has not been explored as yet.

Material and methods: The expression of BBOX1-AS1 was detected by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), colony formation, TUNEL, Western blot, transwell and immunofluorescence assays testified the critical role of BBOX1-AS1 in CC. The relationship between RNAs (BBOX1-AS1, miR-361-3p, HOXC6 and HuR) was analysed by luciferase reporter, RNA Immunoprecipitation (RIP) and RNA pull-down assays.

Results: BBOX1 antisense RNA 1 antisense RNA 1 was revealed to be highly expressed in CC. Decreased expression of BBOX1-AS1 had suppressive effects on CC cell growth and migration. Molecular mechanism assays verified that BBOX1-AS1 had negative interaction with miR-361-3p in CC. Additionally, homeobox C6 (HOXC6) was validated to be a downstream target of miR-361-3p in CC. Furthermore, ELAV-like RNA-binding protein 1, also known as HuR, was uncovered to be capable of regulating the mRNA stability of HOXC6 in CC. More importantly, rescue assays delineated that knockdown of HuR after overexpressing miR-361-3p could reverse BBOX1-AS1 upregulation-mediated effect on CC progression. Similarly, the function induced by BBOX1-AS1 upregulation on CC progression could be countervailed by HOXC6 depletion.

Conclusions: BBOX1 antisense RNA 1 facilitates CC progression by upregulating HOXC6 expression via miR-361-3p and HuR.

Keywords: BBOX1-AS1; CC; HOXC6; HuR; miR-361-3p.

FIGURE 1

BBOX1 antisense RNA 1 (BBOX1‐AS1) expression is significantly elevated in cervical cancer (CC) tissues and cells. A, Venn diagram showed the overlaps of the analysis. B, The expression levels of these long non‐coding RNAs were detected by RT‐qPCR in 3 paired CC tissues and corresponding non‐cancer tissues. C, RT‐qPCR detected BBOX1‐AS1 expression in CC tissues and matched normal tissues. D, BBOX1‐AS1 expression was analysed via RT‐qPCR in early or advanced stages of CC patients. E, The expression of BBOX1‐AS1 in CC cell lines (C33A, CaSki, HeLa and SiHa) and normal cervical epithelial cell line (H8) was examined by RT‐qPCR. F, Subcellular fractionation and FISH assays were adopted for the detection of the subcellular localization of BBOX1‐AS1 in CC cells. ^** P < .01

FIGURE 2

Low expression of BBOX1 antisense RNA 1 (BBOX1‐AS1) represses cervical cancer (CC) progression. A, The efficiency of BBOX1‐AS1 knockdown in SiHa and HeLa cells was measured by RT‐qPCR. B and C, The proliferation ability of SiHa and HeLa cells transfected with sh‐BBOX1‐AS1#1 or sh‐NC was evaluated by CCK‐8 and colony formation assays. D and E, TUNEL and Western blot were carried out to verify the promoting function of BBOX1‐AS1 knockdown on cell apoptosis. F‐H, Cell metastasis in transfected cells was analysed by transwell, Western blot and immunofluorescence. ^** P < .01

FIGURE 3

BBOX1 antisense RNA 1 (BBOX1‐AS1) sponges miR‐361‐3p in cervical cancer (CC). A, Target miRNAs of BBOX1‐AS1 were obtained through searching LncBase and lncRNASNP2. B, The expression of these miRNAs was detected in 100 pairs of CC samples and adjacent normal specimens through RT‐qPCR analysis. C, RNA pull‐down assay was conducted to analyse the binding capacity between BBOX1‐AS1 and above miRNAs in SiHa and HeLa cells. D, MiR‐361‐3p expression in CC cell lines and normal cervical epithelial cell line was detected via RT‐qPCR. E, Pearson's correlation analysis analysed the correlation between BBOX1‐AS1 and miR‐361‐3p. F, A binding site between BBOX1‐AS1 and miR‐361‐3p was displayed. G and H, Luciferase reporter and RIP assays testified the interaction between BBOX1‐AS1 and miR‐361‐3p. I, The efficiency of BBOX1‐AS1 and BBOX1‐AS1 (mut) overexpression was analysed through RT‐qPCR. J‐L, CCK‐8, colony formation assay and transwell assays were, respectively, applied to analyse cell proliferation and metastasis in transfected cells. ^** P < .01

FIGURE 4

Homeobox C6 (HOXC6) is testified to be a downstream target of miR‐361‐3p in cervical cancer (CC). A, Through utilizing starBase, 12 mRNAs that predicted to have binding capacity with miR‐361‐3p in PITA, miRmap, microT and PicTar databases were screened out. B, The expression level of HOXC6 in CC tissues and neighbouring non‐tumour tissues was obtained from GEPIA. C, RT‐qPCR was used to detect the expression of HOXC6 in CC tissues and neighbouring non‐tumour tissues. D, The expression of HOXC6 in CC cells and H8 cells was detected via RT‐qPCR and Western blot analyses. E, Pearson's correlation analysis revealed the correlation between HOXC6 and miR‐361‐3p (or BBOX1 antisense RNA 1 [BBOX1‐AS1]). F, A binding site between miR‐361‐3p and HOXC6 was predicted via starBase. G, The efficiency of HOXC6 overexpression in SiHa and HeLa cells was examined by RT‐qPCR. H and I, The interaction between RNAs (BBOX1‐AS1, miR‐361‐3p and HOXC6) was confirmed by luciferase reporter and RIP assays. J, The expression of HOXC6 in different groups was analysed via RT‐qPCR. ^* P < .05, ^** P < .01

FIGURE 5

BBOX1 antisense RNA 1 (BBOX1‐AS1) promotes the mRNA stability of homeobox C6 (HOXC6) via HuR in cervical cancer (CC). A, The mRNA expression of HOXC6 was detected by utilizing RT‐qPCR after SiHa and HeLa cells were treated with Actinomycin D. B, Through searching starBase, 4 RBPs (U2AF2, ELAVL1, LARP4B and RBM10) were found to be able to bind with BBOX1‐AS1 (or HOXC6). C, RNA pull‐down assay demonstrated that only ELAVL1 (also known as HuR) bound with BBOX1‐AS1 in SiHa and HeLa cells. D, RIP assay verified that HuR could bind with BBOX1‐AS1 (or HOXC6) in SiHa and HeLa cells. E, Efficiency of indicated shRNAs for HuR silencing was identified through RT‐qPCR in SiHa and HeLa cells. F, Stability of HOXC6 mRNA was assessed in transfected cells treated with Actinomycin D was analysed in several different time points. G, RIP assays were utilized to analyse the changes in the interaction between HOXC6 mRNA and HuR under BBOX1‐AS1 silence. H, Effect of BBOX1‐AS1 inhibition on HuR expression was evaluated by RT‐qPCR and Western blot analyses. I, The efficiency of HOXC6 knockdown was measured by RT‐qPCR. J, The expression of HOXC6 in SiHa and HeLa cells transfected with different plasmids was detected via RT‐qPCR. ^** P < .01

FIGURE 6

BBOX1 antisense RNA 1 (BBOX1‐AS1) upregulates homeobox C6 (HOXC6) to accelerate cervical cancer (CC) progression via miR‐361‐3p and HuR. A and B, CCK‐8 and colony formation assays were applied to analyse the proliferation ability of transfected cells. C and E, Cell metastasis in transfected cells was assessed by transwell, Western blot and immunofluorescence. ^** P < .01

FIGURE 7

BBOX1 antisense RNA 1 (BBOX1‐AS1) downregulation inhibits the in vivo tumorigenesis of CC. A‐C, Tumour growth, tumour volume and tumour weight were analysed after transfected SiHa cells were injected subcutaneously into nude mice. D, The expression of BBOX1‐AS1, homeobox C6 (HOXC6), miR‐361‐3p and HuR was detected in transfected cells. E, Immunohistochemistry was applied to detect the expression of Ki67, E‐cadherin, PCNA and N‐cadherin in transfected cells. ^** P < .01