LncRNA MIR4435-2HG drives cancer progression by modulating cell cycle regulators and mTOR signaling in stroma-enriched subtypes of urothelial carcinoma of the bladder

Abstract

Background: The risk for recurrence and metastasis after treatment for urothelial carcinoma of the bladder (UCB) is high. Therefore, identifying efficient prognostic markers and novel therapeutic targets is urgently needed. Several long noncoding RNAs (lncRNAs) have been reported to be correlated with UCB progression. In this study, we found that the subtype-specific lncRNA MIR4435-2 host gene (MIR4435-2HG) plays a novel oncogenic role in UCB.

Methods: RNA-Seq data of TCGA/BLCA were analyzed. The expression of MIR4435-2HG was measured by qRT-PCR in 16 pairs of bladder cancer tissues and adjacent normal tissues. The clinical relecance of MIR4435-2HG was validated via in situ hybridization performed on an in-house cohort of 116 UCB patient samples. RNA pull-down followed by mass spectrometry was performed to identify MIR4435-2HG-binding proteins. To identify signaling pathways involved in MIR4435-2HG activity, comprehensive in vitro and in vivo studies and RNA-Seq assays were performed using UCB cells in which MIR4435-2HG expression was knocked down or exogenously overexpressed. In addition, we performed RNA immunoprecipitation and Western blot analyses to validate the identified MIR4435-2HG-binding proteins and to determine the molecular mechanisms by which MIR4435-2HG promotes UCB progression.

Results: We found that MIR4435-2HG was significantly upregulated in the stromal-enriched subtype of UCB. Increased MIR4435-2HG expression was positively correlated with a high histological grade, advanced T stages, larger tumors, lymph node metastasis and a poor prognosis. In vitro experiments revealed that MIR4435-2HG expression silencing suppressed cell proliferation and induced apoptosis. Inhibition of MIR4434-2HG delayed xenograft tumor growth, while MIR4435-2HG overexpression reversed the MIR4435-2HG silencing-induced inhibition of UCB tumor phenotype acquisition. Mechanistically, we found that MIR4435-2HG positively regulated the expression of a variety of cell cycle regulators, including BRCA2 and CCND1. Knocking down MIR4435-2HG increased the sensitivity of tumor cells to the VEGFR inhibitor cediranib. Furthermore, we found that MIR4435-2HG regulated mTOR signaling and epithelial-mesenchymal transition (EMT) signaling pathways by modulating the phosphorylation of mTOR, 70S6K and 4EBP1. Finally, we confirmed that MIR4435-2HG enhances tumor metastasis through regulation of the EMT pathway.

Conclusions: Our data indicate that upregulated MIR4435-2HG expression levels are significantly correlated with a poor prognosis of UCB patients. MIR4435-2HG promotes bladder cancer progression, mediates cell cycle (de)regulation and modulates mTOR signaling. MIR4435-2HG is an oncogenic lncRNA in UCB that may serve as a diagnostic and therapeutic target.

Keywords: LncRNA; MIR4435-2HG; Molecular subtype; Urothelial carcinoma of the bladder (UCB); mTOR.

Fig. 1

Relative MIR4435-2HG expression in UCB tissues and its clinical significance. A MIR4435-2HG expression in bladder cancer tissues (n = 16) compared with normal tissues (n = 16) examined by qRT-PCR and normalized to GAPDH expression. B Expression of MIR4435-2HG in 116 UCB tissues detected by in situ hybridization. Score from 0 to 3. C Kaplan–Meier overall survival curves according to MIR4435-2HG expression levels. D H-scores of MIR4435-2HG in patients with different tumor grades. E H-scores of MIR4435-2HG in patients with different clinical T stages. F H-scores of MIR4435-2HG in patients with different LN statues. G Expression of MIR4435-2HG in the TCGA/BLCA cohort. H Kaplan–Meier overall survival curves according to MIR4435-2HG expression levels in the TCGA/BLCA cohort.*p < 0.05, ***p< 0.001

Fig. 2

Expression of MIR4435-2HG correlates with the stroma-enriched subtype of UCB. A Immune cell infiltration (red arrows) in stroma-enriched tumors with a high MIR4435-2HG expression. B-C The expression of MIR4435-2HG is significantly elevated in the basal/squamous subtype and luminal-infiltrated subtype of UCB. D-E Scatter plots to illustrate the correlation of the expression of MIR4435-2HG with the stromal score and the immune score of UCB. F-G Scatter plots to illustrate the correlation of MIR4435-2HG expression with stroma markers and immune cell markers. H–K Co-expression network of MIR4435-2HG predicting the survival of the UCB. Note, H indicates the ROC curves for the prediction of the survival in the training set; I indicates the HRs for the high- and low-risk groups in the training dataset; J indicates the ROC curves for the prediction of the survival in the test dataset; K indicates the HRs for the high- and low- risk groups in the test dataset

Fig. 3

Effects of MIR4435-2HG on UCB cell proliferation in vitro and in vivo. A Nuclear fractionation experiment and qRT-PCR detection of the abundance of MIR4435-2HG in the nucleus and the cytoplasm. GAPDH was used as positive control for the cytoplasm, and U6 was positive control for the nucleus. B Downregulation and upregulation of MIR4435-2HG in T24 and UM-UC-3 cells after siRNA or pcDNA3.1( +)-MIR4435-2HG plasmid transfection, respectively. C-D CCK-8 assay-based detection of the viability of si-MIR4435-2HG or pcDNA-MIR4435-2HG transfected T24 and UM-UC-3 cells. E–F Colony formation assay-based determination of the proliferation of transfected UCB cells. Colonies were counted and captured. Values are presented as the mean ± s.d of three independent experiments. G-H T24 cells transfected with empty vector or sh-MIR4435-2HG were injected into the nude mice (n = 10). Tumors before and after inoculation of the nude mice. I Tumor volumes were calculated every 3 days. J Tumor weights were measured after tumor removal. K qRT-PCR was used to examine the average expression of MIR4435-2HG in tumor tissues formed from T24/empty vector and T24/sh-MIR4435-2HG cells. L Ki67 was detected by immunohistochemistry. *p < 0.05, **P < 0.01, ***p < 0.001

Fig. 4

MIR4435-2HG affectes cell cycle progression in UCB cells. T24 and UM-UC-3 cells were transfected with si-NC or si-MIR4435-2HG 1#, 2#. A-B Flow cytometry was performed to determine the cell cycle in T24 and UM-UC-3 cells. C A heatmap representing mRNA expression levels in the T24 and UM-UC-3 cells transfected with control or MIR4435-2HG siRNA for 48 h (U3: UM-UC-3). D Pathways for enrichment analysis of differential genes. E–H The differentially expressed genes detected by RNA-seq were verified in T24 and UM-UC-3 cells by qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

MIR4435-2HG affects the apoptosis in UCB cells. A-B Flow cytometry was performed to determine the apoptotic rates of cells. Values are presented as the mean ± s.d of three independent experiments. C Expression of PARP-1 detected by Western blotting in T24 and UM-UC-3 cells. D-E IC₅₀ of cediranib in T24 and UM-UC-3 cells with or without si-MIR4435-2HG#1,2. F-I Flow cytometry was performed to detect the apoptotic rates of sh-MIR4435 T24 and UM-UC-3 cells with or without stimulation of cediranib. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

MIR4435-2HG promotes the migration and invasion of UCB cells through the EMT pathway. A, B Transwell assay showing changes in the migration ability of T24 and UM-UC-3 cells following knockdown or overexpression of MIR4435-2HG. C, D Transwell assay with Matrigel invasion chambers to evaluate changes in the invasion ability of T24 and UM-UC-3 cells following knockdown or overexpression of MIR4435-2HG. E, F Scratch wound healing assay showing cell migration after knockdown or overexpression of MIR4435-2HG in T24 and UM-UC-3 cells. G Western-blot analysis of the EMT signaling pathway in the MIR4435-2HG-depleted T24 and UM-UC-3 cells. H Western-blot analysis of the EMT signaling pathway in MIR4435-2HG overexpressing UCB cells

Fig. 7

MIR4435-2HG directly interacts with mTOR to play a key role in bladder cancer. A Enrichment analysis of mass spectrometry data of T24 cells. B Kinases associated with cancer pathways among PPI proteins. C RIP was performed in T24 cells after which the co-precipitated RNA was subjected to qRT-PCR for MIR4435-2HG. Expression levels of MIR4435-2HG RNA are presented as fold enrichment in SMARCC1, EIF4B and mTOR relative to IgG immunoprecipitation. D, E Western blotting was performed to detected the expression of proteins in the AKT/mTOR pathway. F Expression of P-S6K and vimentin detected by Western blotting in T24 and UM-UC-3 cells with or without stimulation of rapamycin. G Mechanistic diagram of MIR4435-2HG in UCB