Induction of long intergenic non-coding RNA HOTAIR in lung cancer cells by type I collagen - PubMed (original) (raw)

Induction of long intergenic non-coding RNA HOTAIR in lung cancer cells by type I collagen

Yan Zhuang et al. J Hematol Oncol. 2013.

Abstract

Background: The tumor microenvironment is a crucial determinant in tumor progression. Interstitial extracellular matrix (ECM), such as type I collagen (Col-1), is aberrantly enriched in the tumor microenvironment and promotes tumor progression. Long intergenic non-coding RNAs (lincRNA) are a new family of regulatory RNAs that modulate fundamental cellular processes via diverse mechanisms.

Findings: We investigated whether the expression of lincRNAs was regulated by the tumor promoting Col-1. In a three-dimensional organotypic culture model using the reconstituted basement membrane ECM Matrigel (rBM 3-D), supplementation of Col-1 disrupted acini, a differentiation feature of well-differentiated lung adenocarcinoma cells, and concurrently induced the expression of a tumor-promoting lincRNA, HOX transcript antisense RNA (HOTAIR). Induction of HOTAIR by Col-1 was diminished by a neutralizing antibody against the Col-1 receptor α2β1 integrin. Col-1 activates the expression of a reporter gene controlled by the human HOTAIR promoter. Moreover the expression of HOTAIR and Col-1 was concurrently up-regulated in human non-small cell lung cancer.

Conclusions: Our findings indicate that tumor-promoting Col-1 up-regulates the expression of HOTAIR in NSCLC cells. These initial results warrant further investigation of HOTAIR and other lincRNA genes in lung tumorigenesis.

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Figures

Figure 1

Figure 1

Induction of HOTAIR Expression by Col-1. A) Total cell RNA was extracted from rBM 3-D ± Col-1 cultures of A549 cells. The RNA levels of HOTAIR were measured using qRT-PCR and normalized to the house-keeping gene 36B4. A fold change was obtained setting the values from rBM 3-D culture to one. B) Similar to part A except that the RNA levels of mouse HOTAIR were compared between rBM ± Col-1 3-D cultures of mK-ras-LE cells. C) Similar to part A except that the RNA levels of H19, MALAT1, and XIST were compared between rBM ± Col-1 3-D cultures of A549 cells. D) The RNA levels of HOTAIR were measured using qRT-PCR in rBM ± Col-1 3-D culture of A549 cells with or without an integrin α2β1 neutralizing antibody (mAb-α2β1). A fold change in the HOTAIR transcript was obtained by setting the values from the control IgG group (CTL IgG) to one. E) Similar to part D except that the experiments were carried out in mK-ras-LE cells. Mean and standard deviations were obtained from three independent experiments. * indicated a P value < 0.05 as determined using unpaired two tail student T test.

Figure 2

Figure 2

Activation of the human HOTAIR promoter by Col-1. A) A549 cells were transfected with either pGL3 Basic luciferase reporter (Basic-Luc) or the human HOTAIR promoter-controlled luciferase reporter in the pGL3 Basic backbone (HOTAIR-Luc). The mRNA levels of the firefly luciferase reporter were measured using qRT-PCR and normalized to the co-transfected renilla luciferase reporter. A fold change in the expression of the firefly luciferase reporter was obtained by setting the values of Basic-Luc to one. B) The mRNA levels of the HOTAIR promoter-controlled luciferase reporter were measured in rBM ± Col-1 3-D cultures of A549 cells using qRT-PCR. A fold change in the mRNA levels of the luciferase reporter was obtained by setting the values from the rBM 3-D culture to one. Mean and standard deviations were obtained from three independent experiments. ** indicated a P value < 0.01 as determined using unpaired two tail student T test.

Figure 3

Figure 3

Elevated expression of HOTAIR and Col-1 in human NSCLC. A) The RNA levels of HOTAIR were measured using qRT-PCR in the RNA samples extracted from the cancer and non-cancer tissue specimens of the patients with NSCLC. The RNA levels of HOTAIR were measured using qRT-PCR and normalized to the house keeping gene β-actin. B) Similar to part A except that the mRNA levels of Col-1 were compared between cancer and non-cancer tissues. Means and standard deviations were obtained from 26 pairs of cancer and non-cancer specimens. ** and *** indicated a P value < 0.01 and 0.001 respectively as determined by paired two tail student T test.

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