Date Published: January 25, 2017
Publisher: Public Library of Science
Author(s): Jia Deng, Mengchang Yang, Rong Jiang, Ning An, Xiaoshan Wang, Bin Liu, Aamir Ahmad.
Long non-coding RNAs (lncRNAs) play important roles in the malignant behavior of cancer. HOTAIR, a well-studied lncRNA, contributes to breast cancer development, and overexpression of HOTAIR predicts a poor prognosis. However, the regulatory role of HOTAIR in the cancer stem-like cell (CSC) subpopulation remains largely unknown. Our goal was to determine the regulatory functions of HOTAIR in the processes of self-renewal capacity, tumor formation and proliferation of CSCs derived from breast cancer.
We first enriched and incubated the CSC population derived from breast cancer cell line MCF7 (CSC-MCF7) or MDA-MB-231 (MB231, CSC-MB231). Self-renewal capacity and tumor formation were assessed in vitro and in vivo to determine the stemness of CSCs. We assessed the impact on ectopically upregulated or downregulated expression of HOTAIR in CSCs by soft agar, self-renewal capacity and CCK-8 assays. The functional domain of HOTAIR was determined by truncation. RT-qPCR and semiquantitative Western blotting were performed to detect the expression levels of genes of interest. Chromatin IP (ChIP) was employed to detect the transcriptional regulatory activity of p53 on its target gene.
After the identification of CSC properties, RT-qPCR analysis revealed that HOTAIR, but not other cancer-associated lncRNAs, is highly upregulated in both CSC-MCF7 and CSC-MB231 populations compared with MCF7 and MB231 populations. By modulating the level of HOTAIR expression, we showed that HOTAIR tightly regulates the proliferation, colony formation, migration and self-renewal capacity of CSCs. Moreover, full-length HOTAIR transcriptionally inhibits miR-34a specifically, leading to upregulation of Sox2, which is targeted by miR-34a. Ectopic introduction of miR-34a mimics reverses the effects of HOTAIR on the physiological processes of CSCs, indicating that HOTAIR affects these processes, including self-renewal capacity; these effects are dependent on the regulation of Sox2 via miR-34a. Interestingly, tight transcriptional regulation of p53 by HOTAIR was found; accordingly, p21 is indirectly regulated by HOTAIR, resulting in cell cycle entry.
These results suggest that HOTAIR is a key regulator of proliferation, colony formation, invasion and self-renewal capacity in breast CSCs, which occurs in part through regulation of Sox2 and p53.
lncRNAs, which are typically non-protein coding transcripts longer than 200 nucleotides, can epigenetically interact with transcription factors, transcriptional activators or repressors, and different subunits of complexes, including RNA polymerase (RNAP) II and even duplex DNA, to function as transcriptional or post-transcriptional regulators . As a result of their regulatory roles, lncRNAs strongly influence the malignant behavior of cancer, such as tumorigenesis, proliferation, apoptosis, chemoresistance and invasiveness . For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), an evolutionarily highly conserved and ubiquitously expressed lncRNA, is reportedly highly upregulated in several human malignancies in addition to lung cancer, and it was found to be tightly associated with clinical parameters and promoted invasion and metastasis . However, the functions of thousands of lncRNAs are still unknown, and the extent of their involvement in tumorigenesis is only beginning to be understood.
CSCs are defined as a small fraction of multipotent cells within a tumor that exhibit self-renewal capability and asymmetric cell division [11, 21]. Accumulated evidence has revealed the critical role of CSCs in cancer aggressiveness, metastasis, recurrence and resistance to chemotherapy in several types of cancers. lncRNAs are also reported to play a critical role in cancer progression and metastasis, and one such lncRNAs, HOTAIR, has attracted much attention due to its heightened expression in cancer tissues. For example, in lung cancer and renal cell carcinoma, upregulation of HOTAIR was clearly associated with larger tumor size, advanced pathological stage and extensive metastasis [4, 22, 23, 24]. Overexpression of HOTAIR not only affects tumor formation but also promotes proliferation, migration and invasion in several types of cancers, including gastric, endometrial and lung cancers [9, 25, 26, 27]. The strong association between HOTAIR expression and cancer led us to address whether upregulation of HOTAIR controls cancer by regulating CSCs.