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. 2020 Mar 6:19:546-561.
doi: 10.1016/j.omtn.2019.11.027. Epub 2019 Nov 29.

LINC01413/hnRNP-K/ZEB1 Axis Accelerates Cell Proliferation and EMT in Colorectal Cancer via Inducing YAP1/TAZ1 Translocation

Ling Ji  1 , Xiang Li  2 , Zhenhua Zhou  1 , Zhihai Zheng  1 , Li Jin  1 , Feizhao Jiang  3
Affiliations

LINC01413/hnRNP-K/ZEB1 Axis Accelerates Cell Proliferation and EMT in Colorectal Cancer via Inducing YAP1/TAZ1 Translocation

Ling Ji et al. Mol Ther Nucleic Acids. .

Abstract

Long non-coding RNAs (lncRNAs) are crucial molecules in tumorigenesis and tumor growth in various human cancers, including colorectal cancer (CRC). Studies have revealed that lncRNAs can regulate cellular processes in cancers by interacting with proteins, for example RNA-binding proteins (RBPs). In this study, we recognize a novel lncRNA called LINC01413 that is upregulated in CRC tissues through lncRNAs microarray. Subsequently, we confirmed that an elevated level of LINC01413 expression in CRC tissues was strongly correlated to clinicopathological features, such as tumor size, tumor stage, lymph node metastasis, and distant metastasis, and its association with poor overall survival was also revealed. Additionally, LINC01413 facilitates cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Also, silenced LINC01413 restrains tumor growth in vivo. Moreover, LINC01413 binds with hnRNP-K and induces YAP1 (yes-associated protein 1)/TAZ1 (tafazzin) nuclear translocation to regulate the expression of ZEB1 in CRC cells. Taken together, this research suggested LINC01413 as a positive regulator in CRC progression through the LINC01413/hnRNP-K/TAZ1/YAP1/ZEB1 axis, broadening a new view on CRC treatment.

Keywords: CRC; LINC01413; YAP1/TAZ1; ZEB1; hnRNP-K.

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Figures

Figure 1
Figure 1
LINC01413 Is Upregulated in CRC Tissues and Cell Lines and Indicates Poor Prognosis for CRC Patients (A) High-throughput RNA sequencing of total cellular RNA obtained from three pairs of CRC tissues and corresponding normal tissues was performed to explore the transcriptome changes in CRC. (B) Quantitative real-time PCR results of the expression of the five most altered lncRNAs in CRC cell lines. (C) Relative high level of LINC01413 was determined in CRC cells compared with normal NCM460 cell. Result was obtained using quantitative real-time PCR. (D) Quantitative real-time PCR detected the higher LINC01413 expression in CRC tissues than that in non-tumor tissues. (E) The expression levels of LINC01413 in tissues with or without distant metastasis were accessed by quantitative real-time PCR. (F) LINC01413 expression in different TNM stages of CRC was evaluated by quantitative real-time PCR. (G) Kaplan-Meier analysis (log-rank test) was used to estimate the relationship between LINC01413 expression and overall survival of CRC patients. Error bars show the mean ± SD of more than three independent experiments. *p < 0.05, **p < 0.01 versus control group.
Figure 2
Figure 2
LINC01413 Regulates Cell Proliferation, Apoptosis, Migration, Invasion, and EMT in CRC Cells (A) LoVo cells transfected with shLINC01413 show a relatively low level of LINC01413 (left), and HT-29 cells transfected with pGIPZ/LINC01413 exhibit a relatively high expression of LINC01413 (right). (B) MTT assay revealed that knockdown of LINC01413 inhibits cell survival, whereas overexpression of LINC01413 enhances cell survival, compared to the control groups. (C and D) Flow cytometry analysis was used to determine the apoptosis rate of LoVo (C) or HT-29 (D) cells after transfection. (E-H) Transwell assays demonstrated that cells with higher level of LINC01413 with the higher migratory and invasive ability compared with cells with lower level of LINC01413. (I) Immunofluorescence staining indicates that the dysregulation of LINC01413 affects the distribution of E-cadherin and N-cadherin in CRC cells. (J and K) The expression levels of EMT-associated genes in transfected CRC cells were detected by quantitative real-time PCR. (L) Western blot assays were performed to detect the protein levels of the epithelial and mesenchymal markers in response to LINC01413 inhibition or overexpression. Error bars show the mean ± SD of more than three independent experiments. *p < 0.05, **p < 0.01 versus control group.
Figure 3
Figure 3
LINC01413 Regulates Tumorigenesis and Tumor Metastasis of CRC In Vivo (A) Images of tumors formed from shCtrl or shLINC01413-transfected LoVo cells. (B and C) shLINC01413 transfection decreased the volume tumor (B) and weight (C) in vivo. (D-E) Number of metastasis nodules in shLINC01413 group was more than shCtrl group. (F) Western blot reveals that the level of E-cadherin is remarkably increased while the level of ZEB1 and N-cadherin is decreased in tumors originating from LINC01413-silenced LoVo cells when compared to tumors from shCtrl-transfected cells. Error bars show the mean ± SD of more than three independent experiments. **p < 0.01 versus control group.
Figure 4
Figure 4
LINC01413 Modulates ZEB1 Expression through Promoting YAP1/TAZ1 Complex Translocation into Nucleus (A and B) The expressions of LINC01413, ZEB1, YAP1, and TAZ1 in LoVo cells transfected with shLINC01413 or shCtrl (A) and in HT-29 cells transfected with pGIPZ/LINC01413 or pGIPZ/control (B) were detected by quantitative real-time PCR. (C) The protein levels of ZEB1, YAP1, and TAZ1 in LoVo and HT-29 cells after transfection were estimated by western blot assay. (D and E) Western blot results of the expression of TAZ1 and YAP1 in cytoplasm or nucleus of LoVo (D) and HT-29 (E) cells after silencing or overexpressing LINC01413, respectively. (F) Quantitative real-time PCR analysis of ZEB1 expression in CRC tissues and adjacent non-tumor tissues. (G) Spearman’s correlation analysis suggests a positive correlation between ZEB1 expression and LINC01413 in CRC tissues. (H) The binding site of TEAD4 in the region of ZEB1 promoter was predicted by JASPAR. (I) ChIP assay proves that TEAD4, YAP1, and TAZ1 all bind to the ZEB1 promoter. (J and K) Luciferase reporter assays were performed to evaluate the function of YAP1/TAZ1 (J) or LINC01413 (K) in ZEB1 transcription. Error bars show the mean ± SD of more than three independent experiments. **p < 0.01 versus control group.
Figure 5
Figure 5
LINC01413 Stimulates ZEB1 Expression and the Nuclear Translocation of the YAP1/TAZ1 Complex by Binding with hnRNP-k (A) A western blot assay after a RNA pull-down assay was applied to test protein interaction with LINC01413. (B) RNA pull-down showed the enrichment of hnRNP-k in response to LINC01413 compared with negative control IgG. (C) RIP assay detected the enrichment of LINC01413 in anti-hnRNP-k group compared with anti-IgG. (D) The specific interaction between LINC01413 and hnRNP-K was confirmed by an RNA pull-down assay. (E) The locations of LINC01413 and hnRNP-K in LoVo cells were assessed using FISH. (F) The cross-talk among LINC01413, hnRNP-K, YAP1, and TAZ1 in CRC cells was identified via a coIP assay. (G and H) The impact of the LINC01413/hnRNP-K axis on the nuclear translocation of YAP and TAZ was assessed by IF staining (G) and subcellular fractionation followed by western blot analysis (H). Error bars show the mean ± SD of more than three independent experiments. **p < 0.001 versus control group.
Figure 6
Figure 6
LINC01413/hnRNP-K/TAZ1/YAP1/ZEB1 Axis Stimulates the Formation of a Malignant Phenotype in CRC (A and B) Western blot analysis reveals that LINC01413 affects the expression of ZEB1, YAP1, and TAZ1 (A) as well as the distribution of YAP1 and TAZ1 (B) via an hnRNP-K-dependent pathway. (C-D) MTT assay and flow cytometry analysis showed the cell viability and apoptosis in LINC01413-downregulated -upregulated cells were recovered after overexpression or knockdown of hnRNP-k. (E and F) The migration (E) and invasion (F) capacities of cells under different conditions were evaluated by a Transwell assay. (G) Western blot analysis determines the levels of proteins involved in EMT in CRC cells facing diverse situations. Error bars show the mean ± SD of more than three independent experiments. **p < 0.01 versus control group.
Figure 7
Figure 7
LINC01413 Promotes Nuclear Translocation of YAP1/TAZ1 by Recruiting hnRNP-K and Then Stimulates ZEB1 Expression, Thus Enhancing EMT and Metastasis in CRC
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