HOTAIR induces the ubiquitination of Runx3 by interacting with Mex3b and enhances the invasion of gastric cancer cells
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Long non-coding RNAs (LncRNAs) exert their functions mainly by binding to their corresponding proteins. Runt-related transcription factor 3 (Runx3) is an important transcription factor that functions as a tumor suppressor in gastric cancer. Whether there is an interplay between LncRNAs and Runx3 remains unclear.
RPISeq was applied to screen the LncRNAs that potentially bind to Runx3. The interaction between LncRNA HOX antisense intergenic RNA (HOTAIR) and Runx3 was validated by RNA Immunoprecipitation and RNA pull-down assays. The role of Mex3b in the ubiquitination of Runx3 induced by HOTAIR was assessed by immunoprecipitation. Pearson’s correlation between HOTAIR mRNA expression and Runx3 protein expression was analyzed. Cell migration and invasion were explored by transwell assays.
We found that HOTAIR was bound to Runx3 protein and identified the fragment of HOTAIR spanning 1951–2100 bp as the specific binding site. In addition, mex-3 RNA binding family member B (Mex3b) was an E3 ligase involved in HOTAIR-induced ubiquitous degradation of Runx3. Silencing the expression of HOTAIR or Mex3b attenuated the degradation of Runx3. In human gastric cancer tissues, HOTAIR was negatively associated with the expression level of Runx3 protein (Pearson coefficient − 0.501, p = 0.025). Inhibition of HOTAIR significantly suppressed gastric cancer cell migration and invasion through upregulating claudin1, which could be reversed by co-deficiency of Runx3.
These results uncovered the novel interaction between HOTAIR and Runx3, and provided potential therapeutic targets on the metastasis of gastric cancer.
KeywordsHOTAIR Runx3 Gastric cancer Ubiquitination
Runt-related transcription factor 3 (Runx3) is a transcription factor with 3 different splice variants. Variants 1 and 3 share the same amino sequence, while variant 2 has a shorter amino sequence. Runx3 inhibits the metastasis of malignant tumors, and a list of invasiveness-related genes has been validated as downstream targets [1, 2]. The expression of Runx3 was significantly downregulated in gastric cancer . Both transcription mechanism such as methylation  and post-transcription regulation including ubiquitination change  were responsible for the low expression of Runx3.
Long non-coding RNAs (LncRNAs) are non-protein-coding transcripts (> 200 nt) that have gained widespread attention as a crucial regulator of cancer development . Previous studies have provided evidence that LncRNAs might regulate gene expression and post-transcription modification by multiple mechanisms . Binding to their corresponding proteins, especially transcription factors, is considered as one of the most common mechanisms . However, whether LncRNAs could bind to Runx3 and affect its expression remains unclear.
In this study, HOTAIR (HOX antisense intergenic RNA), an LncRNA localizing in the boundaries of Hox gene cluster, was screened out to be a potential upstream LncRNA regulating Runx3. HOTAIR is an oncogene responsible for malignant behavior in various types of cancer , including the invasiveness of gastric cancer . Here, we identified the interaction between HOTAIR and Runx3, and validated that HOTAIR induced the invasiveness of gastric cancer through Runx3.
This study was approved by the Ethics Committee of the Second Affiliated Hospital, School of Medicine, Zhejiang University. All patients signed the informed consent before surgery. Twenty gastric cancer tissues and paired adjacent noncancerous tissues were obtained. Total RNA and protein were extracted for further analysis.
Seven gastric cancer cell lines (AGS, MKN28, MKN45, BGC823, HGC27, MGC803 and SGC7901) were purchased from Riken Gene Bank (Tsukuba, Japan) and American Type Culture Collection (Manassas, USA), and GES-1, a noncancerous gastric cell line, was obtained from Beijing Institute for Cancer Research (Beijing, China). RPMI 1640 medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, Sijiqing, Huzhou, China) was used to culture those cells in a condition of 5% CO2, 37 °C and 95% humidity.
Construction of mammalian plasmids
Full-length Runx3 CDS, HOTAIR CDS sense/antisense, HOT-A, B, C, D and D1–D4 were amplified by reverse transcription-polymerase chain reaction (RT-PCR). Then those fragments were cloned into the mammalian expression plasmids of either GV141 (XhoI/KpnI, Genechem, Shanghai, China; having an element of 3 × Flag) or GV219 (XhoI/EcoRI, Genechem; having an element of T7 RNA polymerase promoter). The primers consisting enzyme cleavage sites are listed in Online Resource 1 in the electronic supplementary material.
BGC823 and SGC7901 cells were transfected with GV141–Runx3 or GV141 empty vector using Fugene HD (Promega). For siRNA-mediated gene knockdown, gastric cancer cells were transfected with negative control siRNA, HOTAIR siRNA or Runx3 siRNA (Qiagen, Hilden, Germany) using Lipofectamine RNAiMAX Reagent (Invitrogen). All 3 variants of Runx3 have the same 3′-UTR sequence, and could be inhibited by one siRNA.
Screening of potential LncRNAs that bind to Runx3
We input the amino sequence of Runx3 (NM_004350) and the sequence of LncRNAs in Fasta format from LNCiPedia (V3.1) into RPISeq (http://pridb.gdcb.iastate.edu/RPISeq/, a free online software predicting RNA–protein interactions). Two classifiers of random forest (RF) and support vector machine (SVM) for each LncRNA were calculated. Potential Runx3-binding LncRNAs were selected using the following rules: (1) RF ≥ 0.99 and SVM ≥ 0.99; (2) RF ≥ 0.95, SVM ≥ 0.95 and was mentioned in the references from PubMed; (3) RF ≥ 0.90, SVM ≥ 0.90 and was associated with tumor as reported in PubMed; (4) RF ≥ 0.80, SVM ≥ 0.80 and was associated with gastric cancer as reported in PubMed.
Western blot analysis
Quantitative real-time PCR (qPCR)
Total RNAs were either isolated from tumor cells with TRIzol reagent (Invitrogen, Carlsbad, USA) or from tissue samples with PARIS Kit (Thermo Fisher). cDNA was synthesized using RT reagent kit with gDNA Eraser (TaKaRa, Otsu, Japan). SYBR Green Master Mix Kit (Takara) was used to perform qPCR on a LightCycler 480 Instrument (Roche, Penzberg, Germany). GAPDH was used as an internal control for protein-coding genes and U6 was used for LncRNAs. Primers used for PCR reactions are listed in Online Resource 2 in the electronic supplementary material.
RNA immunoprecipitation (RIP)
RIP was performed according to the instructions of Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). Briefly, 3 × Flag–Runx3 overexpressed BGC823 and SGC7901 cells were suspended in RIP lysis buffer and mixed with Flag M2 antibody (F1804, Sigma, Darmstadt, Germany)-coated magnetic beads. Then, the precipitated RNA was purified and converted into cDNA using the reverse transcription kit (Taraka). Quantitative PCR (qPCR) was applied to measure the enrichments of 11 LncRNAs, using U1 RNA as a negative control. The RNAs precipitated by normal mouse IgG antibody and input RNAs were applied as negative and positive control, respectively. For endogenous RIP assay, Runx3 antibody was used to precipitate RNAs in wild BGC823 and MGC803 cells.
Plasmids containing LncRNA HOTAIR or its short fragments were linearized in 3′ end with a restriction enzyme EcoRI. Biotinylated RNA was synthesized in vitro by T7 RNA polymerase using those linearized plasmids as templates, and then purified on G-50 Sephadex RNA Columns (Roche). After validating the size by agarose gel electrophoresis, proper RNA secondary structure was folded from biotin-labeled RNAs following incubation at different temperatures. Meanwhile, nuclear lysates were harvested from gastric cancer cells with addition of prewashed streptavidin–agarose beads. A mixture of folded biotinylated RNAs and nuclear lysates were then incubated with tRNA and prewashed streptavidin–agarose beads in sequence. The pellets were washed and then boiled with loading buffer. The pulled-down Runx3 protein was measured by western blotting.
Cell lysates were extracted from whole cells with IP lysis buffer, mixed with protein G agarose beads and then incubated with Runx3 or Mex3b antibody overnight. Rabbit or mouse IgG was used as negative control. Prewashed protein G agarose beads were then added and shaken gently for 3 h to capture the antigen–antibody complex. The beads were pelleted and washed 3 times with IP lysis buffer before boiling in SDS loading buffer. The pellets were further subjected to immunoblotting with appropriate antibodies.
Cell migration was assessed by modified Boyden transwell chambers (Corning Inc., Corning, USA), and cell invasion was measured in chambers coated with Matrigel (BD Biosciences, Franklin Lakes, USA). In brief, 36 h after being transfected with siRNA targeting HOTAIR or Runx3, gastric cancer cells were trypsinized, resuspended in culture medium containing 1% FBS and then seeded in the upper chamber with culture medium containing 15% FBS added in the lower chamber. After 24 h of incubation in a CO2 incubator, non-migratory cells in the upper chamber were carefully removed with swab cotton. Those cells migrating through the transwell membrane were stained with 1.0 μg/ml DAPI staining solution (Roche) and then counted microscopically (× 400). The cells invading through Matrigel and transwell membrane were incubated with crystal violet (Beyotime, Shanghai, China) and then counted microscopically (× 200).
Screening HOTAIR as a potential LncRNA binding to Runx3
To search for the LncRNAs that potentially bind to Runx3, a RNA–protein binding prediction software RPISeq was applied to perform in silico analysis. Based on RF/SVM classifiers and searching results in PubMed, eleven candidate LncRNAs with the potential to bind to Runx3 were screened (Fig. 1a). To validate these binding LncRNAs, RNAs in gastric cancer cells (BGC823 and SGC7901) were precipitated by RIP assay after transfecting with Runx3 plasmids (see Online Resource 3 in the electronic supplementary material). Q-PCR results showed that HOTAIR, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and Pvt1 oncogene (PVT1) were significantly amplified (over fivefold) in both BGC823 and SGC7901 cells (Fig. 1b). Interestingly, silencing HOTAIR remarkably induced the expression level of Runx3 protein in gastric cancer cells, while knockdown of MALAT1 or PVT1 had no significant effect on the expression of Runx3 (Fig. 1c and Online Resource 4 in the electronic supplementary material). These results suggested that HOTAIR interacts with Runx3 in gastric cancer cells.
To validate the interaction between endogenous Runx3 and HOTAIR, RNAs in wild BGC823 and MGC803 cells which have a relatively high endogenous expression of Runx3 (Online Resource 5 in the electronic supplementary material) were precipitated by RIP assay using the Runx3 antibody. Q-PCR results showed that HOTAIR was significantly amplified in both cell lines (Fig. 1d).
Identification of specific HOTAIR binding sites with Runx3
HOTAIR induces the degradation of Runx3 dependent on Mex3b
To further explore the role of HOTAIR and Mex3b in the ubiquitination of Runx3, we depleted either HOTAIR or Mex3b in both BGC823 and SGC7901 cells, and observed that the ubiquitination level of Runx3 was significantly reduced (Fig. 3e). In addition, the effect of HOTAIR and Mex3b on the stability of Runx3 was evaluated by protein synthesis assay by adding cycloheximide (CHX), a general inhibitor of protein biosynthesis. The degradation half-life time of Runx3 after the treatment of CHX was 2–4 h. In contrast, Runx3 degradation half-life time was extended to 4–8 h by knockdown of either HOTAIR or Mex3b (Fig. 3f). Collectively, our result suggested that HOTAIR induced the degradation of Runx3 by enhancing its interaction with Mex3b.
HOTAIR is negatively associated with Runx3 in gastric cancer tissues
HOTAIR promotes gastric cancer cell invasiveness through Runx3
Both HOTAIR and Runx3 were shown to regulate matrix metalloproteinase (MMP), E-cadherin and other invasion-related genes. However, only Runx3 was reported to be an upstream regulator of claudin1, which is a member of the tight junction protein family and plays a pivotal role in the process of epithelial–mesenchymal transition (EMT). Results revealed that gastric cancer cells with HOTAIR deficiency had an increased expression of claudin1 mRNA and protein, while co-deficiency of Runx3 would partially reverse this phenotype (Fig. 5e, f).
The expression of Runx3 was regulated by several mechanisms including promoter methylation, histone acetylation, miRNAs, etc. Previous studies have reported that the promoter of Runx3 was hypermethylated in gastric cancer . Interestingly, Helicobacter pylori infection and lipopolysaccharide also increase the frequency of Runx3 promoter hypermethylation . miRNA was another regulator responsible for the epigenetic modification of Runx3 . In addition, various post-transcriptional and post-translational regulations are responsible for the expression of Runx3. For example, transforming growth factor-β (TGF-β) could stimulate p300-dependent Runx3 acetylation, thus affecting its degradation and transactivation . In this study, we first reported that LncRNA HOTAIR induced the degradation of Runx3, supporting that HOTAIR is another post-translational regulator of Runx3.
As one of the important members of protein degradation enzymes, E3 ubiquitin ligases could induce the transfer of ubiquitin from E2 conjugating enzyme to the protein molecules for degradation . Recent reports demonstrated that Mex3b and Dzip3 were two certain E3 ligases possessing RNA binding domains, and both were associated with HOTAIR . Mex3b is detectable both in the nuclear and cytoplasm, while Dzip3 is only distributed in cytoplasmic vesicles . As a transcription factor, Runx3 is mainly localized in the nucleus. In this study, we revealed that Mex3b, and not Dzip3, functioned as an E3 ligase binding to Runx3. Either silencing HOTAIR or Mex3b impaired the ubiquitination of Runx3 and enhanced protein stability. Downregulation of Runx3 induced by HOTAIR was partially reversed by siRNA targeting Mex3b, indicating that HOTAIR accelerates the degradation of Runx3 though enhancing its interaction with Mex3b.
In this study, the RNA pull-down assay confirmed that the domain of HOTAIR to which Runx3 binds is located at 1951–2100 bp along the segment spanning nucleotides of HOTAIR. Runx3 contains the fewest number of exons in the Runx family, and the former 3 exons constitute the Runt domain . The binding domain of Runx3 could be identified by RIP with truncated plasmids and site mutations. In addition, ubiquitinated proteins are often conjugated to the Ile44 hydrophobic patch of ubiquitin via specific lysine residues . The interacting domains with Mex3b along the amino sequence of Runx3 is undetermined.
HOTAIR is involved in tumor metastasis through different pathways in several previous studies [20, 21]. Wang et al.  found that knockdown of HOTAIR decreased the secretion of MMP2 and MMP9, thus impairing the metastasis of osteosarcoma. In another study, HOTAIR depletion inhibited the invasion and metastasis of colon cancer stem cells with decreased vimentin and increased E-cadherin . Acquiring mesenchymal characteristics confers a pro-migratory advantage to tumor cells, with altered expressions of E-cadherin, ß-catenin and other EMT-related genes . Claudin1 is a member of the tight junction-related proteins and could block the EMT process. Runx3 promoted the transcriptional expression of claudin1 both in vivo and in vitro , while no previous literature reported the effect of HOTAIR on the expression of claudin1. In this study, knockdown of Runx3 significantly attenuated the decrease of migrated cells and concomitant increase of claudin1 expression induced by siRNA targeting HOTAIR, suggesting the role of HOTAIR–Runx3–claudin1 in the invasiveness of gastric cancer.
In conclusion, we demonstrated that HOTAIR interacted with Runx3 and induced its degradation dependent on the E3 ubiquitin ligase Mex3b, which provided a novel regulatory mechanism responsible for the silencing the expression of Runx3 in gastric cancer. HOTAIR–Runx3 interaction might work as a new therapeutic target for metastatic gastric cancer.
This work was supported by the Natural Science Foundation of Zhejiang Province (LQ17H160010); National Key R&D Program of China (2016YFC1303200 and 2016YFC0107003); National Natural Science Foundation of China (81702308, 81472214); Experimental Animal Science and Technology Program of Zhejiang Province (2015C37085); and Science and technology innovation team of Zhejiang Province (2013TD13).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals and informed consent
All samples were obtained with patients’ informed consent. The Ethics Committee of the Second Affiliated Hospital, School of Medicine, Zhejiang University, approved this study. This study does not involve animals.
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