Long non-coding RNA HAGLR inhibits growth and metastasis and reduces stemness of lung cancer cells through the microRNA-330-3p/SLC34A2 axis CURRENT

Background Lung cancer (LC) remains a leading cause of cancer-related mortality worldwide. Long noncoding RNAs (lncRNAs) are crucial regulatory molecules in diverse pathological processes, including cancer progression. This study was conducted to probe the influence of lncRNA HAGLR on the growth, metastasis and stemness of LC cells. Methods Aberrantly expressed lncRNAs in LC tissues were screened out using microarray analysis. HAGLR expression in LC tissues and cells and in the paired normal ones was determined using RT-qPCR. Overexpression of HAGLR was administrated in H1299 and A549 cells to identify its function in the biological characteristics of LC cells. Sub-cellular localization of HAGLR was determined, and the downstream molecules involved in the HAGLR-mediated events were predicted on a bioinformation system and validated through dual luciferase reporter gene assay. Results HAGLR was poorly expressed in both LC tissues and cells. Overexpression of HAGLR inhibited viability, proliferation and metastasis, and reduced the stemness of H1299 and A549 cells. HAGLR up-regulated SLC34A2 expression through sponging miR-330-3p, leading to further inactivation of the Wnt/β-catenin signaling pathway. Artificial activation of the Wnt/β-catenin pathway recovered the viability and promoted metastasis of LC cells inhibited by HAGLR Conclusion HAGLR serves as a competing endogenous RNA for miR-330-3p to upregulate miR-330-3p expression and inactivate the Wnt/β-catenin pathway, leading to inhibited growth and metastasis and reduced stemness of LC cells.

Background Lung cancer (LC) remains a leading cause of cancer-related mortality worldwide. Long noncoding RNAs (lncRNAs) are crucial regulatory molecules in diverse pathological processes, including cancer progression. This study was conducted to probe the influence of lncRNA HAGLR on the growth, metastasis and stemness of LC cells.
Methods Aberrantly expressed lncRNAs in LC tissues were screened out using microarray analysis. HAGLR expression in LC tissues and cells and in the paired normal ones was determined using RT-qPCR. Overexpression of HAGLR was administrated in H1299 and A549 cells to identify its function in the biological characteristics of LC cells. Sub-cellular localization of HAGLR was determined, and the downstream molecules involved in the HAGLR-mediated events were predicted on a bioinformation system and validated through dual luciferase reporter gene assay.
Results HAGLR was poorly expressed in both LC tissues and cells. Overexpression of HAGLR inhibited viability, proliferation and metastasis, and reduced the stemness of H1299 and A549 cells. HAGLR up-regulated SLC34A2 expression through sponging miR-330-3p, leading to further inactivation of the Wnt/β-catenin signaling pathway. Artificial activation of the Wnt/β-catenin pathway recovered the viability and promoted metastasis of LC cells inhibited by HAGLR Conclusion HAGLR serves as a competing endogenous RNA for miR-330-3p to upregulate miR-330-3p expression and inactivate the Wnt/β-catenin pathway, leading to inhibited growth and metastasis and reduced stemness of LC cells.

Background
Lung cancer (LC), a prevalent and highly invasive malignancy, is one of the most frequent and leading causes of cancer-related death across the globe [1]. In China, LC holds the highest incidence and mortality among all cancer types [2]. Several lifestyle and environmental factorshave been suggested to be linked with the development of LC, of which cigarette smoking, as publicly accepted, is the most relevant and important, accounting for 80-90% ofLC cases [3,4]. Limited accessto early diagnosis and timely treatment are main attributes that leads to poor outcomes of LC patients [5]. Since LC detected at late stages are always accompanying with high metastasis rate and the following less survival opportunity [6]. To date, despite the great progress made in conventionaltherapeutic approaches including surgery, chemotherapy and radiotherapy, the overall 5-year survival rate of non-small-cell lung cancer (NSCLC, the most common type of LC) is reported remaining very low [7]. Identifying novel pathophysiological mechanisms involved in LC progression is of great importance for new treatment strategy development.
Many human diseases are accompanied with mutations identified in a lot of proteincodinggenes; however, most of the discovered genome is "noncoding" but transcribed [8,9]. Besides protein-encoding RNAs, many long and short non-coding RNAs (ncRNAs) have been recognized with advancesin the computational and experimental approaches ofcontemporary biology [10]. Among them, long non-coding RNAs (lncRNAs) are a heterogeneous class that have been discovered to regulate gene expression in different levels and to play important roles in cancer progression [11]. As in human LC, lncRNA LINC00961 [12] and lncRNA CS1-IT1 [5], for example, were documented to suppress LC progression by inhibiting proliferation and metastasis but promoting apoptosis of cancer cells. Besides, another major type of ncRNAs, microRNAs (miRNAs), are crucial forposttranscriptional regulation of gene expression by binding the 3'untranslated region (3'UTR) of target mRNAs through the miRNA-responseelements (MREs), leading to following mRNA degradation [13]. In addition to beingidentified on mRNAs, MREs can also be found on other non-coding transcripts including lncRNAs, by which the ceRNA hypothesis was proposed, which suggests that lncRNAs competes for a common pool of miRNAs and further regulates mRNA expression [14].
This ceRNA network hypothesis has been confirmed in several human diseases including LC. For instance, lncRNA GAS5 was documented to suppress LC cell proliferation and metastasis by serving as a ceRNA for miR-205/PTEN [15]. Likewise, a lncRNA SBF2-AS1/miR-302a/MBNL3 axis was recently identified to influence the radiosensitivity of NSCLC cells [16]. Considering the complex RNA interactions, there remains largely unknown in this field. In light of the above discussion, the study was performed to probe a novel ceRNA network in LC progression. To this end, we firstly determined the aberrantly

Microarray analysis
Microarray analysis was performed to screen out the differentially expressed lncRNAs between tumor and normal tissues. In brief, 0.5 µg total RNA from either tumor tissues or normal tissues was reversely transcribed to cDNA using a GeneChip 3'In vitro Transcription Express Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then the cDNA was hybridized with Human LncRNA Expression Array V4.0 (Arraystar, Rockville, MD, USA), and then the chip was washed and scanned on a GeneChipTM Scanner3000 7G System (Thermo Fisher).
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA from tissues and cells was extracted using a TRIzol Kit (Thermo Fisher). Next, the cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher) according to the RNA template. Then the cDNA was amplified and then diluted for RT-qPCR. The primer sequences are shown in Table 1.
All procedures were conducted in strict accordance with the manufacturer's instructions.
Transwell assays was performed to measure invasion and migration of cells as previously described [17].
Self-renewal assay Immunofluorescence staining Expression of Sex-determining region Y-box 9 (SOX9) and Octamer 4 (OCT4) in H1299 and A549 cells was detected using Immunofluorescence staining. In brief, cells growing on slides were washed 3 times with phosphate buffer saline (PBS), fixed in 4% paraformaldehyde at 4℃ for 15 min, and then treated with 0.5% Triton-100 X for 20 min.
Next, the cell slides were incubated with primary antibodies SOX9 (1:
Statistical analysis SPSS 21.0 (IBM Corp. Armonk, NY, USA) was applied for data analysis.Kolmogorov-SmiRnov checked that data were in normal distribution. Measurement data were exhibited as mean ± standard derivation (SD). Differences between every two groups were compared utilizingthe t test, while those among multiple groups were compared using one-way or two-way analysis of variance (ANOVA) with Tukey's multiple comparisontest used for post hoc test.The survival curve for patients was recorded using the Kalpan-Meier method and analyzed using log rank test. Correlations between molecular expression were compared using Pearson's correlation analysis. The p valuewas acquired from two-tailed tests, and p < 0.05 was considered to present significant difference.

HAGLR is abnormally lowly expressed in LC tissues and cells
LncRNAs with differential expression in LC were determined using microarray analysis based on 5 pairs of LC tissues and para-cancerous tissues and then validated by RT-qPCR.
The results suggested that HAGLR was significantly lowly expressed in LC tissues (Fig. 1A-B). Next, we further assessed HAGLR expression in LC cells and human tracheal epithelial cells, which found that HAGLR was expressed at low levels in LC cells as well, particularly in H1299 and A549 cell lines (Fig. 1C). Moreover, data on the KM-plotter website (http://kmplot.com/analysis/) suggested that LC patients with lower HAGLR expression presented lower survival rate and less median survival time (Fig. 1D). To further identify the function of HAGLR in LC, artificial overexpression of HAGLR was administrated in H1299 and A549 cell lines, and RT-qPCR found the transfection was successfully performed since HAGLR expression was increased in both cell lines (Fig. 1E).

Overexpression of HAGLR inhibits viability of LC cells
The EdU labeling and MTT assay results suggested that overexpression of HAGLR inhibited viability and proliferation while promoted apoptosis of H1299 and A549 cells ( Fig. 2A-C).
In addition, the Transwell assay results suggested that the invasion and migration abilities of LC cells was notably decreased as well following HAGLR overexpression (Fig. 2D-E).
Overexpression of HAGLR reduces the stemness of H1299 and A549 cells Following the findings above, we further explored the role of HAGLR in stemness of H1299 and A549 cells. After 7 d of incubation, the formation of tumor spheres by non-adherent H1299 and A549 cells was evaluated. It was found that overexpression of HAGLR inhibited the size and number of formed tumor spheres by H1299 and A549 cells (Fig. 3A). Next, flow cytometry was performed to measure the number of cancer stem cells (CD44 + /CD133 + ) in H1299 and A549 cells, which suggested that overexpression of HAGLR reduces the stemness of H1299 and A549 cells (Fig. 3B). In addition, the immunofluorescence staining suggested that the expression of SOX9 and OCT4 in both cells was decreased following HAGLR overexpression (Fig. 3C).

HAGLR up-regulates SLC34A2 expression through sponging miR-330-3p
To explore the potential molecular mechanisms involved in the HAGLR mediation, we firstly determined the sub-cellular localization of HAGLR in cells. The data on LncAtlas (http://lncatlas.crg.eu/) suggested that HAGLR is mainly localized in cytoplasm (Fig. 4A), this was further identified by the RNA nuclear-cytoplasm separation assay, where HAGLR was mainly expressed in cytoplasm (Fig. 4B).
This finding indicated that miR-330-3p might regulate growth and stemness of LC through the ceRNA network. Thereafter, the online prediction on StarBase (http://starbase.sysu.edu.cn/) and dual luciferase reporter gene assay identified that HAGLR could directly bind to miR-330-3p (Fig. 5A). Additionally, miR-330-3p was found to be highly expressed in LC patients according to RT-qPCR (Fig. 5B) and was negatively correlated with HAGLR expression (Fig. 5C).
Overexpression of HAGLR up-regulates SLC34A2 expression and inhibits the Wnt/βcatenin signaling pathway.
Next, we assessed the levels of Wnt/β-catenin pathway-related proteins Wnt1 and βcatenin in cells. It was found that over-expression of HAGLR inhibited the Wnt1 and βcatenin expression in H1299 and A549 cells (Fig. 6A). In addition, IQ-1 (1 mg, ab142079), a Wnt/β-catenin signaling pathway agonist, was introduced into H1299 cells and A549 cells following HAGLR overexpression, after which the viability of H1299 and A549 cells was partly recovered, and the migration and invasion abilities of cells were promoted ( Fig. 6B-E).

Discussion
Though it has become a common sense that cigarette smoking is the major cause of LC, LC continues to be one of the most prevalent malignancies and the most common causes of cancer deaths globally, leaving development of novel therapeutic options of great significance to the modern society [3]. HAGLR, also known as HOXD-AS1, whose aberrant expression has been documented to link with cancer progression [21]. Here in this study, we reported that HAGLR could serve as a competing endogenous RNA for miR-330-3p to upregulate miR-330-3p expression and inactivate the Wnt/β-catenin pathway, leading to inhibited growth and metastasis and reduced stemness of LC cells.
Initially, our study found that HAGLR was aberrantly lowly expressed in LC tissues and cells according to microarray analysis and RT-qPCR. Artificial up-regulation of HAGLR inhibited viability and metastasis of H1299 and A549 cells. As aforementioned, uncontrolled metastasis is a major reason leading to death of late-stage LC patients [6].
HAGLR is localized between the HOXD1 andHOXD3 genes in the HOXD cluster and are usually serves as an oncogene in several cancer types [21]. However, its anti-tumor has been documented before, where HAGLR has been demonstrated to inhibit growth and metastasis of colorectal carcinoma by suppressing HOXD3-induced Integrin β3 transcription and the following MAPK/AKT activation [22]. Similarly, low expression of HAGLR was found to be associated with increased cell growth and decreased survival time of patients with lungadenocarcinoma, a major subtype of NSCLC [23]. Moreover, overexpression of HAGLR was found to reduce the stemness of LC cells, presenting as decreased cell number with CD44 + and CD133 + , as well as decreased expression of SOX9 [24] and OCT4 [25] in cells. It is well-known that cancer stem cells are closely correlated with carcinogenesis, tumor progression, relapse and metastasis, leaving reducing stemness of stem cells a great challenge as well as a promising therapeutic approach in cancer treatment [26]. These findings further validated the potential inhibiting role of HAGLR in LC progression.
The findings above triggered us to figure out the potential underlying molecular mechanisms. HAGLR was suggested to be sub-localized in cytoplasm, indicating it might exert functions through the ceRNA networks. Then, online prediction and the dual luciferase reporter gene assay identified that HAGLR could directly bind to miR-330-3p, which was further found to be highly expressed in LC patients. miR-330-3p has been documented to serve as an oncogene in pancreatic cancer [27]. Importantly, previous studies have suggested that miR-330-3p could promote NSCLC LC progression by inhibiting malignant behaviors of LC cells including proliferation, invasion, migration and metastasis [28,29]. Moreover, we further found that miR-330-3p could directly bind to SLC34A2. SLC34A2 was found to be down-regulated in surgical samples of NSCLC as compared to the adjacent normal lung tissues [30]. Quite in line with our study, SLC34A2 was found to be lowly-expressed in LC cell lines, and elevated SLC34A2 was shown to inhibit viability and invasion of LC cells [31]. In addition, SLC34A2 was found as afavorable prognostic marker in LC patients, with higher SCL34A2 levels leading to higher overall survival rates [32]. Furthermore, our study found that the expression of Wnt1 and βcatenin in H1299 and A549 cells was decreased following HAGLR over-expression.
Administration of IQ-1, a Wnt/β-catenin agonist, recovered viability and invasiveness of H1299 and A549 cells. Since HAGLR could promote SCL24A2 expression, and SCL24A2 has been found to negatively regulate the Wnt/β-catenin pathway in NSCLC cells [33]. The Wnt/β-catenin signaling is implicated in multiple physiological processes including cell migration, polarization, as well as in maintenance and proliferation of cancer stem cells in the tumor bulk [34]. These findings suggested that activation of Wnt/β-catenin might be responsible for the growth and metastasis of LC cells.

Conclusion
To sum up, the current study provided evidence that HAGLR could inactivate the Wnt/βcatenin pathway through the miR-330-3p/SCL24A2 ceRNA network, leading to suppressed growth and metastasis and reduced stemness of LC cells. Thus, HAGLR might serve as a novel therapeutic approach for LC treatment. However, though we identified a novel ceRNA network in this study, the specific role of miR-330-3p or SCL24A2alone in LC cell behaviors was not included. Besides, more specific mechanisms by which SCL24A2 inactivates the Wnt/β-catenin pathway remains unelucidated. We hope more studies will be carried out in the near future to validate our findings, and we will focus on more

Consent for publication
Not applicable.

Availability of data and materials
All the data generated or analyzed during this study are included in this published article.

Competing interests
All authors declare that there is no conflict of interestsin this study.

Funding
Not applicable.  in H1299 and A549 cells was assessed by immunofluorescence staining. Data were exhibited as mean ± SD. In panels A, C, D and E, data were analyzed using one-way ANOVA, while data in panel B were analyzed using two-way ANOVA, and Tukey's multiple comparison Figure 4 HAGLR is sub-localized in cytoplasm. A, HAGLR is sub-localized in cytoplasm, as predicted by LncAtlas. B, nuclear and cytoplasmic expression of HAGLR in H1299 cells and A549 cells was determined by RT-qPCR. Data were exhibited as mean ± SD. Data were analyzed using two-way ANOVA, and Tukey's multiple comparison test was applied for post hoc test. Repetition = 3. *, p< 0.05.  Overexpression of HAGLR up-regulates SLC34A2 expression and inhibits the Wnt/ β-catenin signaling pathway. A, levels of Wnt/β-catenin pathway-related proteins Wnt1 and β-catenin in H1299 and A549 cells were determined by western blot analysis. Then, a Wnt/β-catenin signaling pathway agonist IQ-1 was introduced into H1299 cells and A549 cells following HAGLR overexpression. B, proliferating