LOXL1-AS1 Drives The Progression Of Gastric Cancer Via Regulating miR-142-5p/PIK3CA Axis
Authors Li M, Cai O, Tan S
Received 18 July 2019
Accepted for publication 28 September 2019
Published 20 December 2019 Volume 2019:12 Pages 11345—11357
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Prof. Dr. Nicola Silvestris
Ming Li, Ou Cai, Shiyun Tan
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430000, People’s Republic of China
Correspondence: Ming Li
Department of Gastroenterology, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan, Hubei Province 430060, People’s Republic of China
Email [email protected]
Background: Gastric cancer (GC) is a deadly disease, and its incidence is especially high in East Asia including China. Recently, some long non-coding RNA (lncRNAs) have been identified as oncogenes or tumor suppressors. This study aimed to determine the function and mechanism of lncRNA LOXL1-AS1 on the progression of GC.
Methods: RT-PCR was done to measure the expression levels of LOXL1-AS1 and miR-142-5p in GC tissues. The association between pathological indexes and LOXL1-AS1 expression was also analyzed. Human GC cell lines AGS and BGC823 were used as cell models. CCK-8 and colony formation assays were conducted to assess the effect of LOXL1-AS1 on the proliferation of GC cell lines. Transwell assay was conducted to determine the influence of LOXL1-AS1 on cell migration and invasion. Furthermore, luciferase reporter assay was carried out to confirm the relationship of miR-142-5p with LOXL1-AS1. Additionally, Western blot was done to detect the regulatory function of LOXL1-AS1 on PIK3CA, a target of miR-142-5p. In vivo experiment was also performed to validate the roles and mechanism of LOXL1-AS1 on the growth and metastasis of GC cells.
Results: LOXL1-AS1 expression in GC samples was significantly increased, which was correlated with unfavorable pathological indexes. Highly expressed LOXL1-AS1 was closely linked to shorter overall survival time and post-progression survival time of the patients. LOXL1-AS1 markedly modulated the malignant phenotypes of GC cells. Additionally, overexpressed LOXL1-AS1 notably reduced the expression of miR-142-5p, but enhanced the expression level of PIK3CA. In vivo experiments further validated that knockdown of LOXL1-AS1 inhibited the growth and metastasis of GC cells via regulating miR-142-5p and PIK3CA.
Conclusion: LOXL1-AS1 was a sponge of tumor suppressor miR-142-5p in GC, enhanced the expression of PIK3CA indirectly and functioned as an oncogenic lncRNA.
Keywords: LOXL1-AS1, miR-142-5p, PIK3CA, gastric cancer
Gastric cancer (GC) is one of the leading deadly cancer worldwide. More than 70% of diagnosed GC cases are patients from developing countries.1 Although surgery, chemotherapy and targeted drugs offer symptomatic relief and modest improvement in survival for GC patients, the overall survival rate remains unfavorable.2 Unfortunately, a majority of patients are diagnosed at an advanced stage, resulting in a high mortality rate. Moreover, in many cases, traditional therapies fail because of relapsing and distant metastasis due to chemoresistance or radioresistance.3 Hence, an in-depth investigation into the underlying mechanism of GC is highly desirable to explore the novel treatment strategies for GC.
Long non-coding RNA (lncRNA) is a class of non-coding RNA that is more than 200 nucleotides without protein-coding ability. Mounting studies indicate that lncRNA wields an enormous influence in the gene expression regulation at the transcription level, post-transcriptional level, and translation level.4 The role of lncRNAs as oncogenes or tumor suppressors in tumorigenesis and progression has been validated by the accumulating experimental demonstrations.5–7 LOXL1-AS1 is encoded in the complementary strands of LOXL1.8 It conducts a pivotal role in cell stress response and is closely related to Exfoliation Syndrome.9 Besides, high-expressed LOXL1-AS1 facilitates mesenchymal characteristics of glioblastoma via NF-κB pathway10 LOXL1-AS1 was verified as well to modulate the proliferation of medulloblastoma cells through the activation of PI3K/AKT pathway.11 Nevertheless, the defined role of LOXL1-AS1 in GC cells remains poorly understood.
MicroRNA (miRNA) is a class of short-chain non-coding RNA molecules with a length of 21–25 nucleotides.12 Abnormally expressed miRNA is a frequent event in the tumorigenesis and progression of diverse cancers. For instance, miR-214-5p inhibits the progression of mesothelioma via regulating the MDM2-p53 pathway;13 miR-195 restrains the proliferation of colorectal cancer cells;14 miR-221 promotes the migration of hepatocellular carcinoma cells by targeting PHF2.15 Recent studies have revealed that lncRNAs can interact directly with miRNAs and regulate their expression and activity.16,17 In detail, lncRNA ATB has been found to modulate the proliferation of colorectal cancer cells via sponging miR-200c;18 PVT1 was verified to target miR-140 to facilitate the proliferation of ovarian cancer cells;19 NEAT1 accelerates the proliferation of laryngeal squamous cell carcinoma cells via regulating miR-107/CDK6 axis.20
The main goal of our study was to explore LOXL1-AS1 expression and its clinical implications in GC cells, as well as to determine the impact of LOXL1-AS1 on malignant phenotypes of GC cells and its downstream mechanism. Specifically, we demonstrated that LOXL1-AS1 displayed a high expression in GC tissues, and GC patients with highly expressed LOXL1-AS1 had shorter overall survival time and post-progression survival time. In terms of mechanism, LOXL1-AS1 can enhance the proliferation of GC cells via regulating the miR-142-5p/PIK3CA axis. Collectively, this study confirmed the crucial role of LOXL1-AS1/miR-142-5p/PIK3CA in regulating GC progression, thus providing novel clues for GC therapy.
Materials And Methods
All patients involved gave informed consent to the study and signed a written consent form. Tissue collection was conducted in accordance with the Declaration of Helsinki. This study was carried out under the approval of Ethics Review Committee of Renmin Hospital of Wuhan University. GC tissues and matched para-tumorous tissues were procured from 89 cases (49 males and 40 females, 26~70 years old) presented to our hospital from 2014 to 2019. All cancer tissues were pathologically confirmed as GC and no other malignant tumors were found. No patients received neoadjuvant therapy (chemotherapy or radiotherapy) before the operation. The resected tissues were stored in −196 °C liquid nitrogen for the following analysis.
Human GC cell lines (BGC823, AGS, NCI-N87, MGC803 and SGC7901 cells) and normal epithelial cells of gastric mucosa (GES-1) were provided by Shanghai Junrui Biotechnology Co., Ltd (Shanghai, China). All cells were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA), 100U/mL penicillin and 100 μg/mL streptomycin (Gibco, Life Technologies, Carlsbad, CA) in 5% CO2 at 37 °C.
Total RNA was isolated from tissues or cells with RNAiso Plus Reagent (Takara, Dalian, China). About 1 μg total RNA was reversely transcribed to cDNA with SuperScript First Strand cDNA System (Invitrogen, Grand Island, NY, USA). Then, real-time PCR was carried out. The relative expressions were calculated using 2−ΔΔCT method. The specific primer sequences are listed in Table 1.
Table 1 RT-qPCR Primer Sequences
LOXL1-AS1 sequence lacking poly-A tail was procured from Shanghai GeneChem Co., Ltd. (Shanghai, China) and subcloned into pcDNA3.1 plasmid to establish LOXL1-AS1 overexpression cell line. LOXL1-AS1 shRNA was applied to knock down LOXL1-AS1 expression. Empty vector, pcDNA-LOXL1-AS1 vector or LOXL1-AS1 shRNA was transfected into AGS and BGC823 cells cultured in 6-well plate with Lipofectamine® 2000 (Invitrogen, Carlsbad, USA). Twenty-four hours after the transfection, the cells were collected and processed for further analysis.
Cell Proliferation Assay
GC cells in each group were incubated in a 96-well plate for 12 hrs with 5000 cells per well. The culture medium was replaced with a complete medium containing 10 μl CCK-8 reagent (Dojindo, Tokyo, Japan) at a specified time, and then the cells were incubated at 37 °C for 1 hr. Ultimately, Vmax microspectrophotometer (Beyotime, Shanghai, China) was adopted to measure the absorbance at 450nm.
Colony Formation Assay
GC cells with a concentration of 1×103/mL were planted in a 6-well plate. After 2 weeks of culture, the culture medium was discarded and the colonies were carefully washed with PBS twice. The colonies were then fixed with 10% paraformaldehyde for 10 min and stained with 0.1% crystal purple for 15 mins. Afterwards, the number of colonies formed was counted and recorded.
Transwell Migration And Invasion Assay
Cells were inoculated in a 24-well plate equipped with a transwell chamber (8 μm pore size; BD Biosciences, San Jose, CA, USA). GC cells were suspended in the upper chamber (2×105 cells/well) with a serum-free medium, and the lower chamber contained 500 μL RPMI-1640 medium containing 10% FBS. Twenty-four hours later, the cells on the upper surface of the chamber were gently wiped clean with a cotton swab. Subsequently, the cells migrating the lower chamber were fixed using 95% ethanol for 20 mins and then stained by 0.1% crystal violet for 10 mins. Afterwards, the cells were counted under a microscope. In the invasion experiment, except that a layer of matrixgel (BD Biosciences, CA, USA) was coated on the bottom of the transwell chamber, the other steps were the same as the migration experiment.
Luciferase Reporter Assay
A luciferase reporter vector (pMiR-LOXL1-AS1-WT/pMiR-LOXL1-AS1-Mout) was constructed using DNA oligonucleotide and pMiR-Reporter vector. HEK-293T cells were co-transfected with pMiR-LOXL1-AS1-WT or pMiR-LOXL1-AS1-Mut and miR-142-5p mimics or negative control (NC miR). Luciferase activity was measured 24 hrs after transfection using the dual-luciferase reporter assay system (Promega, Madison, WI, USA).
The cells were lysed with RIPA buffer supplemented with protease inhibitors. Total protein extract was separated on 12% SDS-PAGE gels and then transferred to nitrocellulose (NC) membranes. The membrane, after blocked by 5% fat-free milk, was incubated with primary antibodies at 4 °C for 12 hrs. The primary antibodies included anti-PIK3CA (1:1000, Abcam, ab40776), anti-E-cadherin (1:1000, Abcam, ab1416), anti-N-cadherin (1:1000, Abcam, ab18203), anti-Vimentin (1:1000, Abcam, ab92547), anti-ZO-1 (1:1000, Abcam, ab96587) and GAPDH (1:1000, Abcam, ab8245). Following that, the NC membrane was then rinsed with TBST solution and incubated at room temperature for 1 hr with the secondary antibodies (1:2000, Santa Cruz Biotechnology) conjugated to HRP. After that, color rendering was performed using hypersensitive ECL (Hubei Biossci Biotechnology Co, Ltd.). The automatic developing instrument (ChemiDocXRS imaging system) was adopted to detect the signal.
All animal assays were performed under the approval from Animal Care and Use Committee of Renmin Hosptial of Wuhan University. And the experiments were conducted in accordance with UKCCCR Guidelines for the welfare of animals in experimental neoplasia. BALB/c nude mice (female, 4 weeks old) were used in the tumorigenicity assay, and the nursing and use of experimental animals were conducted following the national standard. The mice were randomly divided into the sh-NC group and sh-LOXL1-AS1 group (10 mice in each group). GC cells (1×107 cells) injected subcutaneously to the left (sh-LOXL1-AS1 group) and right side (sh-NC group) of nude mice, and the tumor volume was monitored every 7 days. Thirty-five days later, the tumor was resected and weighed from the sacrificed mice. In the end, RT-qPCR was done to determine the expressions of LOXL1-AS1, miR-142-5p and PIK3CA mRNA, and Western blot was conducted to measure PIK3CA expression. In the lung metastasis study, 1×107 cells (sh-NC group or sh-LOXL1-AS1 group) were injected into a caudal vein of 10 mice in each group. Two weeks later, mice were killed and lung colonization was quantified through pathological examination.
GraphPad Prism 7.0 was adopted for statistical analysis. Students’ t test was carried out to analyze the difference in measurement data. Chi-square test was performed to analyze the correlation between LOXL1-AS1 expression and clinicopathological indexes. The survival curve was plotted by Kaplan–Meier method. All data were presented as Mean ± SD. P<0.05 indicated statistical significance.
The Up-Regulation Of LOXL1-AS1 In GC Samples Was Related To Patients’ Poor Prognosis
First of all, using TCGA data, we analyzed the expression level of LOXL1-AS1 in GC samples, and the results indicated that LOXL1-AS1 was significantly up-regulated in GC tissues compared with normal gastric tissues (Figure 1A). Furthermore, LOXL1-AS1 expression was detected by qRT-PCR in collected 89 GC tissues and adjacent tissues. Consistent with the expression pattern in TCGA, LOXL1-AS1 exhibited a higher expression in GC cells as compared with matched adjacent tissues (Figure 1B). Moreover, compared with normal gastric epithelial cell GES1, all of the GC cell lines showed a higher expression of LOXL1-AS1 (Figure 1C). Based on the above results, we further investigated the relationship between LOXL1-AS1 expression and long-term prognosis with the help of Kaplan–Meier plotter (http://kmplot.com/analysis/). Highly expressed LOXL1-AS1 was proved to be closely linked to shorter overall survival time and post-progression survival time of GC patients (Figure 1D). To sum up, the up-regulation of LOXL1AS1 may conduct a crucial role in GC progression.
The Expression Of LOXL1-AS1 Is Correlated With The Pathological Indexes Of GC Patients
Then, we proceeded to analyze the correlation between LOXL1-AS1 expression and the pathological parameters of the above 89 GC samples (Table 2). Chi-square test signified that the high expression of LOXL1-AS1 in GC cells was closely related to larger tumor (p=0.0165), local lymph node invasion (p=0.0065), distant metastasis (p=0.0084) and higher TNM stage (p=0.0041), but it had no significant correlation with age, gender and tumor differentiation, suggesting that LOXL1-AS1 may facilitate the tumorigenesis of GC.
Table 2 Correlation Between Clinicopathological Parameters And LOXL1-AS1 Expression In 89 GC Patients
LOXL1-AS1 Modulates The Proliferation, Metastasis And Invasion Of GC Cells
The function of LOXL1-AS1 was further explored after we draw the conclusion that LOXL1-AS1 exhibited a significant up-regulation in GC cells. AGS and BGC823 cell lines were selected to construct LOXL1-AS1 overexpression model and knockdown model, respectively (Figure 2A). On this basis, CCK-8 assay was done to detect the proliferation ability of the above cells. The results demonstrated that LOXL1-AS1 overexpression promoted the proliferation of AGS cells. On the contrary, the proliferation ability of BGC823 cells was significantly inhibited in the downregulated LOXL1-AS1 group as compared with the sh-NC group (Figure 2B). In addition, the colony formation ability of GC cells was further explored. The results demonstrated that the overexpression of LOXL1-AS1 increased the colony number of AGS cells, while compared with BGC823 cells transfected with sh-NC, the colony number of sh-LOXL1-AS1 group decreased significantly (Figure 2C). Next, we monitored the impact of LOXL1AS1 on metastasis by transwell assay. The results showed that overexpression of LOXL1-AS1 significantly facilitated the migration and invasion of AGS cells, while the migration and invasion of BGC823 cells transfected with sh-LOX1-AS1 were significantly restrained compared with shNC group (Figure 2D). Since our data suggested that LOXL1-AS1 may be implicated in the metastasis of cancer phenotype, Western blot was done to detect the expressions of biomarkers of EMT including Vimentin, N-cadherin, ZO-1 and E-cadherin. As we expected, the overexpression of LOXL1-AS1 can up-regulate the expressions of Vimentin, N-cadherin while down-regulate the expressions of ZO-1 and E-cadherin. On the contrary, knockdown of LOXL1-AS1 can reduce the expressions of Vimentin, N-cadherin but up-regulate the expressions of ZO-1 and E-cadherin (Figure 2E). These data implied that knockdown of LOXL1-AS1 impedes the migration and invasion of GC cells via inhibiting EMT.
miR-142-5p Is A Key Target Of LOXL1-AS1
To elucidate the downstream mechanism of LOXL1-AS1 in GC, we carried out bioinformatics analysis through StarBase database (http://starbase.sysu.edu.cn/index.php). The data showed that LOXL1-AS1 contained the target site of miR-142-5p (Figure 3A). The results of qRT-PCR suggested that miR-142-5p was down-regulated in GC tissues (Figure 3B). We made a hypothesis that the dysregulation of miR-142-5p was due to the overexpression of LOXL1-AS1. To further verify that miR-142-5p is indeed a target for LOXL1-AS1, we detected the expressions of miR-142-5p in GC cell lines with overexpressed or low-expressed LOXL1-AS1 by qRT-PCR. A decrease in the expression of miR-142-5p was observed in LOXL1-AS1 overexpressed cells whereas an increase in the expression of miR-142-5p was noted in cells with LOXL1-AS1 knocked down (Figure 3C). Additionally, miR-142-5p arrested the luciferase activity of pGL3-LOXL1-AS1 wild type, but had no effect on pGL3-LOXL1-AS1 mutate type (Figure 3D). These results suggested that miR-142-5p could bind to LOXL1-AS1 directly at miRNA recognition sites. Furthermore, the expression of LOXL1-AS1 in GC samples was reversely correlated with miR-142-5p (Figure 3E). Collectively, these data indicated that miR-142-5p was a target of LOXL1-AS1 and negatively modulated by it in GC tissues. To further investigate the interaction between LOXL1-AS1 and miR-142-5p, we co-transfected miR-142-5p in GC cells with overexpressed LOXL1-AS1 (Figure 4A and B). It was observed that overexpressed miR-142-5p attenuated the effects of overexpressed LOXL1-AS1 on the malignant phenotypes of GC cells (Figure 4C and D) (p<0.05). These findings suggested that LOXL1-AS1 accelerates the proliferation, migration and invasion of GC cells via regulating miR-142-5p.
LOXL1-AS1/miR-142-5p Regulates The Expression Of PIK3CA
After confirming that LOXL1-AS1 could modulate miR-142-5p expression, we proceeded to determine the downstream target of miR-142-5p. In non-small cell lung cancer, miR-142-5p exerted the function of tumor suppressor via targeting PIK3CA;21 on the other side, PIK3CA has been validated to regulate the progression of GC.22 Based on the above knowledge, we carried out further study and found that in GC cell lines, LOXL1-AS1 knockdown could notably reduce the expression of PIK3CA both at mRNA and protein level, while overexpressed LOXL1-AS1 played the opposite role (Figure 5A and B). In addition, the high expression of miR-142-5p reduced the expression of PIK3CA and counteracted the effect of the upregulation of PIK3CA induced by LOXL1-AS1 (Figure 5C). Further, qRT-PCR indicated that LOXL1-AS1 was positively related to the expression of PIK3CA (Figure 5D). In short, LOXL1-AS1 could modulate the expression of PIK3CA in GC cells via miR-142-5p.
LOXL1-AS1 Modulates The Progression Of GC Cells In Vivo
To determine whether LOXL1-AS1 affects the growth of tumors in vivo, we constructed a stable LOXL1-AS1 low-expression BGC823 cell with a lentivirus system (Figure 6A). On this basis, a subcutaneous tumorigenesis model was established. As shown, the tumor formed by BGC823/sh-LOXL-AS1 cells had a smaller volume and weight than that of BGC823/sh-NC cells (Figure 6B–D). To determine the influence of LOXL1-AS1 knockdown on cell metastasis in vivo, BGC823/sh-LOXL1-AS1 and BGC823/sh-NC cells were transplanted into the lateral tail vein of nude mice. Two weeks later, mice were sacrificed and lung metastases were monitored. Consistent with the results of in vitro experiments, compared with BGC823/sh-NC cells, lung metastasis of mice injected with BGC823/sh-LOXL1-AS1 cells was significantly hindered (Figure 6E). Western blot and qRT-PCR were also conducted, respectively, to detect the expression level of PIK3CA in the tumors obtained from subcutaneous tumorigenesis model, and PIK3CA in the sh-LOXL1-AS1 group was found to be significantly lower than that of the sh-NC group (Figure 6E). These results indicated that knockdown LOXL1-AS1 can inhibit the growth of GC cells via regulating PIK3CA in vivo.
The impacts of abnormally expressed LOXL1-AS1 on various malignancies have been well documented in a number of studies. For instance, LOXL1-AS1 regulates the proliferation of medulloblastoma cells.11 Moreover, LOXL1-AS1 can facilitate the proliferation, migration and invasion of osteosarcoma cells.23 In the current study, up-regulated LOXL1-AS1 was noted in GC tissues and cells, whose high expression was verified to be closely linked to adverse clinical features. To elucidate the role of LOXL1-AS1 in GC, we overexpressed and knocked down LOXL1-AS1 to verify its effect, and proved its oncogenic function. Besides, it was further validated that LOXL1-AS1 was associated with EMT markers’ expressions in GC cells. These findings confirmed the carcinogenic role of LOXL1-AS1 in the progression of GC.
MiRNA plays a pivotal role in tumor progression. Accumulating evidence have verified that lncRNA can interact directly with miRNA and acts as competitive endogenous RNA (also called “ceRNA”) to modulate its expression and activity.24 For instance, LOXL1-AS1, as a ceRNA of miR-324-3p, enhances the progress of cholangiocarcinoma by regulating ABCA1;25 LOXL1-AS1 mediates the cell cycle of prostate cancer cells through regulating miR-541-3p and CCND1;8 in addition, LOXL1-AS1 mediates the adriamycin resistance of prostate cancer cells by sponging miR-let-7a-5p.26 It is reported that overexpressed miR-142-5p can arrest the progression of GC, but its specific mechanism remains elusive.27 In the present study, we assumed that LOXL1-AS1 can target miRNA to play a role in GC. To confirm this hypothesis, we resorted to a bioinformatics analysis, which predicts the existence of a binding site between miR-142-5p and LOXL1-AS1. We overexpressed and knocked down LOXL1-AS1 to detect miR-142-5p expressions. The results suggested that LOXL1-AS1 reversely regulated miR-142-5p expression in GC cells. In GC cells, LOXL1-AS1 expression was verified to be reversely correlated with miR-142-5p. In addition, luciferase reporter gene assay was carried out to confirm that LOXL1-AS1 can bind directly to miR-142-5p. The results indicated that LOXL1-AS1 could directly target miR-142-5p in GC.
Phosphoinositide 3-kinases (PI3Ks) protein family is involved in the regulation of malignant behaviors of cancer cells.28–30 PIK3CA is a gene encoding phosphatidylinositol-3 kinase (PI3K) p110α catalytic subunit. The abnormal up-regulation of PIK3CA expression can promote proliferation, migration and invasion of cancer cells via enhancing the catalytic activity of PI3K and activating PI3K-AKT signaling pathway.31 In this study, we attempted to explore the downstream target of miR-142-5p in GC cells. Bioinformatics suggested PIK3CA is a key oncogene that can regulate a variety of cell processes in GC and may be one of the candidate targets of miR-142-5p. Previous studies have confirmed that PIK3CA was a target of miR-142-5p in NSCLC cells.21 In this study, we found that the overexpression of miR-142-5p can suppress the expression of PIK3CA, which was consistent with the previous report. Besides, overexpressed LOXL1-AS1 could notably increase the expression of PIK3CA both at mRNA and protein level, and LOXL1-AS1 is positively correlated with PIK3CA in GC tissues. Interestingly, the overexpression of miR-142-5p can partly offset the promoting effect of LOXL1-AS1 on the expression of PIK3CA. These results signified that LOXL1-AS1 could promote the expression of PIK3CA by targeting miR-142-5p in GC.
This study has certain limitations. Other downstream miRNA of LOXL1-AS1 needs to be identified in the following studies. What’ more, in addition to acting as ceRNA, lncRNA also wields its function by regulating mRNA translation or binding to proteins.32 It is quite interesting whether LOXL1-AS1 plays its role via these mechanisms in GC, which also requires further investigation in the future. In summary, our findings highlight a unique lncRNA-regulated mechanism in GC, that is LOXL1-AS1 facilitates the proliferation and metastasis of GC cells by regulating the miR-142-5p/PIK3CA axis.
Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no competing interest.
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