Background
Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cause of cancer-associated death worldwide, with more than 1 million new cases and an estimated 783,000 deaths in 2018 [
1]. GC is an extremely complicated disease with a large number of genetic and epigenetic changes. Despite extensive studies on the pathogenesis of GC in recent decades, the 5-year survival rate of GC remains poor, mainly due to the lack of effective biomarkers for diagnosis of early GC as well as local recurrence and metastasis after operation [
2]. Therefore, continued research into this field is urgently needed to discover novel and more effective biomarkers and therapeutic targets for GC.
Long non-coding RNA (lncRNA) is a type of RNA molecule with a transcript length of more than 200 nucleotides and lacks protein-coding potential [
3]. Initially, lncRNA was regarded as the “garbage” of genome transcription without biological function. Nevertheless, recent studies have shown that lncRNA is involved in various important regulatory processes, such as X chromosome silencing, genomic imprinting, chromatin modification, transcriptional activation, transcriptional interference, intranuclear transport and so on [
4]. The transcripts generated by 4% to 9% of the mammalian genome sequence are lncRNAs (the corresponding protein-encoding RNA is 1%) [
5]. Although the research on lncRNA has progressed rapidly in recent years, the biological functions of most lncRNAs remain largely unknown.
It is well documented that lncRNA is able to tightly regulate gene expression at transcriptional and post-transcriptional levels, which makes it closely related to tumorigenesis and development [
6]. The most widely studied role of lncRNA is that it is capable of functioning as a competitive endogenous RNA (ceRNA) that interacts with and sequesters miRNAs to alleviate the repression of miRNAs on target mRNAs [
7]. For example, Chen et al. [
8] reported that lncRNA ZFAS1 contributed to the progression of colorectal cancer by sponging miR-150-5p to upregulating VEGFA expression. LncRNA CASC2 was proposed to increase PTEN expression via abundantly sponging miR-21 to inhibit pancreatic carcinoma malignancy [
9]. LncRNA CAR10 was found to promote lung adenocarcinoma metastasis by directly binding with and inhibiting miR-30/203 to elevate the expression of SNAI family [
10]. These studies suggest that the ceRNA network plays a vital regulatory role in tumorigenesis and aggressiveness.
Recently, a novel lncRNA, Tubulin Alpha 4B (TUBA4B), has been identified as an important tumor suppressor in various human cancers [
11]. However, its role in GC remains unexplored. In the present study, we aimed to investigate the expression level, clinical implication, biological function and potential regulatory mechanism of TUBA4B in GC.
Materials and methods
Tissues, cell lines and plasma
A total of 83 fresh GC and paired normal tissues were obtained from The Fourth Affiliated Hospital of China Medical University. These tissues were accurately diagnosed as GC by two experienced pathologists and then placed into liquid nitrogen to protect RNA integrity. To assess the diagnostic value of TUBA4B, we also collected plasma samples from GC patients (n = 37) and healthy controls (n = 37). This study was conducted with the approval of the ethics committee of China Medical University. All participants enrolled in this study had signed the informed consent.
To explore the biological function of TUBA4B, a human gastric epithelial GES-1 cells and five GC cell lines (AGS, SGC-7901, BGC-823, MGC-803 and HGC-27) were used. All cells were purchased from ATCC and cultured in DMEM medium with 10% fetal bovine serum. Mycoplasma test was performed on each cell line every 3 months.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
TRIzol reagent (Invitrogen, CA, USA) was employed to extract total RNA from GC tissues, cell lines and plasma. RNA quantification was performed using SYBR Green SuperMix (Roche, Basel, Switzerland) as per manufacturer’s protocols. GAPDH and U3 were used as the internal control for lncRNA/mRNA and miRNAs, respectively. The primer sequences are as follows:
TUBA4B:
Forward (5′ to 3′)-CCCACAGGCTTTAAGGTTGA;
Reverse (5′ to 3′)-AGGCCATAGTGATGGCTGTC
miR-214:
Forward (5′ to 3′)-TGCCTGTCTACACTTGCT;
Reverse (5′ to 3′)-GTCCAGTTTTTTTTTTTTTTTGCAC
mir-216a:
Forward (5′ to 3′)-GCAGTAATCTCAGCTGGCA;
Reverse (5′ to 3′)-TCCAGTTTTTTTTTTTTTTTCACAGT
mir-216b:
Forward (5′ to 3′)-GCAGAAATCTCTGCAGGCA;
Reverse (5′ to 3′)-GGTCCAGTTTTTTTTTTTTTTTCAC
GAPDH:
Forward (5′ to 3′)-TGCACCACCAACTGCTTAGC;
Reverse (5′ to 3′)-GGCATGGACTGTGGTCATGAG
U3:
Forward (5′ to 3′)-TTCTCTGAGCGTGTAGAGCACCGA;
Reverse (5′ to 3′)-GATCATCAATGGCTGACGGCAGTT
Establishment of stable TUBA4B overexpression GC cell lines
The full-length sequence of TUBA4B was synthesized and inserted into pLenti-GIII-CMV-GFP-2A-Puro vector (Applied Biological Materials, BC, Canada), followed by package into lentiviral particles using Lentifectin™ solution (Applied Biological Materials) for high efficiency transduction and stably integrated expression. Next, MGC-803 and HGC-27 cells were transducted with above lentiviral vector at a multiplicity of infection of 25. Two days later, cells were treated with 1.2 μg/mL puromycin (Applied Biological Materials) to select stable TUBA4B overexpression GC cell lines. The overexpression efficiency was determined by qRT-PCR analysis.
Cell proliferation and apoptosis assays
Cell Counting Kit-8 (CCK-8) and colony formation assays were utilized to measure the proliferative ability of MGC-803 and HGC-27 cells after TUBA4B overexpression. For CCK-8 assay, cells with or without TUBA4B overexpression were plated into 96-well plates and then incubated with 10 μL CCK-8 reagent (Sangon Biotech, Shanghai, China), followed by analysis of absorbance. For colony formation assay, MGC-803 and HGC-27 cells with or without TUBA4B overexpression were plated into 6-well plates. After 14 days, cells were fixed by methanol and stained by crystal violet. Cell apoptosis was carried out using Annexin V/7-AAD staining kit (Sino Biological Inc., Beijing, China) as per the standard protocol.
Transwell invasion assay
The invasive ability of GC cells was conducted using the Boyden chambers containing 24-well transwell plates (BD Inc., USA) with 8 mm pore size. MGC-803 and HGC-27 cells were seeded into on the upper surface of the chambers and DMEM medium containing 10% fetal bovine serum was added into the 24-well transwell plates. 18 h later, the invaded cells on the lower surface of the chambers were washed, fixed and stained.
Animal study
To evaluate the effect of TUBA4B on in vivo tumor growth, 5 × 106 control or TUBA4B-overexpressing MGC-803 cells were subcutaneously into the axilla of nude mice (n = 10 in each group), the volume measurement of subcutaneous tumors in each nude mice was conducted every 5 days. On the 30th day, all nude mice were euthanized and the tumors were dissected and weighed. To test the effect of TUBA4B on in vivo tumor metastasis, 1 × 106 control or TUBA4B-overexpressing MGC-803 cells were injected into the nude mice (n = 8 in each group) through the tail vein. Monitoring of lung metastasis was carried out using the IVIS Lumina II system. Five weeks later, all nude mice were sacrificed and the lungs were dissected and metastatic nodules were calculated, followed by H&E staining. All nude mice used were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and grown under specific-pathogen-free condition. The animal study was approved by the Animal Policy and Welfare Committee of China Medical University.
mRNA sequencing
Total RNA from control or TUBA4B-overexpressing MGC-803 cells was extracted by TRIzol reagent (Invitrogen) and subjected to mRNA sequencing. The high-throughput sequencing and subsequent data analysis was performed by GENESKY company (Shanghai, China) using the standard BGISEQ-500 platform. A total of 17,768 genes were detected. The value of differentially expressed mRNA after TUBA4B overexpression was set with fold change ≥ 2 and p < 0.05. Then, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Set Enrichment Analysis (GSEA) were conducted using DAVID v6.8 and GSEA v3.0 software, respectively.
Western blot
Total protein from control or TUBA4B-overexpressing MGC-803 and HGC-27 cells was isolated using 100 μL RIPA lysis buffer and subjected to protein quantification with BCA Protein Assay Kit (Sangon Biotech). Next, the protein was separated on 10% SDS-PAGE gel and then transferred onto PVDF membrane, followed by blockade with 5% dried skimmed milk or bovine serum albumin (for p-PI3K and p-AKT) and incubation with corresponding primary and secondary antibodies. Lastly, the membrane was strictly washed by tris buffered saline tween (TBST) and visualized by ECL western blotting substrate (Invitrogen). The primary antibodies used in this study are as following: anti-PTEN (#22034-1-AP, Proteintech, IL, USA), anti-p-PI3K (#4228, CST, MA, USA), anti-PI3K (#4249, CST), anti-p-AKT (#4060, CST), anti-AKT (#2920, CST), anti-GAPDH (#10494-1-AP, Proteintech).
Biotin pull-down assay
Total protein from MGC-803 and HGC-27 cells were obtained through using lysis buffer and then incubated with control or TUBA4B probe labeled with biotin at 4 °C overnight, followed by incubation with streptavidin-coupled magnetic beads (Invitrogen) on the next day at 25 °C for 2 h. Then, the TUBA4B binding miRNAs were washed and eluted and detected by qRT-PCR analysis.
Luciferase reporter assay
The full-length sequences of TUBA4B and PTEN 3′-UTR with putative wild-type or mutant miR-214/216a/b binding sites were embedded into FL reporter vector (Obio, Shanghai, China), respectively. MGC-803 and HGC-27 cells were seeded into 96-well plates and then co-transfected with a mixture of 5 pmol miR-214/216a/b mimics, 50 ng above FL reporter vectors and 5 ng pRL-CMV Renilla luciferase reporter vectors using Lipofectamine 3000 (Invitrogen). After 2 days of co-transfection, the luciferase activity was detected using Amplite Luciferase Reporter Gene Assay Kit (AAT Bioquest, CA, USA) as per manufacturer’s protocol.
Statistical analysis
Data were shown as mean ± standard deviation (SD) representing at least three effective independent replicates. The differences between groups were analyzed by Student’s t or Chi-square test. The value of TUBA4B in diagnosis and prognosis of GC was assessed by receiver operating characteristic (ROC) curve and Kaplan–Meier plot, respectively. All statistical results were two-tailed and produced by Graphpad 8.0 software. p < 0.05 was considered to be significant.
Discussion
It has been well documented that lncRNA is linked to human diseases, including cancer [
14]. Recently, a novel lncRNA, TUBA4B, was reported to be significantly decreased in breast cancer [
15], non-small cell lung cancer [
16] and ovarian cancer [
17]. However, an in-depth study on its clinical significance and biological function in GC has never been undertaken. Here, we found that TUBA4B was also dramatically downregulated in GC tissues, cells and plasma, which was closely related to malignant clinicopathological features and adverse prognosis. Further studies revealed that TUBA4B was able to abundantly sponge miR-214 and miR-216a/b and upregulate PTEN expression, resulting in dampening oncogenic PI3K/AKT signaling, thereby retarding GC tumorigenesis and aggressiveness (Fig.
5j). Thus, our findings advance the understanding of TUBA4B in human cancers, and demonstrate that TUBA4B is also a anti-tumor factor in GC.
Up to now, numerous studies show that lncRNA is frequently dysregulated in human cancers and can be used as an effective biomarker [
18]. For instance, high lncRNA SNHG1 expression was positively correlated with poor outcome in colorectal cancer patients [
19]. LncRNA MALAT-1 expression in serum was identified as a good distinction between hepatocellular carcinoma patients and healthy controls [
20]. LncRNA CASC11 was shown to be markedly increased in osteosarcoma and predicted dismal survival [
21]. Likewise, some lncRNAs related to the diagnosis or prognosis of GC have been reported, such as FLJ22763 [
22], GMAN [
23], ZEB1-AS1 [
24] and UCA1 [
25]. Herein, we found that GC patients with low TUBA4B expression displayed shorter survival time than patients with high TUBA4B expression, and the AUC value based on plasma TUBA4B expression was 0.8075 (95% CI 0.7103 to 0.9047), implying that TUBA4B is an efficacious diagnostic and prognostic biomarker for GC patients. Further large sample studies are needed to confirm our findings, and it would be worthwhile to clarify the crosstalk between TUBA4B and the above reported GC-associated lncRNAs, and whether TUBA4B can be detected in urine and exosomes.
Accumulating evidence suggests that cytoplasmic lncRNA is capable of altering gene expression via directly interaction with miRNAs, a mechanism known as ceRNA [
26]. Concordantly, by performing luciferase reporter and RNA pull-down assays, we identified that cytoplasmic TUBA4B could serve as an effective sponge for endogenous miR-214, miR-216a and miR-216b in GC cells. Several studies have reported that miR-214 was significantly upregulated in various cancers, including GC [
27‐
29]. However, miR-216a and miR-216b were proposed to be the tumor suppressors in some solid tumors [
30,
31], and the roles of these two miRNAs in GC remain unexplored. In this study, we found that TUBA4B overexpression dramatically reduced the expression of miR-216a and miR-216b, and GC patients with high miR-216a/b expression had worse prognosis than those with low miR-216a/b expression (survival data from Kaplan–Meier plotter database), hinting that miR-216a and miR-216b, like miR-214, are both oncogenes in GC. This notion was also confirmed by subsequent investigation that miR-214 and miR-216a/b could target the 3′-UTR of the well-known tumor suppressor PTEN and inhibit its expression, revealing that miR-214 and miR-216a/b are the mediators of TUBA4B and PTEN. It is widely accepted that PTEN is pervasively decreased in a various of human cancers and most oncogenic phenotypes caused by PTEN loss are attributed to the activation of PI3K/AKT signaling [
32]. In our study, ectopic expression of TUBA4B remarkably increased PTEN expression and decreased p-PI3K and p-AKT expression, and the TUBA4B-induced attenuated aggressive phenotype was significantly rescued by PTEN silencing and AKT activator, suggesting PTEN/PI3K/AKT signaling is responsible for the function of TUBA4B. In all, these above findings indicate that TUBA4B functions as a pivotal negative regulator in GC progression mainly through dampening oncogenic PI3K/AKT pathway via alleviating the inhibitory effect of miR-214 and miR-216a/b on PTEN. Further study is warranted to explore the role of TUBA4B in other cancers. It is noteworthy that nearly 20% of TUBA4B were located in the nucleus. Emerging evidence demonstrates that nuclear lncRNA can modulate gene expression at the transcriptional level via recruiting some key proteins to the promoter regions [
33,
34], it will be interesting to elucidate whether nuclear TUBA4B can also regulate PTEN expression through this mechanism.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.