Background
Gastric cancer (GC) is a cancer which brought pains to patients mind and body with high occurrence rates [
1], while patients in early stage of GC might be cured thoroughly with the efficient methods [
2]. So far, though the chemotherapy and radiotherapy were applied in the treatment, the recurrence rate was still extremely poor due to metastasis and the process of these treatments brought much pain to patient physically and psychologically [
3]. It is urgently needed to discover a novel treatment target to solve this problem.
Mounting articles indicated that long non-coding RNAs (lncRNAs) were actively participating in the process of cancers [
4]. And the abnormal expression of lncRNAs could function as tumor inhibitors or promoters to regulate the biological activities in cancers [
5,
6]. It was found that overexpression of PANDAR predicted poor prognosis and facilitated cell proliferation in cervical cancer [
7]. CCAT1 was discovered to elevate radiosensitivity by sponging miR-148b in breast cancer [
8]. CASC9 overexpression was seen as a tumor promoter in esophageal squamous cell carcinoma by negatively mediating PDCD4 via EZH2 [
9]. However, the role of LINC00511 in GC was unknown to us.
So far, accumulating researches introduced competing endogenous RNA (ceRNA) regulatory system where lncRNAs were treated as rivals to mRNAs to bind with microRNAs (miRNAs) so that those mRNAs lost their ability to translate into protein [
10‐
12]. H19 was reported as a ceRNA to bind to miR-29b-3p in bladder cancer to affect epithelial mesenchymal transition (EMT) [
13]. UCA1 was seen as a ceRNA of Sox4 in esophageal cancer to promote cell proliferation [
14]. HNF1A-AS1 was regarded as a ceRNA to accelerate the metastasis of colon cancer repressing miR-34a/SIRT1/p53 feedback loop [
15]. MiRNAs were a group of non-coding RNAs which were about 18–25 nucleotides in length. Recently, miRNAs have been reported to exert their function in multiple cancers [
16,
17]. miR-195 was seen as a tumor suppressor in non-small cell lung cancer through targeting CHEK1 [
18]. Nevertheless, how miR-625-5p took part in the course of GC needed to be explained. In our study, LINC00511 functioned as a ceRNA of miR-625-5p and fostered the progression of GC by targeting miR-625-5p/NFIX axis.
Our study was designed to explore the function of LINC00511 in GC cells. The results of our study manifested that knockdown of LINC00511 could successfully hinder the progression of GC by targeting miR-625-5p/NFIX axis, hinting that LINC00511 could be taken into account for GC treatment.
Materials and methods
Human tissue samples
Gastric cancer specimens and adjacent normal tissues of 35 participants were gathered from June 2013 to August 2018 from the Second Hospital of Shandong University. Before surgery, no treatment was applied in each patient. The study was allowed by the Institutional Review Committee and every patient signed informed consent before resection. When the surgical resection was finished, tissues were frozen instantly in liquid nitrogen, then removed and long-stored in a refrigerator at − 80 °C.
Cell culture
Human gastric mucosa epithelial cell (GES1) and gastric cancer cells (HGC27, BGC823, MGC803 and SGC7901) were bought from Chinese Academy of Sciences (Beijing, China). The above cells were incubated in DMEM (Gibco-BRL, Grand Island, NY, USA) containing 10% FBS (Gibco-BRL) plus 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were developed in a humidified condition with 5% CO2 at 37 °C. Medium for incubation was changed every 3 days.
Cell transfection
Transfection of HGC27 or BGC823 cells was processed in line with the protocol of Lipofectamine 3000 kit (Invitrogen). The duplex specific short hairpin RNAs (shRNAs) to LINC00511 (termed sh-LINC00511#1 and sh-LINC00511#2) and their corresponding nonspecific shRNAs as negative control (NC; termed sh-NC), were designed and synthesized by Genechem (Shanghai, China). The targeting sequences of LINC00511-specific shRNAs were as follows: sh-LINC00511#1, 5′-TACCGCGACACAAGTCTCCGTCCTCCTT-3′; sh-LINC00511#2, 5′-TCCGATGACGGGAGACGGGGTTCTGTCC-3′; sh-NC, 5′-TACCTCTGGATTCTCTCCCGCTCTCTGA-3′. For overexpressing LINC00511 or NFIX, cells were transfected with the pcDNA3.1 vector targeting LINC00511 or NFIX for 48 h, with cells treated with the empty pcDNA3.1 vector (Genechem) used as NC. The overexpression and silencing of miR-625-5p was achieved by transfecting cells with miR-625-5p mimics/inhibitors or NC mimics/inhibitors (all, Gene Pharmacy, Shanghai, China). 48 h post-transfection, cells were reaped for subsequent analysis.
Quantitative real-time PCR
Total RNA was obtained by TRIzol reagent (Invitrogen). Total RNA was reverse-transcribed into cDNA by use of a Reverse Transcription Kit (TaKaRa, Dalian, China). Then, qRT-PCR was progressed in Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA) by using a SYBR-Green Real-Time PCR Kit (Takara). 2− ∆∆Ct method was chosen for calculating fold expression changes. In addition, GAPDH/U6 was the internal reference.
CCK-8 assay
Transfected HGC27 or BGC823 cells were inoculated in fresh 96-well plates and cultured over specific time points (0, 24, 48, 72 and 96 h), followed by incubation with CCK-8 solution (Dojindo, Kumamoto, Japan) for another 4 h. Finally, the absorbance was measured under a microplate reader (Bio-Tek Instruments, Hopkinton, MA, USA) at 450 nm. Bio-triple repeats were required for CCK-8 assay.
TUNEL assay
Cell apoptosis of transfected HGC27 or BGC823 cells was determined by TUNEL apoptosis kit (Roche, Mannheim, Germany). Biologic coloring agent of DAPI (Haoran, Shanghai, China) was applied for dying the above cells. An EVOS FL microscope (Invitrogen) was employed for detecting the relative fluorescence intensity. Bio-triple repeats were required for TUNEL assay.
Transfected HGC27 or BGC823 cells were cultured in each well of a 6-well plate. After 2 weeks of cultivation, colonies were fixed in 4% PFA (Solarbio, Beijing, China) for 10 min and dyed with crystal violet (Solarbio) for 5 min. Colonies were counted manually later. Bio-triple repeats were required for colony formation assay.
Flow cytometer
The Annexin V/PI staining apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) was applied for measuring the proportion of apoptotic cells. Transfected HGC27 or BGC823 cells were loaded in 6-well plates, after which were washed twice using PBS (Sigma-Aldrich, St. Louis, MO, USA) and re-suspended in 1X Annexin V binding buffer (Invitrogen). Thereafter, cells were added to Annexin V-FITC and PI in a tube to incubate for 15 min. 400 µL of 1 × binding buffer was subsequently supplemented to each tube, followed by implementation of flow cytometry within 1 h. Final fluorescence was obtained on BD FACSCANTO II flow cytometer (BD Biosciences). Bio-triple repeats were required for flow cytometry analysis.
Western blot
Western blot was carried out in the light of prior method [
19]. Primary antibodies against cleaved caspase-3 (ab2302), total caspase-3 (ab13847), Bax (ab32503), Bcl-2 (ab32124), NFIX (ab101341) and GAPDH (ab9485) were all gained from Abcam (Cambridge, USA) and applied individually. Bio-triple repeats were required for western blotting.
Subcellular fractionation
The cytoplasmic and nuclear RNA purification kit (Norgen, Ontario, Canada) was employed for separating and purifying cytoplasmic and nuclear RNA based on specifications. Expression levels of LINC00511, GAPDH (cytoplasmic control) and U6 (nuclear control) were examined using qRT-PCR assays.
RNA immunoprecipitation (RIP)
RIP was undertaken employing the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA). HGC27 or BGC823 cells were re-suspended in RIP lysis buffer, followed by incubation with RIP buffer containing magnetic beads (Invitrogen) conjugated with anti-Ago2 antibody (Abcam) or anti-IgG antibody (Abcam) and rotated at 4 °C overnight. The abundance of LINC00511 or miR-625-5p or NFIX was assayed by qRT-PCR.
Luciferase reporter assay
LINC00511-WT/MUT or NFIX-WT/MUT was sub-cloned into the pmirGLO dual-luciferase vector (Promega, Madison, WI, USA) so as to generate pmirGLO-LINC00511-WT/MUT or pmirGLO-NFIX-WT/MUT. The pmirGLO-LINC00511-WT/MUT was co-transfected into HGC27 or BGC823 cells with miR-625-5p mimics or NC mimics. The pmirGLO-NFIX-WT/MUT was co-transfected into HGC27 or BGC823 cells with miR-625-5p mimics or miR-625-5p mimics + pcDNA3.1/LINC00511 or NC mimics. Following co-transfection for 48 h, dual luciferase reporter assay system (Promega) was applied.
RNA pull-down
Cell lysates of HGC27 or BGC823 cells were incubated with Bio-miR-625-5p-WT/MUT or Bio-NC. Magnetic beads were later added. At length, qRT-PCR was undertaken for assaying.
Xenograft models
The nude mice were gained from Vital River Laboratory (VRL, Beijing, China). GC cells transfected with sh-LINC00511#1 and sh-NC were reaped. Afterward, cells were injected into mice, subcutaneously. Volume of tumor was recorded by measuring length and width every 4 days. 4 weeks later, mice were killed, and the tumor weight was taken down.
Immunohistochemistry (IHC)
Tumor tissues obtained from xenograft models were embedded in paraffin (Sigma-Aldrich) and subjected to IHC. Tissues were deparaffinized and hydrated, followed by permeabilized for 10 min using 0.5% Triton X-100 (Sigma-Aldrich) in PBS. IHC for anti-Ki67 (Abcam) was carried out by the indirect avidin biotin-enhanced horseradish peroxidase method (Vector Laboratories, Burlingame, CA, USA). Sections were observed via a microscope (Nikon, Tokyo, Japan) and assayed using the Image-Pro Premier software offline program (Media Cybernetics, Rockville, MD, USA).
Statistical analysis
Data were denoted as mean ± SD. GraphPad Prism 7 software package (GraphPad Software, La Jolla, CA, USA) was used for analyzing the experimental data. One-way ANOVA or Student’s t-test was used to confirm the difference in two or multiple groups. P < 0.05 had statistically significance. Most importantly, this experiment was made in triplicate.
Discussion
GC brings a lot of pain and trouble to patients and it is necessary to cure it in early stage. When it developed into end-stage, the recurrence rate of treatment became increased due to metastasis [
20,
21]. Thus, early diagnosis is extremely vital for the treatment of GC. Recently, emerging studies surrounded the topic of ceRNA regulatory system where lncRNAs would bind to miRNAs competitively against mRNAs. Then, mRNAs could be translated into proteins and play biological function in in physiological and pathological process [
22,
23]. It was observed that ROR played as a ceRNA to modulate Oct4, Sox2 and Nanog expression by sponging miR-145 in colonic cancer stem cell [
24]. The GBAP1 pseudogene worked as a ceRNA via sponging miR-22-3p [
25]. In a word, these examples verified that lncRNAs modulated the development of cancer through ceRNA system.
In our study, LINC00511 was discovered highly expressed in GC tissues and cells, which had the same results as it was shown in osteosarcoma [
26]. Then, we detected the expression of LINC00511 in I/II and III/IV stage. The outcomes revealed that LINIC00511 was powerful in expression among I/II and III/IV stage but there is no striking difference between them. Knockdown of LINC00511 could repress cell proliferation while promoted apoptosis in GC. Based on these data, LINC00511 was verified to have crucial functions in GC progression. But these data were not sufficient to illustrate it did exert tremendous effects in early stage of GC. The relationship between LINC00511 and early development of GC would be explored in our future research. Besides, nuclear–cytoplasmic fractionation was used and the results showcased that LINC00511 amassed in cytoplasmic. A recent research also manifested that LINC00511 localized in cytoplasm in pancreatic adenocarcinoma [
27]. And with the help of starBase database, we found that some miRNAs had binding sites with LINC00511. And qRT-PCR assays were used to detect these miRNAs expression and we found that miR-625-5p was in a low level of expression in GC cell lines. The previous study confirmed that miR-625-5p was a tumor inhibitor in glioma [
28]. Moreover, overexpression of miR-625-5p could suppress the cell proliferation while enhance apoptosis of GC. LINC00511 was reported to function as a ceRNA by sponging miR-29b-3p in previous study. While we found that LINC00511 served as a sponge of miR-625-5p and miR-625-5p inhibitor could recover the effect of sh-LINC00511#1. Subsequently, we used starBase database to find out the downstream target of miR-625-5p and the results of RNA pull down assays manifested that NFIX bound to miR-625-5p evidently while others depicted no conspicuous change. So, NFIX was selected as the target of miR-625-5p. NFIX was introduced to have oncogenic function [
29]. Then, the results of rescue assay proved the effectiveness of LINC00511/miR-625-5p/NFIX axis in the course of GC cell. Overexpression of NFIX could counteract the effects of LINC00511-induced on cell proliferation and apoptosis. These results strongly confirmed LINC00511 role in ceRNA system in GC cell.
To further understand the role of LINC00511 in GC cell, we also conducted mice experiments to verify the effect of LINC00511. Compared with others study the role of LINC00511 in tongue squamous cell carcinoma without vivo experiments [
30], the data from our study stayed more convincing. The data collected from the tumor revealed that knockdown of LINC00511 could inhibit tumor growth and the volume and weight of tumor were evidently declined by sh-LINC00511#1. The expression of NFIX and Ki67 were also cut down by sh-LINC00511#1while the expression of miR-625-5p expression was enhanced.
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