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Clinical Medical Research Center, and Department of Oncology, Inner Mongolia People's Hospital, No. 20 Zhaowuda Road, Saihan District, Hohhot 010017, PR China
Clinical Medical Research Center, and Department of Oncology, Inner Mongolia People's Hospital, No. 20 Zhaowuda Road, Saihan District, Hohhot 010017, PR China
Clinical Medical Research Center, and Department of Oncology, Inner Mongolia People's Hospital, No. 20 Zhaowuda Road, Saihan District, Hohhot 010017, PR China
Clinical Medical Research Center, and Department of Oncology, Inner Mongolia People's Hospital, No. 20 Zhaowuda Road, Saihan District, Hohhot 010017, PR China
Clinical Medical Research Center, and Department of Oncology, Inner Mongolia People's Hospital, No. 20 Zhaowuda Road, Saihan District, Hohhot 010017, PR China
Clinical Medical Research Center, and Department of Oncology, Inner Mongolia People's Hospital, No. 20 Zhaowuda Road, Saihan District, Hohhot 010017, PR China
Clinical Medical Research Center, and Department of Oncology, Inner Mongolia People's Hospital, No. 20 Zhaowuda Road, Saihan District, Hohhot 010017, PR China
Increasing evidence showed that Heart and Neural Crest Derivatives Expressed 2 antisense RNA 1 (HAND2-AS1) was involved in the progression of several cancers, but its expression and function in gastric cancer (GC) was rarely reported. HAND2-AS1 expression in GC tissues and cells was detected at first. Cell function assays were performed to investigate the biological roles of HAND2-AS1 in GC cells. Moreover, the genes regulated by HAND2-AS1 in GC were investigated. Downregulation of HAND2-AS1 was found in GC tissues and cell lines. HAND2-AS1 overexpression inhibited GC cell proliferation, invasion, and arrested cell cycle at G0/G1 phase, whereas HADN2-AS1 knockdown significantly promoted cell proliferation and invasion. Bioinformatic analysis showed there is a potential HADN2-AS1/microRNA-769–5p (miR-769–5p)/transcription elongation factor A like 7 (TCEAL7) axis in GC. Luciferase activity reporter system was used to confirm this link. Taken together, our study showed that HAND2-AS1 exerts its tumor suppressive role in GC via regulating miR-769–5p/TCEAL7.
Worldwide, the incidence of gastric cancer (GC) ranks the fifth place, while its mortality ranks as the second place among all the cancer types in 2018 [
]. However, we still not fully understand the mechanisms behind the initiation and progression of GC, and hence further investigates are urgently needed.
The human genome project indicated that only 2% of total genome are protein-coding genes, which implied most of these genes are unable to code proteins [
]. In the past decades, numerous lncRNAs have been identified to be aberrantly expressed in human cancers, however, there were still many of them remained to be functionally characterized. For instance, lncRNA small nucleolar RNA host gene 16 was revealed to be highly expressed in GC, and affected cancer progression in vitro and in vivo via regulating microRNA-628–3p (miR-628–3p)/neuropilin 1 [
]. Wang et al. reported that lncRNA cancer susceptibility 19 was elevated expression in advanced GC tissues, and correlated with late tumor stages and poor overall survival [
]. Another work conducted by Guo and co-workers indicated that lncRNA prostate cancer-associated transcript 1 was upregulated in cisplatin resistant GC tissues and cells [
]. Also, they showed knockdown of prostate cancer-associated transcript 1 promotes the response of GC cells to cisplatin via regulating miR-128 and zinc finger E-box binding homeobox 1 [
Previous studies indicated that Heart and Neural Crest Derivatives Expressed 2 antisense RNA 1 (HAND2-AS1) was aberrantly expressed in human cancers and functioned as tumor suppressor [
]. However, the role and mechanism of HAND2-AS1 was far from fully elucidated in cancers. microRNA-769–5p (miR-769–5p) is a miRNA that reported to be decreased expression in non-small cell lung carcinoma, and regulated the cancer cell behaviors via targeting TGFBR1 [
]. Moreover, miR-769–5p was reported to function as a sponge for LINC00460 to regulate the expression of epidermal growth factor receptor and eventually to affect drug resistance of cancer [
Long Noncoding RNA LINC00460 Promotes the Gefitinib Resistance of Non-small Cell Lung Cancer Through Epidermal Growth Factor Receptor by Sponging miR-769-5p.
In this study, we investigated the expression pattern and biological roles of HAND2-AS1 in GC tissues and cells. Importantly, the molecular mechanisms behind HAND2-AS1 regulates GC cell events were deeply investigated.
2. Materials and methods
2.1 Cell lines and cell culture
GC cell lines (HGC-27 and MGC-803) and normal gastric epithelial cells (GES-1) were acquired from Cell culture collection center, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium in the appearance of 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The temperature of incubator was maintained at 37 °C and contained 5% CO2.
2.2 Cell transfection
HAND2-AS1 or miR-769–5p silenced cell was constructed by transferring small interfering RNA against HAND-AS1 (si-HAND2-AS1, 5′-CCGAGGUGCUCCAAUAUUATT-3′, GenePharm, Shanghai, China) or miR-769–5p inhibitor (5′-AGCUCAGAACCCAGAGGUCUCA-3′). HAND2-AS1, transcription elongation factor A like 7 (TCEAL7), microRNA-769–5p (miR-769–5p) overexpressed cells were built through transferring pcDNA3.1 contains HAND2-AS1 (pHAND2-AS1, Generay, Shanghai, China), TCEAL7 (pTCEAL7, Generay) or, miR-769–5p mimic (5′- UGAGACCUCUGGGUUCUGAGCU-3′). For transfection, cells were incubated to about 90% confluence, and 5 × 105 cells were transfected with these molecules using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the recommended instructions.
RNA was extracted from cells using Trizol, quantified at NanoDrop-1000,and reverse transcribed into complementary DNA using cDNA synthesis kit (Takara, Dalian, Liaoning, China). RT-qPCR was performed at ABI 7500 system (Applied Biosystems, Foster City, CA, USA) using the following primers: HAND2-AS1: 5′-GGGTGTTTACGTAGACCAGAACC-3′ (forward) and 5′-CTTCCAAAA-GCCTTCTGCCTTAG-3′ (reverse); TCEAL7: 5′-ACATCATGCAAAAACC-CTGCAA-3′ (forward) and 5′-ACGGTCCCGAGAATGCCTAT-3′ (reverse); β-actin: 5′-GACCTCTATGCCAACACAGT-3′ (forward) and 5′-AGTAC-TTGCGCTCAGGAGGA-3′ (reverse); miR-769–5p: 5′-TGAGACCTCTGG-GTTCTGAGCT-3′ (forward) and 5′-GTGCAGGGTCCGAGGT-3′ (reverse); U6 snRNA: 5′-AGAGCCTGTGGTGTCCG-3′ (forward) and 5′-CATCTTCAAAGCACTTCCCT-3′ (reverse). PCR conditions were: 95 °C for 40 s, followed by 40 cycles at 95 °C for 15 s and 55 °C for 45 s. β-actin was used as internal control for HAND2-AS1 and TCEAL7, while U6 snRNA was used as endogenous control for miR-769–5p.
2.4 Western blot analysis
Total protein was isolated from cells using RIPA lysis buffer, separated by 10% SDS-PAGE (30 µg/lane), and transferred to polyvinylidene fluoride membrane. Membranes were blocked by fat-free milk for 1 h at room temperature, and incubated with antibodies (rabbit anti-TCEAL7, LS-C146100, LSBio, Seattle, WA, USA, rabbit anti-GAPDH: ab181602, Abcam, Cambridge, MA, USA) at 4 °C overnight. After that, membranes were washed with PBST and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (ab6721) for 1 h, followed by development using an BeyoECL kit.
2.5 Wound healing assay
Cells in 6-well plate were cultured to about 90% confluence at the serum-free medium. Pipette tip was used to create scratch at cell surface. Then, phosphate-buffered saline (PBS) was used to remove cell debris. At 0 and 24 h after wound creation, cell images were captured and observed under microscope to measure wound width.
2.6 Cell invasion assay
Transwell invasion assay was performed to detect cell invasion ability. Briefly, cells were incubated in Matrigel (Corning, NY, USA) pre-coated 8 μm insert (Millipore, Billerica, MA, USA). The upper chamber was filled with RPMI-1640, while the bottom chamber was filled with FBS contained medium. After 48 h of transfection, non-invasive cells were removed by cotton swab, while invasive cells in the lower chamber was fixed with methanol, stained with crystal violet, and counted under microscope.
2.7 Cell apoptosis assay
Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) kit purchased from Beyotime (Haimen, Jiangsu, China) was used to detect cell apoptosis rate. Cells were collected, treated with cold PBS, and incubated with FITC/PI at dark from 15 min. Then, cells were subjected to flow cytometry analysis (BD Biosciences, San Jose, CA, USA).
2.8 Luciferase reporter assay
DIANA LncBASE prediction tool was used to explore the targets of HAND2-AS1, and the results showed that miR-769–5p contains putative binding site. Then, targetscan was used to analyze targets for miR-769–5p and results showed TCEAL7 contains putative binding site for miR-769–5p. Wild-type (wt) sequence of HAND2-AS1 or TCEAL7 contains putative binding region for miR-769–5p was cloned into psiCHECK-2 vector (Promega, Madison, WI, USA) to generate wt-HAND2-AS1 or wt-TCEAL7. Mutant (mt) luciferase reporter vectors, mt-HAND2-AS1 or mt-TCEAL7, were built from wt-HAND2-AS1 or wt-TCEAL7 using site-direct mutagenesis kit (Takara). For luciferase activity analysis, cells were co-transfected with luciferase vectors and miRNAs using Lipofectamine 2000. Relative luciferase activity was measured using Renilla activity as internal control after 48 h of transfection.
2.9 Expression of HAND2-AS1, miR-769–5p, and TCEAL7 in GC tissues as analyzed at StarBase
The expression levels of HAND2-AS1, miR-769–5p, and TCEAL7 in GC tissues and normal tissues were explored at StarBase (http://starbase.sysu.edu.cn/index.php), an open-source platform for studying the miRNA-ncRNA, miRNA-mRNA, ncRNA-RNA, RNA-RNA, RBP-ncRNA and RBP-mRNA interactions from CLIP-seq, degradome-seq and RNA-RNA interactome data..
2.10 Xenografts in mice
The study protocol was approved by ethic committee ofp Inner Mongolia People's Hospital. Cells with HAND2-AS1 overexpression were injected into the lower right flank of BALB/C nude mice (5 per groups). Width (W) and length (L) of tumor were measured with caliper every 5 days. The formula (W2 × L)/2 was used to estimate tumor volume. At 4 weeks after injection, mice were killed to measure tumor weight.
2.11 Statistical analysis
Data obtained from at least three experiments were analyzed with SPSS17.0 (IBM Corporation, Armonk, NY, USA) and presented as mean ± SD. Difference in the expression level of HAND2-AS1, miR-769–5p, and TCEAL7 in tumor tissues and normal tissues was analyzed using the build-in algorithm at ENCORI website. Student's t-test was employed to analyze differences in 2 groups, while one-way ANOVA and Tukey test was used to analyze differences in 3 or above groups. Difference was regarded as significant when P value less than 0.05.
3. Results
3.1 HAND2-AS1 expression in GC tissues and cells
The analysis of HAND2-AS1 expression in GC tissues and normal tissues was performed at StarBase. The results presented in Fig. 1A revealed that HAND2-AS1 expression was decreased in GC tissues compared with normal tissues. Moreover, RT-qPCR revealed that HAND2-AS1 expression level in GC cells was lower than that in normal cell line (Fig. 1B).
Fig. 1Expression of HAND2-AS1 in GC tissues and cells. (A) Downregulate expression of HAND2-AS1 was observed in GC tissues compared with normal tissues. (B) Expression level of HAND2-AS1 was found lower in GC cells than in normal cells. HAND2-AS1: Heart and Neural Crest Derivatives Expressed 2 antisense RNA 1; GC: gastric cancer.
3.2 Knockdown of HAND2-AS1 promotes GC cell migration, invasion but inhibits apoptosis
Expression level of HAND2-AS1 in GC cell after si-HAND2-AS1 introduction was analyzed with RT-qPCR. We showed HAND2-AS1 expression level was significantly decreased by si-HAND2-AS1 (Fig. 2A). Wound-healing assay showed cell migration ability was promoted after HAND2-AS1 knockdown (Fig. 2B). Transwell invasion assay revealed that invasive cell numbers were elevated by si-HAND2-AS1 (Fig. 2C). The analysis of cell apoptosis percentage after si-HAND2-AS1 transfection showed that cell apoptosis was significantly suppressed compared with siR-NC transfection (Fig. 2D).
Fig. 2Decreased HAND2-AS1 expression promoted GC cell migration, invasion but inhibited apoptosis. (A) HAND2-AS1 expression, (B) Cell migration, (C) Cell invasion, and (D) Cell apoptosis in GC cell with si-HAND2-AS1 or siR-NC transfection. HAND2-AS1: Heart and Neural Crest Derivatives Expressed 2 antisense RNA 1; GC: gastric cancer; siR-NC: negative control small interfering RNA; si-HAND2-AS1: small interfering RNA targeting HAND2-AS1.
3.3 Overexpression of HAND2-AS1 inhibits GC cell migration, invasion but promotes apoptosis
Furthermore, gain-of-function experiments were performed to analyze the functions of HAND2-AS1. The transfection of pHAND2-AS1 in GC cell was verified by RT-qPCR (Fig. 3A). Wound-healing assay and transwell invasion assay revealed that overexpression of HAND2-AS1 inhibits GC cell migration and invasion (Fig. 3B, C). In addition, flow cytometry assay showed that cell apoptosis in GC cell was promoted by pHAND2-AS1 (Fig. 3D).
Fig. 3Elevated HAND2-AS1 expression inhibited GC cell migration, invasion but promoted apoptosis. (A) HAND2-AS1 expression, (B) Cell migration, (C) Cell invasion, and (D) Cell apoptosis in GC cell with pHAND2-AS1 or pcDNA3.1 transfection. HAND2-AS1: Heart and Neural Crest Derivatives Expressed 2 antisense RNA 1; GC: gastric cancer.
3.4 HAND2-AS1 exerts its role via targeting miR-769–5p
Targets for HAND2-AS1 were predicted at DIANA LncBASE and miR-769–5p was selected for analyses as it ranks top among all potential targets and it was previously reported to have crucial roles in cancer progression [
Long Noncoding RNA LINC00460 Promotes the Gefitinib Resistance of Non-small Cell Lung Cancer Through Epidermal Growth Factor Receptor by Sponging miR-769-5p.
. The binding region between HAND2-AS1 and miR-769–5p was shown in Fig. 4A. Luciferase activity reporter assay showed that overexpression of miR-769–5p inhibits the relative luciferase activity of cells transfected with wt-HAND2-AS1 (Fig. 4B). miR-769–5p was revealed to be elevated expression in GC tissues (Fig. 4C). Wound-healing assay, transwell invasion assay, and flow cytometry assay revealed the effects of pHAND2-AS1 on GC cell behaviors via regulating miR-769–5p (Fig. 4D-F).
Fig. 4HAND2-AS1 directly target miR-769–5p in GC. (A) Binding region between HAND2-AS1 and miR-769–5p. (B) Relative luciferase activity in cells transfected with wt/mt-HAND2-AS1 or miR-769–5p mimic or miR-NC. (C) Expression of miR-769–5p in GC tissues and normal tissues. (D) Cell migration, (E) Cell invasion, and (F) Cell apoptosis in GC cell with miR-769–5p mimic, miR-769–5p inhibitor, miR-NC, pHAND2-AS1 and mi-NC, or pHAND2-AS1 and miR-769–5p mimic transfection. HAND2-AS1: Heart and Neural Crest Derivatives Expressed 2 antisense RNA 1; GC: gastric cancer; wt: wild-type; mt: mutant; miR-NC: negative control microRNA; miR-769–5p: microRNA-769–5p.
Furthermore, the targets of miR-769–5p were predicted by TargetScan. The analysis result showed TCEAL7 was a putative target for miR-769–5p (Fig. 5A). Luciferase activity reporter assay confirmed the interaction of miR-769–5p and 3′-UTR of TCEAL7 (Fig. 5B). Analysis of TCEAL7 expression in human tissues showed that TCEAL7 expression level was elevated in GC tissues compared with normal tissues (Fig. 5C). In addition, TCEAL7 protein level was found to be decreased expression in GC cells compared with normal cells (Fig. 5D). Functional experiments showed that overexpression of TCEAL7 inhibits GC cell migration, invasion but promotes apoptosis (Fig. 5D-F). Moreover, we showed the introduction of miR-769–5p mimic partially reversed the effects of pTCEAL7 (Fig. 5D-F).
Fig. 5miR-769–5p directly target TCEAL7 in GC. (A) Binding region between TCEAL7 and miR-769–5p. (B) Relative luciferase activity in cells transfected with wt/mt-TCEAL7 or miR-769–5p mimic or miR-NC. (C) Expression of TCEAL7 in GC tissues and normal tissues. (D) Expression level of TCEAL7 was found lower in GC cells than in normal cells. (E) Cell migration, (F) Cell invasion, and (G) Cell apoptosis in GC cell with pTCEAL7, pcDNA3.1, pcDNA3.1 and miR-769–5p mimic, pcDNA3.1 and miR-769–5p inhibitor, or pTCEAL7 and miR-769–5p mimic transfection. TCEAL7: transcription elongation factor A like 7; GC: gastric cancer; wt: wild-type; mt: mutant; miR-NC: negative control microRNA; miR-769–5p: microRNA-769–5p.
Furthermore, we examined the effects of HAND2-AS1 overexpression on tumor growth in vivo. As indicated in Fig. 6A, tissues obtained in groups with HAND2-AS1 overexpression were significantly smaller than those without. Similarly, tumor volume and weight were also smaller in HAND2-AS1 overexpression than the control groups (Fig. 6B, C).
Fig. 6HAND2-AS1 inhibits GC tumor growth in vivo. (A) Tumors removed from HAND2-AS1 overexpression and control groups were presented. (B) Tumor volume, and (C) Tumor weight in groups with or without HAND2-AS1 overexpression were presented. HAND2-AS1: Heart and Neural Crest Derivatives Expressed 2 antisense RNA 1; GC: gastric cancer.
In this work, by analyzing HAND2-AS1 expression in GC tissues or cells using StarBase or RT-qPCR. HAND2-AS1was found to be significantly decreased in GC tissues and investigated cell lines compared with normal tissues and cells. Furthermore, in vitro experiments uncovered that overexpression of HAND2-AS1 could inhibit GC cell migration, invasion but promote apoptosis, while the knockdown of HAND2-AS1 caused the opposite effects. The in vivo experiments showed forcing the expression of HAND2-AS1 could suppress GC tumor growth. Collectively, our results suggested a tumor suppressive role of HAND2-AS1 in GC progression, which is similar to its role in cancers including endometrioid endometrial carcinoma, colorectal cancer, and non-small cell lung cancer [
Furthermore, the ceRNA theory was applied to investigate the biological functions of HAND2-AS1 in GC. Through bioinformatics tools and mechanical experiments, we showed miR-769–5p exhibited strong binding potential to HAND2-AS1. Here, we showed miR-769–5p expression level was elevated in cancer tissues compared with normal tissues. Recue experiments validated miR-769–5p as a functional target of HAND2-AS1. A recent work indicated that high miR-769–5p expression was correlated with tumor-node-metastasis (TNM) stage, lymph node metastasis, and infiltration depth of GC patients [
]. In this work, we analyzed the targets of miR-769–5p using TargetScan and found TCEAL7 was a putative target. This interaction was further validated by luciferase activity reporter assay and rescue experiments. TCEAL7 was revealed as a tumor suppressor gene to regulate the NF-kappaB pathway [
]. Here, we presented a novel regulatory mechanism for roles of HAND2-AS1 in GC progression. Another difference of this current work with our previous work is that we analyzed the roles of HAND2-AS1 on GC tumor growth in vivo using animal model. The limitation of this work was no GC patients were enrolled, and thus we failed to explore the correlations of HAND2-AS1 with clinicopathological features of GC patients.
In summary, our results identified HAND2-AS1 was decreased expression in GC tissue sand cells. Besides, HAND2-AS1 could regulate the migration, invasion, and apoptosis of GC cells via regulating the miR-769–5p/TCEAL7 axis. Our results may provide novel therapeutic targets for GC.
CRediT authorship contribution statement
Lan Yu: Formal analysis, Writing - original draft. Wei Luan: Formal analysis, Writing - original draft, Writing - review & editing. Zongqi Feng: Formal analysis, Writing - original draft, Writing - review & editing. Jianchao Jia: Formal analysis, Writing - original draft, Writing - review & editing. Zhouying Wu: Formal analysis, Writing - original draft, Writing - review & editing. Min Wang: Formal analysis, Writing - original draft, Writing - review & editing. Feng Li: Formal analysis, Writing - original draft, Writing - review & editing. Zhiying Li: Formal analysis, Writing - original draft, Writing - review & editing.
Availability of data and materials
Not applicable.
Ethics approval and consent to participate
Animal study protocol was approved by Inner Mongolia People's Hospital.
Consent for publication
Not applicable.
Conflicts of interest
None declared.
Acknowledgments
This work was supported by National Natural Science Foundation of China (Grant No. 81560405), Research Foundation of Inner Mongolia People's Hospital (Grant No. 201415), Inner Mongolia Natural Science Foundation (Grant No. 2018MS08060), Scientific Research Project Foundation of Inner Mongolia Health Commission (Grant No. 201702003), and Ph.D. Science Foundation of Inner Mongolia People's Hospital (Grant No. BS201805).
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Long Noncoding RNA LINC00460 Promotes the Gefitinib Resistance of Non-small Cell Lung Cancer Through Epidermal Growth Factor Receptor by Sponging miR-769-5p.
Recently, an interesting study entitled “Long non-coding RNA HAND2-AS1 inhibits gastric cancer progression by suppressing TCEAL7 expression via targeting miR-769–5p” was published in Digestive and Liver Disease [1]. In this study, the authors revealed that HAND2-AS1 was downregulated in gastric cancer (GC) tissues and cell lines. They further showed that HAND2-AS1 overexpression inhibited GC cell proliferation, invasion and arrested cell cycle at G0/G1 phase via regulating the miR-769–5p/TCEAL7 axis, whereas HAND2-AS1 knockdown caused the opposite effects.
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