lncRNA ABHD11-AS1 drives gastric cancer development by targeting miR-1301-3p/PDPK1 signaling CURRENT POSTED

Background Long noncoding RNAs (lncRNAs) has long been reported to associate with multitudinous kinds of cancer. The ABHD11-AS1, a newly identified lncRNAs, has been demonstrated as a new marker of gastric cancer (GC). In this study, a significant upregulation of ABHD11-AS1 was observed in GC, compared to adjacent normal tissues. Method Immunohistochemical analysis for ABHD11-AS1, 3-phosphoinositide dependent protein kinase 1 (PDPK1), miR-1301-3p was performed on gastric tumor and adjacent normal tissues. The gene expression was evaluated by qRT-PCR. The proliferation was assayed by CCK8 and Annexin V/PI assay. ABHD11-AS1 is highly expressed in compared with normal tissues( p<0.01) Knock-down of ABHD11-AS1 suppress the aberrant proliferation of GC cells si-Scramble( p<0.01). a for miR-1301-3p in GC miR-1301-3p/ABHD11-AS1 regulated the expression of PDPK1 in GC cells compared with control p<0.01). miR-1301-3p/ ABHD11-AS1 co-mediated proliferation and apoptosis of GC compared with si-Scramble/Mock and si-ABHD11-AS1/Mock p<0.01) may exert oncogenic effect by negatively regulating miR-1301-3p expression in GC. Then bioinformatics analysis was performed to predict the latent downstream targets of miR-1301-3p. The analysis revealed that PDPK1 may be a downstream target of

3 Background Gastric cancer(GC) is one kind of common tumors with high morbidity around the world [1].
Although great progress has been made in diagnostic methods as well as treatments for GC, there are still a growing number of GC patients with poor prognosis [2][3][4]. Even worse, a large proportion of patients with GC remain undiagnosed until late stage which is incurable. The pathogenic mechanism mediating the fast proliferation of GC is still not fully understood yet. Long noncoding RNAs (lncRNAs), comprising more than 200 nucleotides and belonging to noncoding RNAs, play a vital role in physical progress and in many human diseases [5,6]. Aberrant expression of lncRNAs has been reported to mediate the initiation and metastasis of tumor, which can also serve as a prognostic marker of many kinds of disease. [7][8][9].
LncRNA ABHD11-AS1, known as long noncoding RNA ABHD11 Antisense RNA 1, has been reported to associate with the oncogenesis and metastasis of gastric tumor [6,10]. The expression of ABHD11-AS1 was significantly increased in tumor tissues compared with adjacent normal tissues. miRNAs are a group of automatically occurring single-stranded short 21-23 nt non-coding RNAs, which exist in eukaryotic organisms [11,12]. miRNA can prevent the transcription of targeted mRNAs and by which the expression of target gene is inhibited [13,14]. LncRNAs can serve as miRNA sponges, diminishing the regulatory effects of miRNAs on mRNAs [15,16]. 3-phosphoinositide dependent protein kinase 1 (PDPK1) is a serine/threonine kinase with many substrates including PKA, AKT, PKC-zeta, and p90S6K, functioning as a major control point by activating a rang of signaling pathways involved in cellular proliferation and apoptosis [17,18]. The downstream molecules of PDPK1 are usually hyper-activated in many kinds of cancer cells and the overexpression of PDPK1 has been observed in a many kinds of human cancer cell lines, including GC [19][20][21]. Therefore, PDPK1 is expected to function as an oncogenic protein. 4 In this study, we demonstrated that the overexpression of ABHD11-AS1 could serve as a clini-copathological factor for GC patients. We pioneeringly identified that miR-1301-3p acts as a key mediator for the regulation of ABHD11-AS1 on the expression of PDPK1.

Patients and tissue samples
GC tissues and adjacent normal tissues were collected from 120 volunteers at jiangshu Province People's Hospital from January to December,2017. Each specimen was snapfrozed in liquid nitrogen and stored temporarily in a −80℃ freezer before RNA isolation and qRT-PCR analysis. All specimens were collected before patients received either chemotherapy or radiotherapy. Written informed consents were obtained from all patients for the agreement regarding the use of tissue samples in our research. Selection bias of GC samples collection in our study was avoided.
Cells were transfected with Lipofectamine 3000 (Life Technologies) according to the manufacturer's guide. The ABHD11-AS1 sequences were as follows:  Total RNA from tissues and cells was extracted by TRIzol reagent (InvitrogenCarlsbad,CA) and cDNA was reverse-transcripted using SuperScript First-StrandSynthesis System (Invitrogen) according to the manufacturer's direction. Quantitative RT-PCR was conducted with ABI 7500 system (Applied Biosystems, CA, USA) to measure the relative expression of target genes. GAPDH or U6 was used as the internal normalizer for target genes or miRNA, respectively. All the primers were designed and synthesized by Shanghai Sheng gong Biotechnology (Shanghai). The primer sequenc es were as follows: GAPDH,F:TCGGAGTCAACGGATTTGGT;R:TTCCCGTTCTCAGCCTTGAC U6, F: CTCGCTTCGGCAGCACA; R: AACGCTTCACGAATTTGCGT 2.5 Cell proliferation assay CCK8 assay was used to measure the cell proliferation activity by a cell counting kit (CCK8, MedChem Express) kit according to manufacturer's recommend protocol.

Colony formation assay
For clonogenic assay, 200 cells per well of CRC cells were seeded in six-well plates and incubated for 2 weeks until visible colonies formed (cell number more than 50). Colonies were washed, fixed with 4% formaldehyde, stained with 0.1% crystal violet, and counted.

Cell apoptosis detection assay
For cell apoptosis, active caspase 3 human ELISA kit (Invitrogen) was used to evaluate the cell apoptosis according to the manufacture's recommended protocol. Annexin V/PI assay was performed to measure the apoptosis percent of GC. The procedure was the same as 6 the previous description
Subsequently, mutated or wild-type pMir Reporter luciferase vectors and miR-1301-3p mimic or NC-mimic were co-transfected into MGC803 and BGC823 cell lines in 96-well plates for 24 h with Lipofectamine 3000 (Invitrogen,Carlsbad,CA,). The luciferase activity was detected by the luciferase reporter assay system (Promega) according to the manufacture's protocol.

RNA immunoprecipitation (RIP) assay
We utilized the Magna RIP RNA-binding protein immunoprecipitation kit (Sigma) to perform RIP assay according to the manufacturer's recommended procedure. Cells were lysed by RIP lysis buffer. Then, whole cell lysate was co-incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Sigma), and negative control normal mouse IgG (Sigma). Immunoprecipitated RNA was acquired and then the expression levels of miR-133a-3p and ABHD11-AS1, which emerged in the sediments, was analyzed by qRT-PCR analysis.

Western blot analysis
Cells were collected and lysed with RIPA lysis buffer supplemented with proteinase inhibitors on ice (Roche). And then, the lysates were collected and treated with ultrasoni -7 cation and centrifugation at 15000 rpm for 8 min. The supernatants of cell specimen were the kept as purified target pro tein. And target proteins were seperated by electrophoresis in a 10% sodium-dodecyl sulfate polyacrylamide gel (SDS-PAGE) and blotted onto PVDF membrane. After blocking with 5% BSA buffer for 1h, the blots were incubated with primary antibodies for 2 h at room temperature. The primary antibodies used in this study included β-actin antibody (Rabbit, Sigma) and PDPK1 antibody (Rabbit, Cell Signaling Technology). After subsequent incubation with secondary antibody, the bands were scanned by using LI-COR-Odyssey scanner (LI-COR). Signal intensity of bands were further analyzed by Image J software.

Statistical methods
All the statistical analyses were performed with GraphPad Prism 5.0. The data are presented as the means ± standard deviation (S.D.). Student's t-test or one-way analysis of variance (ANOVA) were used to analyze the difference between groups for expression of apoptosis percent, colony formation, cell proliferation, target gene. The log-rank test and Kaplan-Meier method was conducted to evaluate PFS or OS. two-tailed P-values less than 0.05 were considered statis tically significant. ( *P <0.05, **P <0.01, ***P <0.01 ).

ABHD11-AS1 is highly expressed in GC tissues
To prove oncogenic role of lncRNA ABHD11-AS1 in GC, we firstly analyzed the relationship between the expression level of ABHD11-AS1 and survival duration of GC patients in open databases (Figure1A,B). We found a worse prognosis of gastric cancer patients was correlated with a higher level of ABHD11-AS1.
Based on the analysis, we further explore whether the level of ABHD11-AS1 was significantly increased in GC specimens and cell lines. Firstly, we determined the 8 expression level of ABHD11-AS1 in GC tissues and adjacent normal tissues with RT-PCR analysis, and the gene expression was normalized to GAPDH. As expected, the expression level of ABHD11-AS1 was significantly upregulated in GC tissues compared with normal counterparts (*p<0.01, Firgure 1C). Moreover, to further validate whether the expression level of ABHD11-AS1 was significantly increased in GC, we measured the expression of Meanwhile, the results of Annexin V-FITC staining assay proved that the knock-down of ABHD11-AS1 promoted the apoptosis percent of GC cell lines compared to si-Scramble ( *p<0.01, Figure2H,I). Since apoptosis-promoting gene Bax and enzyme caspase-3 could mediate the process of cell apoptosis [23][24][25], their protein levels were measured by RT-PCR and Elisa assay in our study. The results of Elisa assay suggested that inhibition of ABHD11-AS1 enhanced the level of apoptosis-promoting enzyme caspase-3 compared to si-Scramble (*p<0.01 ,Figure2J ) and the RT-PCR analysis showed that Bax was remarkably higher in ABHD11-AS1 knock-down group comapred to si-Scramble ( *p<0.01, Figure2k ).
In conclusion, ABHD11-AS1 could be a potential therapeutic target to suppress the growth of GC.  01,Figure3A,B ). Therefore, we suspected miR-1301-3p as a potential target molecule interacting with ABHD11-AS1 in this research. To further explore whether ABHD11-AS1 functioned by sponging miRNA or acting as competing endogenous RNAs (ceRNAs), bioinformatics tools (microRNA.org and miRcode) were utilized to identify the microRNA binding sites of ABHD11-AS1( Figure 3C). qPCR assay showed miR-1301-3p expression level was significantly decreased in GC tissues compared with adjacent normal tissues (*p<0.01 , Figure 3D ).

miR-1301-3p/ABHD11-AS1 regulated the expression of PDPK1 in GC cells
By searching public database (starbase), Pearson correlation analysis suggested that there was a strong negative correlation between miR-1301-3p and PDPK1 expression in GC tissues (Firgure 4A). Bioinformatics analysis by TargetScan and miRBase predicted that PDPK1 had matched binding bases with PDPK1 ( Figure 4B). Luciferase reporter assay revealed that the over-expression of miR-133a remarkably suppressed PDPK1 3'-UTR activity while the activity of the mutant PDPK1 3'-UTR was unaffected compared with mock (*p<0.01 , Figure 4C ). Moreover, the up-regulation of miR-133a had a significant negative effect on the expression of PDPK1 at the levels of both mRNA and protein, compared to mock treatment (*p<0.01 , Figure4D,E ). To determine whether ABHD11-AS1 competitively inhibited the binding of miR-1301-3p with PDPK1, luciferase reported assays were conducted. The results suggested that ABHD11-AS1 overexpression could offset the suppressing effect of miR-1301-3p on endogenous ABHD11-AS1 in GC cell lines (*p<0.01, Figure H,I ). In addition, q-PCR analysis and western blot assays demonstrated that there was a mutual antagonism between miR-1301-3p and ABHD11-AS1 on binding to PDPK1 ( Figure 4J,K,L ). In short, miR-1301-3p/ABHD11-AS1 behave as counteract regulators on the expression of PDPK1 in GC lines.

miR-1301-3p/ ABHD11-AS1 co-mediated proliferation and apoptosis of GC cell
Finally, we showed that ABHD11-AS1 could promote GC through regulating the binding

Discussion
Great progress has been made in high-throughput gene sequencing analysis , which updated our comprehension that only less than 2% of human genome have been proved to be transcribed, and many non-coding RNAs with limited or no protein-coding capacity exist in human genome [26][27][28]. Long non-coding RNAs (lncRNAs) are known as a class of noncoding RNA tran-scripts and are associated with some key process, such as, cellar proliferation and apoptosis. Growing number of evidences have demonstrated that lncRNAs may contribute to the tumorigenesis of GC[29-31]. LncRNA ABHD11 Antisense RNA 1 (ABHD11-AS1) has been demonstrated to serve as diagnostic and prognostic biomarker in ovarian cancer, bladder cancer, gastric cancer [6,32,33]. However, the underlying molecular mechanisms of ABHD11-AS1 was still elusive in GC.
In this study, we firstly showed that the expression of lncRNA ABHD11-AS1 was upregulated in GC and ABHD11-AS1 expression could be one prognostic marker for GC proved that biotin-labeled ABHD11-AS1 remarkably pull-down miR-1301-3p. Moreover, a negative correlation between miR-133a and ABHD11-AS1 was identified in this study.
Simultaneously, luciferase reporter assays also revealed that miR-1301-3p and ABHD11-AS1 shared same binding site. Lastly, we found an endogenous interaction between miR-1301-3p and ABHD11-AS1 by using RIP with anti-Ago2 antibody. Although the antagonism relationship between ABHD11-AS1 and miR-1301-3p has not been reported in GC cells, nevertheless, it has been reported that miR-1301-3p contributed to the expansion of However, increased expression of ABHD11-AS1 has been found in GC [33]. Therefore, ABHD11-AS1 may exert oncogenic effect by negatively regulating miR-1301-3p expression in GC. Then bioinformatics analysis was performed to predict the latent downstream targets of miR-1301-3p. The analysis revealed that PDPK1 may be a downstream target of miR-1301-3p. In previous studies, PDPK1 has been identified as a marker of tumor [19].
In conclusion, our results firstly demonstrated that the overexpression of ABHD11-AS1 was correlated with a poor prognosis of GC patients. ABHD11-AS1 could serve as a ceRNA to promote PDPK1 expression by neutralizing miR-1301-3p, consequently contributing to the proliferation and apoptosis of GC. These results demonstrated that ABHD11-AS1 could be a potential therapeutic target for GC treatment.

Ethics approval and consent to participate
The study has been approved by the ethics committee ofThe Affiliated Cancer Hospital of Nanjing Medical University All procedures performed in our study were in accordance with the ethical standards of the institutional committee and with the 1964 Helsinki declaration and its later 14 amendments or comparable ethical standards.Written informed consent was obtained from all individual participants included in the study.

Consent for publication
We have obtained written consent to publish from the participants.

Availability of data and materials
Not applicable

competing interest
All authors certify that they have no affiliations with or involvement in any organization or entity with any financial

Funding
The authors declare that there are no sources of funding to be acknowledged.  The expression of PDPK1in GC cell lines is regulated by the antagonism effect between miR-1301-3p and ABHD11-AS1 The correlation between PDPK1 and Mir-