miR-96-5p enhances cell proliferation and invasion via targeted regulation of ZDHHC5 in gastic cancer

Objective: To explore the biological function and mechanism of miR-96-5p in gastric cancer. Methods: The expression of DEMs related to GAC prognosis was identified in GAC tumor samples and adjacent normal samples by qRT-PCR. A target gene miR-96-5p was selected using TargetScan, miRTarBase, miRDB databases. The combination of miR-96-5p and ZDHHC5 was verified by luciferase receptor assay. To further study the function and mechanism of miR-96-5p ， we treated MGC-803 cells with miR-96-5p inhibitor and si-ZDHHC5, then detected cell viability, apoptosis, migration and invasion ability, as well as the expression of ZDHHC5, Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9 and COX-2 by western blot. Results: Compared with adjacent normal samples, the levels of miR-96-5p, miR-222-5p, and miR-652-5p were remarkably increased, while miR-125-5p, miR-145-3p, and miR-379-3p were significantly reduced in GAC tumor samples ( P < 0.01), which were consistent with bioinformatics analysis. Furthermore, ZDHHC5 was defined as a direct target gene of miR-96-5p. miR-96-5p silence significantly reduced cell viability, increased cell apoptosis, and suppressed cell migration and invasion, as well as inhibited the expression of Bcl-2 and COX-2 and promoted Bax, cleaved caspase-3 and cleaved caspase-9 level in MGC-803 cells ( P < 0.01). Notably, ZDHHC5 silence reversed the inhibiting effects of miR-96-5p on MGC-803 cells growth and metastasis suggested that downregulated miR-96-5p might inhibit tumor cell growth and metastasis via increasingZDHHC5 expression enhance MGC-803 cell apoptosis, as well as decrease MGC-803 cell metastasis.


Introduction
Gastric adenocarcinoma (GAC) is the most common malignant tumor originating in the stomach, with approximately 951,000 diagnosed cases and 723,000 deaths in 2012 [1,2]. At present, the common and effective treatment method is to combine surgery with adjuvant radiotherapy or chemotherapy to improve the 5-year survival rate of GAC [3]. However, due to atypical symptoms of early GAC, most patients with proximal or distal metastases find delayed diagnosis, which leads to poor treatment and prognosis [4]. Thus, it is essential to reveal novel diagnostic and therapeutic targets for GAC.
It is well known that a major challenge of GAC treatment is poor prognosis, and environmental exposures and genetic mutations have been identified to be associated with this outcome [5]. A large body of evidence indicates that the poor prognosis of GAC is significantly associated with many molecular biomarkers such as microRNA (miRNA) [6,7]. miRNAs, as endogenous non-coding small-molecule RNAs, widely exist in severe conditions [8]. It has been revealed that a large number of miRNAs are involved in various biological functions such as cell differentiation, apoptosis, migration, invasion, and proliferation in human diseases through post-transcriptional regulation of gene expression [8,9]. Previous studies have demonstrated that miRNA disorders can significantly influence the prognosis of patients with gastric cancer, such as miR-203 [10], miR-21 [11], and miR-25 [12]. Imaoka et al. [10] have reported that a low serum miR-203 expression is related to poor prognosis, and which is considered as a noninvasive biomarker for prognosis of gastric cancer patients.
Simonian et al. [11] also have evidence that circulating miR-21 can be considered as a diagnostic and prognostic biomarker for gastric cancer. In addition, Li et al. [12] revealed that miR-25 is associated with the prognosis of gastric cancer, and can induce cell migration and proliferation by targeting transducer of ERBB2, 1.
Therefore, it is important to find novel miRNAs related to the prognosis of GAC. May contribute to the development of GAC diagnosis. In the current study, the miRNA expression data of GAC based on The Cancer Genome Atlas (TCGA) were analyzed to screen differently expressed miRNAs (DEMs) and DEMs related to GAC prognosis. In addition, DEMs were identified in clinical samples and the mechanism of DEM was investigated in vitro. Based on this, we aimed to search for new therapeutic targets for GAC, and provide some useful insights in improving the prognosis of GAC patients.

DEMs screening related to prognosis
The overall survival (OS) time was individually extracted from clinical information.
Then, combined with the OS times and the expression levels of DEMs, DEMs related to prognosis were screened using KMsurv package of R, with the threshold of log-rank P<0.05.

Clinical validation sample collection
This study obtained ethical approval from the ethics committee of DongDa Hospital of ShanXian, and the study was performed according to the Helsinki Declaration.
Twenty paired adjacent normal tissues and GAC tumor tissues were collected from October 2017 to October 2018 in our hospital. All samples were confirmed by HE staining and stored in RNAlater. Meanwhile, peripheral blood from these GAC patients and 20 paired healthy subjects were obtained. The clinical information, including age, weight, gender, distant metastasis, lymph node metastasis, depth of invasion, and TNM stage are shown in Table 1.

Predicting target genes of DEMs
Target genes of DEMs related to prognosis were predicted using the three online analysis databases, including miRDB, miRTarBase, and TargetScan. Overlapping target genes among the three tools were selected to make the bioinformatic analysis more reliable. Then, the Venn diagram online tool was used to obtain the intersection of predicted target genes between the three databases, and the target genes that overlapped in the three databases were considered as potential target genes for DEM.   Table 2. U6 was used as the internal control for measuring miRNA level, and GAPDH was served as the internal control of for measuring ZDHHC5 expression. Data were analyzed with 2 -ΔΔCt method.

Luciferase reporter assay
The target gene of miR-96-5p was verified using the luciferase reporter assay. The

Statistical analysis
Statistical analysis was conducted using SPSS Statistics software 22.0 (Chicago, IL, USA). Continuous variables were expressed as mean ± standard derivation (SD) and analyzed by independent-Samples t test. Categorical variables were expressed as percentages and assessed by two-sided chi-square test. The differences of multiple groups were performed by one-way ANOVA following with post-hoc of Dunnett t test. P < 0.05 was considered to be statistically significant.

DEMs between GAC sample and normal sample based on TCGA
According to the selective criteria, a total of 299 DEMs were identified between GAC and normal control samples, including 225 upregulated and 74 downregulated miRNAs. As shown in Figure 1A and B, volcano plots and heat maps were conducted for these 299 DEMs.

DEMs related to prognosis based on TCGA
Based on these 299 DEMs, the relationship between patient OS and miRNA expression was evaluated, and the results showed that 35 DEMs were significantly

DEMs identification in clinical samples
A total of 20 GAC patients and 20 healthy subjects were included in this study. There were no significant differences in age, weight, and gender between GAC patients and healthy subjects (  Figure 3A), which was consistent with bioinformatics analysis results by TCGA. Moreover, miR-96-5p levels were detected in the blood of GAC patients and healthy subjects, while no significant difference were found ( Figure 3B).

Target gene prediction and identification of miR-96-5p
Considering that miR-96-5p has the highest correlation with GAC prognosis, the function of miR-96-5p was investigated in the following experiments. It was found that a total of 39 overlapping target genes existed in the TargetScan, miRTarBase, and miRDB databases ( Figure 4A). Based on this bioinformatics analysis, ZDHHC5 was considered as a potential target gene of miR-96-5p ( Figure 4B). Luciferase receptor assay showed that after co-transfection with ZDHHC5-WT and miR-96-5p mimic, the relative luciferase activity was reduced compared with co-transfection with miR-96-5p NC, while significant difference was not found after ZDHHC5-MUT treatment ( Figure 4C), which suggested ZDHHC5 as a direct target gene of miR-96-5p. In addition, the mRNA level of ZDHHC5 was obviously decreased in cells with miR-96-5p mimic, and remarkably increased after transfection with miR-96-5p inhibitor (p < 0.01, Figure 4D).

Discussion
In this study, based on miRNA expression profile data from TCGA, a total of 299 , and it is oncogene that promotes cell proliferation. It has been reported that miR-652-5p is associated with non-small cell lung cancer [18], esophageal adenocarcinoma [19], and breast cancer [20], while the mechanism of miR-652-5p was not elaborated. Current research on miR-222-5p has focused on the role of angiogenesis in the endothelium [21,22], and few studies investigated the effect of miR-222-5p in cancers. As down-regulated miRNAs in GAC, miR-125-5p was identified as a tumor suppressor in glioblastoma [23], cervical in cancers, such as bladder cancer [26], lung squamous cell carcinoma [27], gallbladder cancer [28], and head and neck squamous cell carcinoma [29], which is also considered as a tumor suppressor. However, few studies have investigated the role of miRNA-379-3p; only one recent study reported that miRNA-379-5p exerted an antitumor effect by regulating tumor invasion and metastasis in hepatocellular carcinoma [30]. Unfortunately, the effect of these miRNAs on GAC has not been reported. Therefore, it is important to further reveal the mechanism and prognostic significance of these miRNAs in GAC. In line with predictions, our study showed that ZDHHC5 was identified as a target gene of miR-96-5p. ZDHHC5 is a member of the ZDHHC protein family. It encodes a DHHC type with five zinc fingers and is recognized as palmitoyl S-acyltransferase (PAT) [31]. It has been suggested that S-palmitoylation is closely associated with cancer development, and ZDHHC enzymes are the key enzymes responsible for palmitoylation [32]. Individual ZDHHC enzymes exert different effects on various cancers, either tumor suppressors or oncoproteins [32].
Previous studies have documented that high expression of ZDHHC5 is associated with the poor prognosis of glioma [33]. In addition, the report of Tian et al. [34] suggested that DHHC5 knockdown can dramatically inhibit cell proliferation and invasion in non-small cell lung cancer. The present study revealed that ZDHHC5 silence partly reversed the effects of miR-miR-96-5p down-regulation on tumor cell growth and metastasis, which indicated that miR-96-5p silence inhibited tumor cell In conclusion, six prognosis-related miRNAs discovered from this work include miR-96-5p, miR-125-5p, miR-145-3p, miR-222-5p, miR-379-3p, and miR-652-5p in GAC samples. Furthermore, downregulated miR-96-5p markedly inhibited tumor cell growth and metastasis through targeting ZDHHC5. Current findings provide a potential molecular mechanism of miR-96-5p in GAC; however, further study is needed to investigate the mechanism and prognostic significance of these miRNAs in GAC. Informed consent statement: All participants in our study provided informed consent.

Declarations of Interest
All authors declare no conflict of interest.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.