Long non-coding RNA ZNF674-AS1 antagonizes oxaliplatin resistance of gastric cancer via regulating EZH2-mediated methylation of CHST7

Chemoresistance leads the cause of poor outcome of patients with gastric cancer (GC). Long non-coding RNAs (LncRNAs) is intimately involved in the regulation of tumorigenesis and progression. Here, we demonstrated ZNF674-AS1 was down-regulated in oxaliplatin (OXA)-resistant tissues and cell lines, lower level of ZNF674-AS1 predicted poor prognosis of GC patients. Besides, forced expression of ZNF674-AS1 not only reduced cell viability, colony formation, expression of drug-resistant markers but also promoted cell apoptosis of OXA-resistant GC cells, exposed to oxaliplatin. Silence of ZNF674-AS1 exhibited an opposite effects on OXA resistance of GC cells. Further mechanistic research showed that ZNF674-AS1 interacted with EZH2, led to higher methylation level of target gene CHST7. In addition, functional experiments verified that depletion of CHST7 re-sensitized OXA-resistant GC cells to OXA. Thus, our results indicated that ZNF674-AS1 suppressed OXA resistance of GC through EZH2-mediated inhibition of CHST7, providing potential theoretic basis and therapeutic strategy for chemoresistant GC.


INTRODUCTION
Gastric cancer (GC) seriously threatens people's life for the very unsatisfactory prognosis globally [1,2]. Compared to targeted therapy and surgery, chemotherapy is still the preferred routine treatment strategy for advanced GC patients to reduce postoperative recurrence and prolong survival [3]. For example, oxaliplatin (OXA) is a common agent for the therapy of multiple cancers, GC is no exception [4,5]. Nevertheless, the occurrence of OXA resistance greatly reduces the therapeutic efficiency of chemotherapy [6]. Therefore, the molecular mechanisms of OXA resistance in GC need to be urgently explored.
EZH2, one core member of the Polycomb Repressive Complex 2 (PRC2), acts as a critical regulator in epigenetic mediation of genes via catalyzing trimethylation of Lys-27 of histone H3 (H3K27me3), which serves as a crucial mediator in cancer development and aggressiveness. Elevated EZH2 has been observed in multiple solid tumor types GC included; high level of EZH2 predicts poor outcome of cancer patients [12,13]. Increasing studies indicate LncRNAs participate in EZH2-mediated H3K27me3, resulting in inhibition of target gene epigenetically AGING [14,15]. Here, we concerned a less-studied LncRNA, ZNF674-AS1, which was reported in hepatocellular, non-small cell lung cancer [16] and glioma cancers [17,18]. However, the specific functional mechanisms of ZNF674-AS1 in GC are still vague.
In the present work, we show the tumor-suppressive phenotypes of ZNF674-AS1 in GC, inhibition of which enhanced OVA resistance of GC cells. Mechanism studies indicated the inhibitory effects of ZNF674-AS1 on OVA resistance depended on EZH2-induced epigenetically inhibition of CHST7 expression.

Clinical tissues
70 GC tumor sample tissues (31 from OVA-resistant GC patients and 39 from OVA-sensitive GC patients) were collected from the Department of General Surgery, the Second Affiliated Hospital of Anhui Medical University. Informed consent has been signed by each enrolled patient before surgery. Sample collection and usage were performed according to the relevant guidelines.

Quantitative real-time PCR
Trizol reagent (Takara, Dalian, China) was used for extraction of total RNA from GC tissue specimens and cells as the recommendation of manufacturer's manual. qRT-PCR was carried out as previously described [23].

Western blotting
RIPA buffer (Beyotime, Shanghai, China) pretreated with protease inhibitors (Beyotime, Shanghai, China) was used to extract total proteins from GC cells. Western blotting was conducted as previously described [23]. Primary antibodies against β-actin, EZH2 and CHST7 were purchased from Abcam (Cambridge, United Kingdom).

Cell function assays
Cell proliferation of GC cells was detected with CCK-8 kit (Solarbio, Beijing, China) following the instruction. The apoptosis of GC cells was performed by using the Caspase-3 Colorimetric Assay kit from Promega Corporation. Colony formation assay was conducted as previously described [24].

Isolation of nuclear and cytoplasmic RNA
RNA cellular sublocalization was analyzed using a Purification Kit (NorgenBiotek, Canada) according to the manuals of manufacturers.

RNA pull-down assay
The detailed protocol of RNA pull-down assay was described previously [25]. Probes against ZNF674-AS1 were listed as below: AGING

RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP)
RIP and ChIP assay were performed as previously described [26]. Antibodies targeting EZH2 and control IgG were purchased from Abcam.

Statistical analysis
All data are presented as mean ± standard deviation (SD). Chi-squared test was used for analysis of association between ZNF674-AS1 expression and clinicopathological characteristics of GC patients. Student's t-test and twoway ANOVA were used for statistical analysis between groups. Kaplan-Meier's analysis was employed for statistical comparison of survival rate. P < 0.05 was considered to be statistically significant.

Ethics approval and consent to participate
The study was approved by the ethics committee of the Second Affiliated Hospital of Anhui Medical University, Anhui Medical University.

Data statement
Data will be provided on reasonable request.

Reduced ZNF674-AS1 correlated to oxaliplatin resistance and worse survival in gastric cancer
To explore the potentially dysregulation of ZNF674-AS1 in OVA-resistant gastric cancer, we first assessed ZNF674-AS1 expression in OVA-resistant GC samples and the OVA-sensitive counterparts by qRT-PCR. A significantly lower ZNF674-AS1 level was observed in OXA-resistant samples than that in OXA-sensitive ones ( Figure 1A). We analyzed the survival rate of 70 GC patients, which indicated patient with a lower ZNF674-AS1 expression exhibited a worse outcome ( Figure 1B). Further, pathological examination in Table 1 indicated elevated ZNF674-AS1 in gastric cancer was negatively associated with advanced WHO stage (P=0.0312) and lymphovascular invasion (0.0147). In keeping with the results of GC tissues, ZNF674-AS1 was remarkably down-regulated in OVA-resistant GC cells, as compared with their parental ones ( Figure 1C). Collectively, these data suggested expression inhibition of ZNF674-AS1 in GC may be mechanistically correlated to OXA resistance and worse clinical outcomes. On the other hand, endogenic ZNF674-AS1 was knockdown in parental AGS and MKN-45 cells by stably transfection of shRNAs against ZNF674-AS1 ( Figure 3A). Silence of ZNF674-AS1 obviously increased cell viability, IC50 value and colony formation capacity of AGS and MKN-45 cells with treatment of OXA ( Figure 3B-3F). Cell apoptosis was remarkably decreased in ZNF674-AS1-depleted AGS and MKN-45 cells ( Figure 3G). Knockdown of ZNF674-AS1 resulted in increased expression of drug resistance-related genes ( Figure 3H, 3I). Taken together, our data suggested ZNF674-AS1 obviously enhanced chemosensitivity of GC cells. AGING

ZNF674-AS1 negatively regulated the expression of CHST7
Previous studies have reported LncRNAs mediated their neighboring genes expression via EZH2-mediated epigenetic regulation [27,28]. To determine the molecular mechanisms underlying ZNF674-AS1induced OVA resistance of GC cells, the neighboring CHST7 was concerned. Both protein and mRNA levels of CHST7 were decreased in AGS/OXA and MKN-45/OXA cells transfected with ectopic ZNF674-AS1 plasmid (Figures 4A-4C). In contrast, silence of ZNF674-AS1 markedly led to increased expression of CHST7 ( Figure 4D-4F). More importantly, CHST7 mRNA level was notably elevated in OXA-resistant GC tissues, as compared with the OXA-sensitive counterparts ( Figure 4G). Furthermore, CHST7 level was remarkably increased in six GC cell lines (AGS, AGS/OXA, MKN-45, MKN-45/OXA, MKN-28, MKN-28/OXA cells) as compared with GES-1. In addition, A higher level of CHST7 was observed in OXA-resistant GC cells than that in their parental GC cells ( Figure 4H). Summarily, we proved that ZNF674-AS1 suppressed the expression of CHST7.

DISCUSSION
Gastric cancer is an aggressive malignant tumor accompanied by high mortality and poor prognosis [29]. Chemotherapy has been employed in GC patients to reduce the risk of metastasis and postoperative recurrence [30]. However, acquisition of drug resistance seriously impairs the therapeutic effect of chemotherapy in GC. In the present study, we constructed OVA-resistant GC cells and explored the functions and mechanisms of ZNF674-AS1 in oxaliplatin resistance of GC. AGING Increasing evidence suggested that LncRNAs participated in several biological processes of multiple cancer types including proliferation, epithelialmesenchymal transition (EMT), drug resistance and metastasis [31,32]. Despite it has been previously reported to serve as a tumor suppressor in hepatocellular and glioma cancers [17,18], the detailed expression, functions and regulatory mechanisms of ZNF674-AS1 in GC remains to be explored. In the current work, a notably lower level of ZNF674-AS1 was observed in OVA-resistant GC samples and cells, which implied ZNF674-AS1 may participate in the mediation of oxaliplatin resistance in GC.
EZH2 inhibits expression of target genes by catalyzing the formation of H3K27me3 [33]. More and more evidence has suggested that LncRNAs acted as a crucial role in EZH2-mediated epigenetic regulation in multiple cancer types. For example, elevated LINC01088-induced proliferation advantage depended on EZH2-mediated epigenetically inhibition of p21 expression in human non-small cell lung cancer [34]. Another study suggested that LINC01535 enhanced distant metastasis via mediating EZH2-targeted miR-214 in cervical cancer [35]. In addition, a recent study has reported that STXBP5-AS1 inhibited cancer stem cell phenotypes of pancreatic cancer cells through EZH2/ADGB pathway [26]. Here, we demonstrated ZNF674-AS1 directly bond to EZH2, led to expression inhibition of its neighboring CHST7 epigenetically, resulting in impairment of oxaliplatin resistance in GC. However, how does ZNF674-AS1 recruit EZH2, it is still needed to further determine other factors involved. Carbohydrate Sulfotransferase 7 (CHST7) belongs to the CHST family, which undertakes the function to transfer sulfate to carbohydrate groups in glycoproteins [36]. Fewer studies has reported CHST7 was associated with carcinogenic process. A previous literature uncovered that serum CHST7 was significantly in-creased in all overall stages of primary lung tumors [37]. In addition, Bi et al. reported that methylation level of CHST7 in white blood cells was correlated to colorectal cancer risk [38]. In the current study, we firstly revealed the expression pattern and functions of CHST7 in oxaliplatin resistance of gastric cancer.

CONCLUSIONS
In our work, we demonstrated that ZNF674-AS1 and CHST7 exhibited two opposite expression patterns and functions during regulation of oxaliplatin resistance in GC, which was due to ZNF674-AS1-induced inhibition of CHST7 at transcriptional level ( Figure 7). Our study potentially provided novel therapeutic targets and strategies for GC patients suffering from oxaliplatin resistance.

AUTHOR CONTRIBUTIONS
KY performed the experiments. YW analyzed the experiments and wrote the manuscript. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest. epigenetically silences CHST7 expression via binding to EZH2, which inhibits oxaliplatin resistance of GC.