lncRNA ZFAS1 Promotes HMGCR mRNA Stabilization via Binding U2AF2 to Modulate Pancreatic Carcinoma Lipometabolism

Being one of the most lethal malignant tumors worldwide, pancreatic carcinoma (PC) shows strong invasiveness and high mortality. In tumorigenesis and progression, the role played by long-chain noncoding RNAs (lncRNAs) cannot be ignored. This article mainly probes into the function of lncRNA ZFAS1 in PC. ZFAS1 expression in PC and normal counterparts retrieved from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Atlas (TCGA) database was analysed by GEPIA2. Its expression profile in clinical specimens and human PC cell strains was quantified using qRT-PCR. Measurements of BxPC-3 cell multiplication and invasiveness employed CCK-8, plate clone formation test, and Transwell chamber assay. ZFAS1's impact on lipid content in BxPC-3 cells was detected. RNA pulldown and RIP assays analyzed the interaction of ZFAS1 with U2AF2 and HMGCR in BxPC-3 cells. Finally, the impacts of U2AF2 and HMGCR on the biological behavior of BxPC-3 were observed. ZFAS1 was kept at a high level in PC tissues versus the normal counterparts. ZFAS1 gene knockout remarkably suppressed PC cell multiplication and invasiveness and decreased the contents of free fatty acids, total cholesterol, triglycerides, and phospholipids. Mechanistically, ZFAS1 stabilized HMGCR mRNA through U2AF2, thus increasing HMGCR expression and promoting PC lipid accumulation. Meanwhile, reduced PC cell viability and invasiveness were observed after downregulating U2AF2 and HMGCR. As an oncogene of PC, ZFAS1 can modulate lipometabolism and stabilize HMGCR mRNA expression by binding with U2AF2 in PC, which is a candidate target for PC diagnosis and treatment.


Introduction
With a five-year survival under 10%, pancreatic carcinoma (PC) is an extremely fatal and malignant digestive tract tumor [1]. Surgical resection is the most effective means for PC treatment. Nonetheless, due to inconspicuous early symptoms, the disease has frequently advanced locally to arteriovenous invasiveness and/or early distant metastasis, leaving only a small percentage (5-10%) of patients with tumors that can be surgically removed with a chance of cure [2][3][4]. And despite the relatively effective treatment, fewer than 4% survive for ten years or more [5]. Given the current situation of PC treatment, it is urgent to clarify the potential molecular mechanisms of PC and work on finding feasible biomarkers and therapeutic targets to provide tailored medical services and enhance patient outcomes.
Recently, mass evidence has demonstrated the strong connection between lipometabolism disorders and tumor cells' malignant biological behavior [6,7]. Lipids are essential in maintaining normal cell function and homeostasis. In addition to their critical roles as important components of cell membranes, they provide precursors for vital molecules involved in the pathways responsible for growth and differentiation [8,9]. One of the most distinctive metabolic abnormalities among cancer-associated disorders is dysregulation of lipometabolism [10]. Cancer cells use lipometabolism to get energy, biofilm components, and signal molecules required to proliferate, survive, invade, metastasize, and respond to tumor microenvironment and cancer treatment [11]. Lipids can sufficiently stimulate PC cell multiplication [12]. Lipogenic enzymes are often overexpressed in various cancers, including PC [13][14][15]. Therefore, finding a reliable molecule involved in lipometabolism regulation and OC progression is also pressing. Long-chain noncoding RNAs (lncRNAs) longer than 200 nucleotides are essentially a kind of ncRNAs with diverse and nonspecific biological functions.
In recent years, lncRNAs have been revealed as key regulators in tumorigenesis [16]. Meanwhile, mass studies have shown their aberrant expression in PC, participating in biological processes such as multiplication, metastasis, and chemotherapy resistance [17][18][19]. lncRNAs generally play different roles in cytoplasm and nucleus [20]. In mammals, it is generally believed that the glucose-fatty acid-(FA-) protein metabolism balance is essential, and the break of the balance can induce various diseases and even tumors [21].
A large amount of evidence shows that in various diseases, especially during cell carcinogenesis, metabolic patterns involving mitochondrial oxidative phosphorylation, glycolysis, and FA oxidation, a phenomenon researchers called tumor cell metabolic reprogramming, alter greatly [22,23]. In tumor cells, many key enzymes associated with lipolysis and lipid synthesis are over-expressed [24]. lncRNAs have been reported to interfere with lymph node metastasis of cervical cancer, which promote FA metabolism reprogramming and cervical cancer cell metastasis via modulating FABP5-a carrier for FA uptake and transport [25].
Located on chromosome 20q13. 13, ZNFX1 Antisense RNA 1 (ZFAS1) is a transcript of the encoding gene identified as a regulator of breast alveolar and epithelial cell differentiation [26]. ZFAS1 was initially reduced in breast cancer tissue, suggesting that this transcript has tumor-suppressive effects [27]. Subsequent studies, however, report that ZFAS1 exerts an opposite function during the progression of most cancer types. Besides its role in tumor pathogenesis, ZFAS1 is involved in the molecular cascade leading to a series of diseases, such as osteoarthritis [28], epilepsy [29], and atherosclerosis [30]. But the molecular mechanism ZFAS1 participates in metabolic reprogramming remains uncharacterized. Precursor messenger RNA (pre-mRNA) splicing is a key step in processing gene transcripts that encode most eukaryotic proteins and is a part of a series of ordered reactions catalyzed by large RNA-protein complexes called spliceosomes [31][32][33]. Alternative splicing may generate different mRNAs and proteins from individual transcript, which is critical in development, differentiation, and multiple human diseases, including cancer [34,35]. The U2 Auxiliary Factor complex, one of the components of spliceosomes, comprises a small (U2AF1) and a large (U2AF2) subunit. As essential proteins, U2AF1 and U2AF2 work together to bind U2 snRNP to the 3 splice site of introns in most eukaryotes [36]. Recently, U2AF1 somatic mutations have been repeatedly demonstrated in a number of human tumors [37,38]. HMGCR, abbreviated from 3hydroxy-3-methyl-glutarylcoenzyme-A reductase, is a ratelimiting enzyme of cholesterol (CHOL) biosynthesis and has been found to have carcinogenic effects in gastric cancer, glioblastoma, and prostate cells. Based on the above information, the novelty and motivation of this paper is to explore ZFAS1 expression and its mechanism in cases with PC and try to clarify whether ZFAS1 can influence PC progression through lipometabolism for the first time.

Clinical Specimen Collection. Between February 2020 and
December 2021, 43 paired PC and adjacent equivalents specimens were collected from PC patients who received no preoperative chemoradiotherapy but surgical treatment at the Department of General Surgery of Ningbo Medical Center Lihuili Hospital. PC tissues and normal counterparts resected intraoperatively were isolated for immediate liquid nitrogen storage until analysis. All postoperative specimens were pathologically confirmed, and all patients signed informed consent, agreeing to donate pancreas and PC tissues for relevant experimental detection. Our hospital has ethically ratified this research. The Gene Expression Profiling Interactive Analysis (GEPIA2) public database (https://gepia2.cancer-pku.cn/ index) was responsible for analyzing related molecule expression in PC specimens in The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/).

Cell Cultivation, Treatment, and Cytoplasmic and
Nuclear RNA Isolation. Supplied by Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS) cell bank, BxPC-3, PANC-1, SW1990, PaCa-2 (human PC cell strains), and HPNE (human pancreatic ductal epithelial cells) were conventionally cultivated by immersing in DMEM (Gibco, USA)+10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) in a 37°C incubator (5% CO 2 ). GenePharma (Shanghai, China) was responsible for designing and synthesizing the siRNA targeting ZFAS1, U2AF2, and HMGCR and the negative control si-NC. On the day before transfection, logarithmic growth-phase BxPC-3 cells were seeded into the wells of a 6well plate and for siRNA and si-NC transfection following the manufacturer's instructions of Lipofectamine 3000 (Invitrogen, USA) when the cell density reached 40%.
2.3. Cytoplasmic and Nuclear RNA Isolation. ZFAS1 distribution in BxPC-3 cell's cytoplasm and nucleus was determined by referring to the PARIS kit recommendations (Life Technology, USA) by referring to the supplier's manuals. In general, cells after being washed with PBS were lysed and centrifuged (300 g, 5 min); separate the supernatant (cytoplasm) and remaining pellet (nuclear) after being washed by PBS 5 times.

qRT-PCR.
After being isolated using TRIzol (Invitrogen, USA), cell and tissue total RNA were reverse-transcribed using a PrimeScript RT Reagent Kit (Takara, Japan). Following that, using the ABI Step One Real-time PCR System (Thermo Fisher Scientific, USA), a PCR reaction was performed on the cDNA according to the SYBR Green Kit recommendations (Takara, Japan). Shanghai Sangon synthesized the specific primers (Table 1) used in qRT-PCR. lncRNA ZFAS1 expression and U2AF2 and HMGCR expression normalized with β-actin and GAPDH, respectively, were computed using 2 -ΔΔCt . The average value was obtained after running the experiment three times. were used to obtain a biotin-labeled RNA transcript, which was then mixed with resuspended streptavidin-labeled magnetic beads and incubated overnight (4°C) after in vitro transcription of ZFAS1 using T7RNA polymerase (Ambio life, USA). Then, they were incubated indoor in lysate for 1 h, together with the RNase inhibitor. WB was carried out after bead washing with a wash buffer to determine the eluted proteins.

Western Blotting (WB
2.11. RNA-Binding Protein Immunoprecipitation (RIP) Assay. Immunoprecipitation analysis of RNA binding proteins was made by referring to the EZ-Magna RIP Kit (Millipore USA) instructions. Cells were treated with cold PBS rinsing and cleavage with RIP lysis buffer. U2AF2 antibody (1 : 50, Cell Signaling, USA) and nonspecific control antibody IgG were used for immunoprecipitation. RIP lysate was incubated with a magnetic bead binding antibody overnight (4°C). Following that, proteinase K was used for immunoprecipitated protein degradation. The bound RNA was separated from the supernatant, and the RNA concentration was measured using NanoDrop (Thermo Fisher Scientific, USA). Furthermore, the purified RNA was analyzed by RT-qPCR to detect ZFAS1 and HMGCR mRNA levels.
2.12. Stability Analysis of RNA. BxPC-3 cells were treated with 6 and 12 h of incubation with 2.5 mg/mL actinomycin D (ActD) after 48 h of transfection, while those without ActD intervention served as the negative control. HMGCR mRNA stabilization was detected by qRT-PCR after isolating the total RNA.
2.13. Statistical Processing. SPSS 22.0 was adopted for data analysis and GraphPad prism 8.0 for rendering and presentation. Quantitative variables were given as mean ± standard deviation ðMean ± SDÞ, and double-tailed P < 0:05 was deemed statistically significant. Each assay was repeatedly determined 3 times. The difference in ZFAS1 expression  3 Journal of Immunology Research between PC patients and normal controls was identified using the nonparametric Mann-Whitney U test. The comparison methods for quantitative variables were independent sample t-test (intergroup) and one-way ANOVA plus Bonferroni posttest (multigroup).

ZFAS1 Keeps at a Higher Level in PC and PC Cells.
We first analyzed PAAD transcriptome data from TCGA database using GEPIA2, combined with its expression data in normal tissue from the GTEx database and found that ZFAS1 was kept at a higher level in PC (Figure 1(a)). Subsequently, we used qRT-PCR to analyze ZFAS1 in 43 PC tissue specimens and normal counterparts and found notably higher ZFAS1 in cancerous tissue specimens (P < 0:05, Figure 1(b)). Furthermore, upregulated ZFAS1 was determined in human PC cell strains (BxPC-3, PANC-1, SW1990, and PaCa-2) versus HPNE (P < 0:05, Figure 1(c)).

Discussion
In the tumor microenvironment, tumor cells' nutrition availability constantly changes as the tumor progresses, 7 Journal of Immunology Research and cancer cells use lipometabolism to sustain their rapid proliferation, survival, migration, invasiveness, and metastasis [39]. A cancer hallmark has been increasingly identified as a reprogrammed FA metabolism characterized by increased fat production [40]. Enhanced fat production includes de novo FA synthesis [41] and CHOL biosynthesis [42]. Recently, there has been renewed interest in studying lipid reprogramming pathways in tumor cells [43]. But further research is required, given the incomplete understanding of the mechanisms of enhanced FA and CHOL synthesis in tumor cells.
Meanwhile, lncRNAs are critical in cell metabolism by reprogramming tumor cell metabolic pathways [44]. And via integrating vicious transformation and metabolic reprogramming of cells, they regulate a variety of metabolic enzymes [45]. Reportedly, lncRNA HAGLROS regulates lipometabolism reprogramming in intrahepatic cholangiocarcinoma via mTOR axis. ZFAS1 is upregulated in multiple cancers, including gastric [46], cervical [47], and pancreatic carcinomas [48]. This study found upregulated ZFAS1 in PC tissues and cell strains, and ZFAS1 gene knockout statistically suppressed PC cell multiplication and invasiveness in vitro.  Journal of Immunology Research Similarly, Liu et al. [49] found that lncRNA ZFAS1 promoted pancreatic adenocarcinoma metastasis via sponge aspiration of the miR-3924-mediated RHOA/ROCK2 pathway. The existing studies focus on the involvement of ZFAS1 in the multiplication and metastasis of cancer cells by regulating miRNA-mediated pathways [50,51], while herein, we reveal a novel role of ZFAS1 and the potential molecule mechanism in PC. We found that ZFAS1 can affect PC lipometabolism, but its underlying mechanism remains to be clarified. Decreased HMGCR expression was observed after knocking out ZFAS1 in PC cells, which made us speculate that ZFAS1 could modulate HMGCR and reduce FFAs, TC, TGs, and PLs in PC cells. HMGCR, the rate-limiting enzyme synthesized by CHOL, is reported to be upregulated in gastric cancer, thus promoting the malignant phenotype of cancer cells [52].
Moreover, HMGCR interferes with cisplatin resistance of ovarian cancer cells, and inhibiting its expression has antimetastasis and antitumor effects [53]. Our study also found that inhibiting HMGCR can inhibit PC cell multiplication and invasiveness. Therefore, we believe that ZFAS1 promotes PC cell growth via upregulating HMGCR to promote CHOL biosynthesis. Subsequently, we found that ZFAS1 does not directly regulate HMGCR expression but through binding to U2AF2 protein through RNA pulldown assay. Further, it was found that downregulating ZFAS1 and U2AF2 expression reduced HMGCR mRNA stabilization in PC cells, and ZFAS1 knockout reduced the interplay of   [54]. Similarly, this study found for the first time that lncRNA ZFAS1 enhanced HMGCR mRNA stabilization via binding to U2AF2 to participate in PC lipometabolism, thus regulating the progression of PC. In addition to this, lncRNA SNHG1 and RNA binding protein hnRNPL were found to form a complex and coregulate CDH1 to enhance prostate cancer growth and metastasis [55]. A similar mechanism was presented in our study. In Palangat et al.'s study [56], U2AF, a splicing factor and a heterodimer of U2AF1 and U2AF2, performs the recognition and binding to the 3 ′ splice site, which is a key initiation step in spliceosome assembly [57]. U2AF1 interacts with its binding partner, U2AF2, to bind to mature RNAs in the cytoplasm and acts as a translational repressor, directly interacting with hundreds of spliced and polyadenylated mRNAs in the cytoplasm.

Conclusion
To summarize, we discovered abnormally high lncRNA ZFAS1 expression in PC, and ZFAS1 increased PC cell multiplication and invasiveness by regulating lipometabolism. Its main mechanism of action is that ZFAS1 binds to U2AF2 and promotes its interaction with HMGCR mRNA to reprogram lipometabolism, thus promoting CHOL synthesis and ultimately promoting PC cell growth. However, this study also has some shortcomings. For example, the effect of ZFAS1 on PC through this pathway has not been verified in vivo and its mechanism in vivo is still unknown. Therefore, we will conduct further in vivo validation of its mechanism of action in subsequent studies. We collectively believe that ZFAS1 is a promising diagnostic marker for PC, and its mechanism of affecting lipometabolism reprogramming can provide a new direction and target for PC treatment.

Data Availability
The labeled dataset used to support the findings of this study is available from the corresponding author upon request.

Conflicts of Interest
The authors declare no competing interests.