Lnc-RNA UCA1 Promotes TGF-β-Mediated Epithelial-Mesenchymal Transition via Inhibiting miR-204 in Gastric Cancer Cells

(lncRNA) Two gastric cancer cell lines are chosen, MNK45 and SGC-7901. Transforming growth factor-β (TGF-β) is used to promote epithelial-mesenchymal transition (EMT) by using cancer cell invasion assay. The transmembrane cell quantities are counted and ZEB1, slug, vimentine and E-Cadherin gene expression levels are measured by quantitative PCR assay. siRNA of UCA1 and miR-204 are used to conrm crosstalk among TGF-β, UCA1 and miR-204.


Introduction
Cancer is basically a genetic modi cation disease resulting in aberrant cellular homeostasis and successive growth. The discovery of protein-coding genetic mutations established our principles of understanding how these exome aberrances drove pathogenesis of tumor. However, only protein-coding sequence mutations cannot solely explain why and how cancer is generated and developed. Since the coding sequences account for only 2% of the whole genome, it is reasonable to assume that the noncoding sequences play major roles on the cancer phenotypes.
All genes in human are transcribed into RNA, which dominantly are noncoding RNAs [1,2]. Long noncoding RNAs (lncRNA) are transcripts > 200 nucleotides with no protein translation potentials [3,4]. It is recognized that lncRNAs are delicately regulated and restricted to certain cell types [5]. The biological functions of majority of the lncRNAs remain un-discovered. MicroRNAs (miRNAs) bind to 3'-untranslated region (3'UTR) of mRNAs of target genes, resulting in the degradation of mRNAs or the suppression of translation process [6,7]. The involvement of miRNAs in regulating tumors malignancies had been reported by plenty of researchers.
Gastric cancer is the most common subtype of gastrointestinal cancer worldwide. It is the fth most common type of cancer and the third in mortality comparing with all other cancer types [8]. According to report in 2015 of National Central Cancer Registry of China (NCCRC), gastric cancer is the second both in incidence rate and mortality in China [9]. The ve-years survival rate of gastric cancer is below 30% [10,11]. Patients with gastric cancer are often diagnosed at the intermediate or even terminal stages of the disease with liver, lymph nodes or lung metastasis, which hinders e ciency of the treatment and contributes to the low ve-years survival rates. The transforming growth factor-β (TGF-β) is the key factor in the gastric tumor micro-environment. By stimulating vascular endothelial growth factor C (VEGF-c), TGF-β signaling pathway stimulates lymph-angiogenesis, increases invasion ability of the tumor cells and promotes epithelial-mesenchymal transition (EMT) [12][13][14]. Slug, ZEB1, Vimentine, and E-cadherin are proved to be related with the EMT process [15]. To understand how the TGF-β signaling pathway delicately regulates metastasis of gastric cancer would de nitely provide strong evidence to support excise therapy in clinic.
LncRNA urothelial cancer associated 1 (UCA1) is highly expressed in variant tumor cells, such as bladder cancer and oral squamous cell carcinoma, and associated with bad prognosis of the diseases [16][17][18].
But its impact on gastric cancer is unclear. As a possible target of UCA1, recent studies have shown that miR-204 expressed signi cantly low in several tumors including colorectal cancer [19]. However, the potential role of miR-204 in gastric cancer is largely unknown. The purpose of this study is to investigate whether and how lncRNA UCA1 and miR-204 participate in the TGF-β stimulated EMT in two gastric cancer cell lines.

Cell Culture
Gastric cancer cell lines, MNK-45 and SGC-7901 were purchased from ATCC. Cells are cultured in DMEM medium with 10% fetal bovine serum, 100U/ml penicillin, 100µg/ml streptomycin, at 37°C incubator with 5% carbon dioxide. Cell expansion and splitting process is restrictedly performed according to the manufacture's protocol.

Tumor Cell Invasion Assay
When the culture ask is 70-80% con uent, the gastric cancer cells are cultured with DMEM medium without fetal bovine serum overnight. Recombinant human TGF-β1 is purchased from PeproTech (Cat:100-21c). 10ng/ml TGF-β1 is added into the cell culture medium and incubated for 48 hours.
After being digested with trypsin, the gastric cells are calculated and put in ice for further usage. 600µl DMEM medium with 15% fetal bovine serum is added into the bottome of the transwells, subsequently cell suspension is added into the membrane at 2x10 5 concentration. Incubate at 37°C incubator overnight. After getting rid of the oating cells at the bottom of the transwells, the cells adherent to the bottom of transwells are xed with 50% methanol for 15 minutes, and washed with PBS solution for three times. Then stained with crystal violet solution for 30 minutes. After being air-dryed, the transwells are observed and six zones are chosen randomly under microscope.

Quantitative RT-PCR
Trizol is used to extract total RNA from the gastric cancer cells according to standard protocol [14]. cDNA is synthesized by using PrimeScript RT kit from Takara

Statistics Analysis
Average and standard deviation data were analyzed in Excel. T test is used for signi cance study. P < 0.05 considered as signi cance. Univariate analysis of variance is used by using SPSS 10.0.

TGF-β enhances gastric tumor cell invasion
After being incubated with 10ng/ml TGF-β, Transwell-Matrigeal trans-membrane assay is performed, and subsequently cells stayed at the bottom of the transwells are stained with crystal violet. Multiple negative controls are used. Six random visions under microscope are took into account, and the numbers of stained cells are calculated and compared.
As shown in Fig. 1, for the MNK-45 and SGC-7901 cell lines, there are both signi cant more cells went through membrane once treated with TGF-β than non-treated cells (p < 0.05).

TGF-β treatment promotes EMT
We use the MNK-45 cell line to study how the EMT related genes change their expression levels due to its signi cant enhanced transmission ability by TGF-β treatment. After being treated with TGF-β for 24 hours, in the MNK-45 cells, the slug, ZEB1, vimentine, and E-Cadeherin gene expression levels are 3.93±0.35, 5.10±0.17, 3.67±0.21, 0.50±0.10 times comparing with the negative control cells (P < 0.05) (Fig. 2). By using quantitative PCR assay, we demonstrate that TGF-β treatment can increase slug, ZEB1 and vimentine gene levels and decrease E-Cadherin expression levels signi cantly (P < 0.05).

Blockage of UCA1 inhibits TGF-β induced activation of EMT
To analyze whether lncRNA UCA1 interferes with the TGF-β signaling pathway, several siRNAs of UCA1 (si-UCA1) and negative control siRNAs of UCA1 (NC-si-UCA1) are tested upon MNK-45 cells. The si-UCA1 chosen to use in the subsequent experiments can block 70% of UCA1 expression (0.30±0.05). The NC-si-UCA1 does not interfere with UCA1 expression at all (data not shown).

Discussion
In this study, we demonstrated that TGF-β can signi cantly increase gastric cancer cell transmission ability and remarkably enhance EMT related gene expression levels. This result is correlated with previous Recent studies demonstrated aberrant lncRNA UCA1 expression existed in variant carcinomas including bladder cancer, gastrointestinal tumor, neural blastoma and breast cancer [20]. UCA1 attracts miRNA in a competitive way, so called "sponge", in order to free target genes of the miRNA to perform their functions.
Interestingly, UCA1 plays through variant pathways. In renal cancer, UCA1 plays a critical regulatory role in proliferation and progression of renal cancer cells by interacting with miR-182-5p/DLL4 axis [21]. In gastric cancer, UCA1 works with miR-7, 495, 498 to promote tumor-genesis [22,23]. In MNK45 gastric cancer cell line, we demonstrated that UCA1 sponges miR-204 to free its target gene ZEB1 (Fig. 6). It is still early to assume that UCA1 works in a tissue speci c way to promote metastasis and thoroughly studies are required to nd out its fundamental mechanisms.
Interconversion between epithelial and mesenchymal is highly conserved process during embryogenesis. Epithelial-mesenchymal transition (EMT) is regulated by environmental signals, such as Wnt, TGF-β, FGF family members and intracellular signaling pathways [24]. EMT related transcription factors (EMT-TF) include zinc nger proteins (e.g., SNAI1, SNAI2), helix-loop-helix transcription factors (e.g., E47), zinc nger and homeodomain protein ZEB1 (also called TCF8 or DeltaEF1) and ZEB2 (also called SIP1). ZEB1/2 may trigger the repression of epithelial genes, such as E-Cadherin, to damage adhesion and tight junctions and the stimulation of mesenchymal factors, such as vimentine, to facilitate transdifferentiation process. In this study, ZEB1 gene expression level is signi cantly enhanced and E-Cadherin expression level is nearly at the half level after TGF-β treatment in gastric cancer cells. This suggests that TGF-β de nitely promotes EMT process. Besides ZEB1, vimentine, slug and E-Cadherin, other EMT related genes will be tested in the subsequent studies. Page 7/9 In this study, we demonstrated the cross-talk among TGF-β, lnc-UCA1 and miR-204 in gastric cancer cells as shown in Fig. 6. miR-204 inhibits TGF-β function, and UCA1 sponges miR-204 to stop its functions. We reasonably suspect that miR-204 expression level could be measured and used to predict prognosis of the gastric cancer. Meanwhile, UCA1 inhibitors might be considered as potential genetic medical drugs, although further and wider explorations are required in the future.

Declarations
Ethics approval and consent to participate

Authors' contributions
Dr. Ding-Fu Zhong did most of the lab works. Dr. Dan Chen and Dr. Ying Nie analyzed the data and did the microscope job. Dr. Hong-Ying Zhang and Dr. Yi Yang discussed with Dr. Ding-Fu Zhong and provided very useful suggestions. Dr. Li-Yu Hu wrote the manuscript and the mastermind behind all the lab works.