Preliminary Study on the Relationship Between LncRNA GAPLINC Expression and Clinical Pathology of Esophageal Squamous Cell Carcinoma and Its Metastasis Mechanism

Background Long non-coding RNA (lncRNA) GAPLINC (gastric adenocarcinoma predictive long intergenic noncoding RNA) plays a carcinogenic role in a variety of different tumor types. It is deserved to explore the biological function of LncRNA GAPLINC in the development of esophageal cancer. Tissues of 40 patients undergoing esophageal squamous cell carcinoma (ESCC) radical surgery were collected, including ESCC tissues and corresponding adjacent normal tissues. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of LncRNA GAPLINC and evaluate the relationship between its expression and ESCC clinicopathology. The expression level of LncRNA GAPLINC in human ESCC cell line (TE11) was detected by qRT-PCR. After specic siRNA interference, the expression of LncRNA GAPLINC was detected. The effects of LncRNA GAPLINC on ESCC cell proliferation, migration and invasion were detected by ow cytometry, cell counting kit-8 (cck-8) and Transwell, respectively. 40 cases of ESCC tissues and normal tissues adjacent to cancer. The results of qRT-PCR assay showed that LncRNA GAPLINC had a signicant high expression in ESCC. Combining with clinical pathological data analysis, we found that LncRNA GAPLINC expression was no related to the patient's age, sex, history of tobacco and alcohol, tumor location, esophageal lesion length, depth of invasion, lymph nodes metastasis but closely related to tumor differentiation degree. The study revealed the correlation between LncRNA expression level and tumor differentiation degree, indicating the potential of LncRNA GAPLINC as a biomarker for diagnosis in ESCC. In order to further explore the function of LncRNA GAPLINC in esophageal tumors, we used qRT-PCR technology to detect ve human esophageal squamous carcinoma cell lines (KYSE-450, KYSE-510, KYSE-150, TE-10 and TE- 11). Compared with the normal esophageal epithelial cell line (HET-1A), LncRNA GAPLINC expression was up-regulated in these ve esophageal squamous carcinoma cell lines, which was similar to the ESCC tissue. We selected TE-11 cell lines to knock down and overexpress LncRNA GAPLINC. The apoptosis was detected by ow cytometry assay and the proliferation ability by CCK-8 test. At the same time, the migration and invasion of cancer cell was also detected. The Annexin V apoptosis test had shown that knocking down of GAPLINC lead to a signicant difference of apoptosis. But overexpression of GAPLINC had no effect on apoptosis. Knocking down LncRNA GAPLINC lead to a decrease on cell proliferation, migration and invasion. Another side, overexpressing LncRNA GAPLINC led to an increase on cell proliferation, migration and invasion. It was conrmed that downregulation of LncRNA GAPLINC could signicantly inhibit the proliferation, migration and invasion ability of ESCC cells

Background esophageal squamous cell carcinoma (ESCC) has become one of the six most common malignant tumors in the world, with about 572,000 new cases and 509,000 deaths in 2018, ranking 7th and 6th among all tumors in the world, respectively 1 . China has the highest incidence and mortality rate of ESCC in the world, accounting for 70% 2 . It should be noted that more than 95% of patients with ESCC diagnosed for the rst time are in advanced stage, with a poor prognosis and a 5-year survival rate of only about 30% 3,4 . The current researches cannot completely explain the pathogenesis of ESCC 5 . There are many factors related to the occurrence and development of ESCC 6 . Therefore, it is of great signi cance for the prevention and treatment of ESCC to further study its pathogenesis, clarify its biological behavior, identify its susceptible genes and speci c biomarkers.
LncRNA is a kind of long non coding RNA with more than 200 nucleotides in length, which is mainly transcribed by RNA Poymerase II and widely distributed in the human genome. The current studies have discovered a huge quantity of LncRNAs widespread existing in each kind of malignant tumor. These LncRNAs could regulate the expression of oncogenes and tumor suppressor genes in different ways and play an important role in the occurrence, development, metastasis and recurrence of tumors 8 . Another side, the LncRNAs are stable in circulating body uids such as plasma and urine, providing a new basis for the diagnosis and treatment of malignant tumors. Therefore, searching new LncRNAs as diagnostic biomarkers and therapeutic targets for malignant tumors will become a research hotspot.
The GAPLINC is 924bp in length, located on the short arm of human chromosome 18, and the RNA length after transcription is 643nt 9 . LncRNA GAPLINC is rst con rmed to be signi cantly increased in gastric cancer tissues, and its high expression could reduce cancer cell apoptosis, promote proliferation and invasion ability. Its expression level is positively correlated with tumor volume, distant metastasis, lymph nodes proportion and clinical grade. The expression level of GAPLINC is strongly correlated with CD44 and its role as a proto-oncogene is partly depended partly on CD44 10 . In the study of colon cancer, GAPLINC is also observed to play the role of oncogene. At present, there are few researches reporting on LncRNA GAPLINC. It is deserved to explore the biological function of LncRNA GAPLINC in the development of ESCC.

Patients and tumor tissue
In this study, 40 pairs of specimens were collected, including ESCC cancer tissues and normal esophageal tissues. These tissue were from the patients under thoracic surgery in 2018. Basic information and clinicopathological data of ESCC patients were collected. The normal tissue adjacent to the cancer required ≥5cm from the edge of the tumor tissue.
Clinical and pathological data: Of the 40 patients with esophageal squamous cell carcinoma in this study, 34 were male (85%) and 6 were female (15%); 31 were over 60 years old (77.5%) and less than 60 years old 9 patients (22.5%); 13 Page 4/25 patients with lymph node metastasis (32.5%), 27 patients without lymph node metastasis (67.5%); 11 patients with T1 to T2 (27.5%), 29 patients with T3 to T4 (accounting for 72.5%) ( Table 1). Tissue samples: After taking out fresh samples, freeze them with liquid nitrogen as soon as possible and store at -80 degrees Cell sample: Digest the cells using a cell scraper or trypsin, and then centrifuge at 12000rpm/min for 5 minutes. Collect the cells and store at -80 degrees.
Other samples: Save the samples as soon as possible -80 degrees.
Total RNA extraction: After cutting the tissue sample into small pieces, liquid nitrogen is ground (50mg) into a powder and transfer to a 1.5ml tube without RNase (cell samples and other liquid samples do not need to be ground, skip to the next step directly).
700ul of lysate per tube, 200ul of sample to be extracted/ground sample, vortex for 10s until completely mix. Add 20ul proteinase K solution and 20ul magnetic beads to obtain puri ed RNA. Short-term storage at -20 degrees, long-term storage at -80 degrees.
Reverse transcription of RNA into cDNA: Genomic DNA removal reaction: Prepare the reaction solution for removing genomic DNA according to the ingredients in Table 2 on ice. The reaction mixture (Master Mix) should be prepared according to the number of reactions +2, and then transfer into reaction tubes. Finally, RNA samples are added to perform various reactions. Reverse transcription reaction: Prepare the reverse transcription reaction solution according to the ingredients in Table 3 on ice. First prepare the reaction mixture (Master Mix) according to the reaction number +2, and then dispense 10μl into the reaction tube to ensure the accuracy of the solution preparation. Perform a reverse transcription reaction immediately after gently mixing.

CCK-8 assay
Collect cells in different groups, adjust the cells to 106 cells/ml, and add 100μl of cell suspension to the 96-well plate. Adjust the incubator to 37°C, 5% CO2 conditions, and then put into the culture plate, incubate for 72h. Add 10μl CCK-8 solution to each well and continue to incubate the plate in the incubator for 1-4 hours. The absorbance at 450 nm was measured with a microplate reader.

Transwell assay
Resuspend cells in basal medium and count, adjust the number of cells to 5*105 power/ml. According to different groups, inoculate 5*104 cells in the transwell chamber, add 500μl of fresh medium containing 20% FBS in the lower chamber, gently shake the cells to distribute them evenly, and incubate at 37 degrees 5% CO2 for 12h. Take out the transwell chamber, carefully wipe off the cells that have not passed through the chamber with a cotton swab, and x it with 4% paraformaldehyde for 10 minutes. Dye with crystal violet dyeing solution for 5 minutes, and wash until the basement membrane is transparent.
Observe, photograph, count and analyze under the microscope.

Cell preparation
The cell suspension is prepared by digesting the cultured cells with trypsin. Take out 10μl of cell suspension for cell counting, and then dilute with complete medium to a concentration of 100,000 cells/ml. Cell suspension was added to the 6-well culture plate with a volume of 2 ml per well. When the cell growth reached 70% con uence, the cell culture uid was discarded and the cells were collected after 16 hours of culture.
Annexin V-Alexa Fluor647 cell apoptosis Move the cell culture uid into a suitable centrifuge tube, wash the adherent cells once with PBS, and digest the cells with an appropriate amount of trypsin cell digest solution (which may contain EDTA). Incubate at room temperature. When the adherent cells are gently blown down, remove the pancreatin cell digest. It is necessary to avoid excessive digestion of pancreatin during the process. Add the cell culture uid collected in step 2A and mix well. Transfer to 1000g in a centrifuge tube and centrifuge for 5 minutes, then discard the supernatant. After collecting the cells, resuspend the cells gently with PBS and count. Take 50,000-100,000 resuspended cells, centrifuge at 1000g for 5 minutes, discard the supernatant and add 195μl Annexin V-FITC binding solution to resuspend the cells gently. Add 5μl Annexin V-Alexa Fluor647 and mix gently. Add 10μl propidium iodide staining solution and mix gently. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes, then place in an ice bath. Immediately perform ow cytometry.

qRT-PCR Assay
The expression levels of LncRNA GAPLINC in ESCC tissues, paracancerous tissues and ve human esophageal squamous carcinoma cell lines were detected by qRT-PCR technology. After qRT-PCR ampli cation, the single-peak dissolution curves were obtained and the CT value was between 25 and 35 cycles, indicating that the ampli cation effect was reliable, no pollution, and no primer dimer formation.

Expression of LncRNA GAPLINC in ESCC tissues and adjacent tissues
Real-time uorescence quantitative PCR was used to detect the expression of LncRNA GAPLINC, a longnon-coding RNA in 40 pairs of ESCC tissues and corresponding adjacent tissues. The relative expression of LncRNA GAPLINC in normal tissues was 1.669±0.028, and the expression of tumor tissues was 1.748±0.026. LncRNA GAPLINC expression in ESCC tissue was signi cantly higher than normal tissues adjacent to cancer ( Figure 2) and the difference was statistically signi cant (*p <0.05).

Relationship between abnormal expression of LncRNA GAPLINC in esophageal squamous cell carcinoma and its clinicopathology
Based on the clinical and pathological data of 40 patients, we further analyzed the relationship between LncRNA GAPLINC expression and clinical factors (gender, age, history of tobacco and alcohol, tumor location, length of esophageal lesion, depth of invasion, degree of differentiation, lymph node metastasis, etc). The analysis of statistical results showed that the relative expression of LncRNA GAPLINC was signi cantly related to the degree of differentiation (P<0.05), and was not related to the patient's gender, age, history of tobacco and alcohol, tumor location, esophageal lesion length, in ltration depth, and lymph node metastasis (Table 5).  The lentivirus was prepared, and then TE-11 cells were infected for subsequent experiments.

Knockdown and overexpression of LncRNA GAPLINC could affect cell proliferation
The cellular viabillity of TE11 cell line after LncGAPLINC-Knockdown and LncGAPLINC-Overexpression were shown in Figure 4. We found that LncGAPLINC-Knockdown decreased the cell proliferation rate, and LnGAPLINC-Overexpression increased the cell proliferation rate (P<0.05).

Knockdown and overexpression of LncRNA GAPLINC could affect cell migration and invasion
Transwell cell migration and invasion experiment was used to detect TE11 cell migration and invasion ability after LncGAPLINC-Knockdown and LncGAPLINC-Overexpression. Compared with the control group, the number of migration and invasion decreased after knocking down and increased after overexpressing (P <0.05) ( Figure 5 and Figure 6).  (Table 6).

Discussion
Esophageal cancer is one of the most common malignant tumors. It has ranked sixth among the causes of tumor-related mortality worldwide, and the morbidity has ranked seventh 11 . The regional differences in esophageal cancer incidence are obvious. year survival rate is not obvious. At present, the clinical prognosis of ESCC mainly depends on the TNM staging. But the prognosis of patients is very different in the same TNM stage. It is not comprehensive enough to rely on the clinical pathological staging to evaluate the prognosis. The inherent molecular biological mechanism is also worth exploring.
Long-chain-non-coding RNA is long as more than 200 nucleotides and does not encode protein.
According to the position, LncRNAs can be divided into 5 types: sense LncRNA, antisense LncRNA, bidirectional LncRNA, intron LncRNA and intergenic LncRNA 13 . Although LncRNAs cannot encode proteins, they can directly interact with DNA, RNA and proteins in order to regulate variety of mechanisms 14 .
In the recent years, studies have found that genetic abnormalities are one of the main causes of ESCC, indicating that ESCC occurrence might be related to environmental factors and genetic factors. In order to achieve the accurate early diagnosis of ESCC and the better prognosis evaluation, we need to further explore its pathogenesis at the genomic level. Previous studies have con rmed that the genesis and development of esophageal cancer involved many genes, such as C-MYC, EGFR, INT-2 and P53. The research on LncRNA in esophageal cancer is not rare, but it is still one of the hot spots in current tumor research. Compared with miRNA, LncRNA is much more abundant at function and mechanism and it may be the core factors in RNA regulation. More and more studies have shown that LncRNA is differentially expressed in tumors 15 . Several studies have shown that LncRNA has a relatively stable secondary structure in body uids, which can be detected in plasma or other body uids 16  LncRNA GAPLINC is rst con rmed to highly express in gastric cancer tissues. Both RT-qPCR and ISH assay have con rmed that GAPLINC expression in gastric cancer tissues is signi cantly higher than corresponding normal gastric mucosal tissues, indicating GAPLINC may be a major risk factor for poor prognosis. GAPLINC is an important predictor of patient prognosis analysis, which is independent of AJCC stage. Its high expression can reduce the apoptosis of cancer cells and promote the proliferation and invasion ability. The expression level is positively correlated with tumor volume, distant metastasis, tumor involvement, lymph nodes proportion and clinical grade. Studies have shown that the p53 mutants will lost the function of wildtype p53s and obtain the function of different oncogenes. The p53 mutant R248W can combine with the GAPLINC promoter sequence to promote its expression. There is a strong correlation between GAPLINC expression level and CD44. The proto-oncogene function of GAPLINC partly depends on CD44 10 . Similar results have been observed in colon cancer study. These researches have indicated that LncRNA GAPLINC could act as an oncogene in gastric and colon cancer. The function and mechanism of LncRNA GAPLINC in esophageal squamous cell carcinoma is unknown. This study collected 40 cases of ESCC tissues and normal tissues adjacent to cancer.   LncRNA GAPLINC expression levels in ESCC tissues and adjacent normal esophagus tissues (*p<0.05). The cell proliferation of LncGAPLINC-Knockdown cell line, LncGAPLINC-Overexpression cell line (*p<0.05).

Figure 7
The cell apoptosis of LncGAPLINC-Knockdown cell line, LncGAPLINC-Overexpression cell line.