Mechanism Research On The Influence Of REV3L In TLS On ESCC Invasiveness And Chemoresistance

Research background:Esophageal squamous cell carcinoma (ESCC) is one of the fatal malignancies with a relatively high incidence in the world. Clinical evidences indicated that the etiology of ESCC includes multiple factors containing both environmental and genetic determinants. Although a great numbers of genetic and epigenetic alterations were reported in ESCC, molecular markers for early diagnosis and prognosis remain to be discovered. The majority of ESCC deaths are due to invasive disease, therefore identification of novel genes involved in the tumorigenesis and development of ESCC could contribute to improving the outcomes of this disease. Translesion DNA synthesis (TLS) is one type of DNA damage tolerance mechanisms that allows continuing DNA synthesis even in the presence of DNA damage. REV3L, the catalytic subunit of the DNA Pol ζ, is well known to participate in error-prone TLS with less stringent and lower processivity. REV3L maintains the genomic integrity through inserting substitute nucleotide in the opposite DNA adducts, which increases mutation rate and contributes to carcinogenesis. REV3L gene polymorphisms were correlated with the risk of lung cancer and breast cancer. Pol ζ was reported to promote tumor formation and be significantly associated with poor progression in cervical cancer. Thus, REV3L may play an important role in carcinogenesis and tumor progression. The REV3L gene appears to be ubiquitously expressed in normal and tumor tissues, while its expression pattern remains contentious in different cancer tissues. REV3L expression was down-regulated in colon, lung, gastric and renal cancer tissues as compared to adjacent tissues, whereas it was up-regulated in human glioma tissues. Our previous study indicated that the mRNA level of REV3L was significantly elevated in ESCC when compared with normal controls, but its role in ESCC development is unclear. In this study, we analyzed the expression of REV3L in ESCC and adjacent normal tissues, as well as its association with clinicopathological parameters. Furthermore, we elucidated the role of REV3L in ESCC progression and drug-resistance using well-established ESCC cell lines.Methods:The shRNA construct against REV3L (REV3L-shRNA, shREV3L) and the control plasmid (shNC) were purchased. The shREV3L construct or control vector were transfected into ECA-109 and TE-1 cells by using Lipofectamine. Stable clones were selected in medium containing G418. Individual clones were isolated and expanded for further characterization. Total RNA was extracted using TRIzol reagent. For reverse transcription, RNA sample was reverse transcribed. Reverse transcriptase (RT-PCR) was conducted. The PCR products were separated by electrophoresis and the quantification of each band was performed using Quantity One software. Cells were fixed, washed and permeabilized. Samples were blocked and incubated with the REV3L antibody. After washing, the Rhodamine-labeled goat anti-rabbit antibody was added and incubated. Nuclear counterstaining was performed using DAPI. Cells were visualized under a fluorescence microscope. Cell Counting Kit-8 (CCK-8) was used to measure cell proliferation. Cells was seeded in plates for 1,2,3,4,5 or 6 days. Then, CCK-8 solution was added to each well, and incubated for 2h. The optical density was measured at 450 nm with a microplate reader. The viability index was calculated as the experimental OD value/the control OD value. For 5-Fluorouracil (5-FU) experiments, cells were initially plated in plates.24 hours later, the cells were treated with various concentrations of 5-FU for 48 h. Cell viability was measured as described above. The invasive potential of cells was evaluated using Transwell inserts. The inserts were pre-coated with Matrigel. Then, cells in serum-free medium were added to the upper chambers. The lower chambers were filled with medium. After incubation for 24 h, the inserts were fixed and stained. The invading cells in the lower chambers were photographed under a microscope. Cells were seeded in plates.24 hours later, the cells were treated with 50 μM and 100 μM 5-FU for 24 h or 48 h. Cell cycle analysis was performed using the propidium iodide (PI) single staining method. Cells were collected, fixed, washed and re-stained. The cell cycle profiles were assayed using a FACScan flow cytometry, and data were analyzed using MultiCycle software. Cell apoptosis was measured using PE Annexin V Apoptosis Detection kit. After 48h of culture, cells were harvested and processed and analyzed on a FACScan flow cytometry. Western blot was done on whole-cell extracts which were lysed in RIPA lysis buffer. The proteins were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 h, the membranes were incubated with primary antibodies targeting β-actin, Survivin, CyclinD1 and PARP. After washing three times with TBST, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody for 2h. The proteins were visualized using enhanced chemiluminescence.Results:The expression of REV3L in ESCC tissues was significantly higher than that in adjacent tissues (P< 0.05). REV3L mRNA expression was positively correlated with lymph node metastasis and clinical stages (Ⅱb and Ⅲ). In contrast, REV3L mRNA expression was not correlated with pT, gender, ages, and histological grades among the groups of patients. These findings indicated that overexpression of REV3L in ESCC is associated with lymph node metastasis and tumor progression.To study the potential role of REV3L in esophageal cancer, stable cell lines with REV3L knockdown were established using the ECA-109 and TE-1 cells. RT-PCR and immunofluorescence revealed that REV3L expression at mRNA and protein levels was significantly lower (P<0.05) in the cells stably transfected with shREV3L as compared to the cells transfected with control vector (shNC), indicating an effective knockdown of the REV3L expression. To examine whether the modulation of REV3L expression affects the tumorigenic properties of the esophageal cancer in vitro, we used CCK-8 assay and Transwell analysis respectively and found the cell proliferation and invasion were both decreased in REV3L knockdown cells compared to control cells (P<0.05). These results suggest that REV3L plays an important role in esophageal cancer progression. To further identify the mechanisms by which silencing REV3L inhibited esophageal cancer cell proliferation and invasion, we analyzed the expression level of CyclinDl and Survivin proteins. Western blot analysis revealed that REV3L knockdown significantly down-regulated CyclinDl and Survivin protein expression in esophageal cancer cells. Several concentrations of 5-FU were used to treat ECA-109 and TE-1 cells for 48 h. a dose-dependent inhibition of cell growth was observed in 5-FU-treated TE-1 and ECA-109 cells, and the REV3L knockdown cells were more sensitive to the cytotoxic effect of 5-FU. The results from cell cycle analysis showed that the amount of cells at G1 phase in ECA-109/shREV3L cells was significantly more than those in ECA-109/shNC cells after treatment with different doses of 5-FU for 24 h. Apoptotic rates in ECA-109/shREV3L cells were significantly higher than in ECA-109/shNC cells in response to different doses of 5-FU for 48 h. We then performed western blot to analyze the cleavage of PARP protein, and found that ECA-109 REV3L knockdown cells had increased PARP cleavage compared with control cells after treatment with 50 μM or 100 μM 5-FU for 48 h.Conclusions:REV3L is the catalytic subunit of the Pol ζ. REV3L expression is significantly up-regulated in ESCC tissues compared with adjacent tissues, and positively correlated with lymph node metastasis and clinical stages. Inhibition of REV3L expression reduces ESCC cell proliferation and invasion at least in part through suppressing CyclinDl and Survivin expression. REV3L functions to confer chemoresistance to 5-FU treatment via regulation of cell cycle and apoptosis. These findings illustrate a role of REV3L in human ESCC progression and chemoresistance, and provide a potential new diagnostic marker and therapeutic target for ESCC.