Expression And Significance Of Long Non-coding RNA MEG 3 In Esophageal Cancer Cells

Objective: Esophageal cancer is malignant cancer arising from the esophagus mucous membrane surface. It's one of the most commonly diagnosed cancer and the eighth-most common cancer worldwide. In 2012, there were about 456,000 new cases, accounting for 3% of the total cancer cases globally. Esophageal cancer is the sixth most common cause of cancer-related deaths in the world. It caused about 400,000 deaths in 2012, 5% of the total deaths worldwide. China is one of the countries having the highest national incidence of the esophageal cancer, with more than 220,000 new cases annually and about 200,000 deaths. Up to now, over 90% of esophageal cancer patients have developed into medium- to advanced- stage upon diagnosis, with poor life quality. The overall five-year survival rate is below 20%. As science advances and a lot of researchers continue studies into it, we have gained a deeper understanding of the esophageal cancer. However, it remains an arduous and pressing task to improve treatment level of the cancer in China. Thus, studying development mechanism of esophageal cancer, experimenting early detection index, and exploring targeted therapy is playing an important role in clinical treatment and also leaving a far-reaching significance in promoting the development of the human society.Latest research findings indicate the genesis and development of esophageal cancer is related to abnormal changes of various genetics and epigenetics. Yet so far, molecular mechanism of the cancer development has not been completely clarified, in need of effective molecular targets and markers for prognosis. Long non-coding RNAs?long nc RNAs, lnc RNA? are non-protein coding transcripts longer than 200 nucleotides, abundantly existing in human genome. They impact expression and functionality of coding genes in different ways, and also regulate biological progresses of cells.Maternally expressed gene 3?MEG3? is one of the recognized Inc RNA and mainly used to express normal brain, marrow, breast, womb, lung and the gastrointestinal tract tissues while the expression in malignant tumors is obviously decreased or lost. Some studies show significant down-regulation of expression of MEG3 in lung cancer. Thus, over-expression of MEG3 will be capable of inhibiting proliferation of the lung cancer cells. However, the role of MEG3 expression in esophageal cancer remains unknown. The study is to detect expression of Inc RNA MEG3 in esophageal cancer, explore effects of expression change of MEG3 upon esophageal cancer tissues, and to provide new markers and new targets for treatment interventions.Methods:1 Expression of MEG3 in esophageal cancer tissues and cells1.1 Thirty-two paired esophageal cancer tissues and adjacent normal esophageal tissues were obtained from patients who hadn't received any chemo-radiotherapy before surgery and were diagnosed esophageal cancer after surgery. All of the specimens were cleaned by PBS liquid and immediately frozen in liquid nitrogen for 24 hours, and then stored at-80°C fridge.1.2 qRT-PCR detects MEG3 expression in esophageal cancer tissues and adjacent normal esophageal tissues: First of all, total RNA was extracted from tissues or cultured cells with Trizol reagent. Secondly, using agarose-gel electrophoresis?AGE? evaluated RNA quality to ensure completeness of RNA. Thirdly, designed sense/anti-sense primers of reverse transcription kit for Inc RNA MEG3, using primerprimer5 software and based on genes of MEG3 and GAPDH, respectively, according to Gene Bank's primer sequences, and normalized to GAPDH. The isolated RNA was then reverse transcribed. The expression of MEG3 was quantified by q RT-PCR.1.3 Purchased Eca-109 cell lines and HEEC cell lines.1.4 Cell culture: Both were cultured in RPMI-1640 supplemented with 10% fetal bovine serum?FBS?, 100 units/ml penicillin and 100 ?g/ml streptomycin. The cells were incubated at 37°C in a humid atmosphere with 5% CO₂. Every 1-2 days fresh DMEM was replaced. Sub-culture was conducted when cell fusion reached 80%-90%.1.5 qRT-PCR tested MEG3 expression in Eca-109 cell lines and HEEC cell lines:First of all, total RNA was extracted from tissues or cultured cells with Trizol reagent. Secondly, using agarose-gel electrophoresis?AGE? evaluated RNA quality. The last step was to use q RT-PCR to test expression levels of Inc RNA MEG3.1.6 Statistical analysis:Data were analyzed using SPSS 20.0 software. The measurement data were expressed as the mean X±S. Comparison among groups adopted t tests, statistical analysis expressed as ratio. Comparison among groups adopted ?² tests. Statistical differences were established at p ? 0.05.2 Effect of MEG3 on esophageal cancer tissue proliferation, migration and invasion2.1 Primer compound: The same methods used in the above tests2.2 Plasmid construction: Firstly, amplified designed primers and examined upon completion of amplification with agarose-gel electrophoresis?AGE?. After that, we created eukaryotic expression vector pc DNA3.1-lnc RNA MEG3. Then, verified the gene through double enzyme digestion and AGE, using blank vector pc DNA as negative control.2.3 Plasmid transfection: All plasmid vectors?pc DNA-MEG3 and blank vector? for transfection were restructured via Midiprep extraction. According to the manufacturer's instructions, pc DNA-MEG3 or blank vector were transfected by Fu GENE6 reagent into cells cultured on 6-well plates. Results were tested after 48 hours.2.4 G418 selected cells: Firstly, we set the concentration of G418. Then, selected considerably stably transfected and over-expression Inc RNA MEG3 cell strain, Eca109-p CDNA3.1-lnc RNA MEG3. And we used RT PRC to test target gene expression levels so as to confirm whether the cell strains had been constructed successfully, with transfected blank vector as negative control and compared with transfection reagent added blank control.2.5 Cell proliferation assay in vitro: Using MTT cell proliferation assay kit conducted cell tests of over-expression esophageal cancer cells in accordance with the instructions to examine cell proliferation ability, with transfected empty vector as control.2.6 Cell invasion assay in vitro: Using Transwell assay tested invasion of over-expression esophageal cancer cells, with transfected empty vector as control.2.7 Cell migration assay: Migration assay was performed on over-expression esophageal cancer cells to test cell migration, with transfected empty vector as control.2.8 Western blot assay: Cell lysis was conducted on transfected esophageal cancer cells to extract total protein. Using coomassie blue staining?Bradford? tested protein level, with 50 ?g of polyacrylamide gels?PAGE? and PVDF membranes that were blocked non-specific binding with nonfat milk. Then, I and II antibodies were incubated. At last, ECL chromogenic substrate was used to visualize the expression of p53 in cells. GAPDH was used as a control. Results were analyzed by Image J gray-scale value.2.9 Statistical analysis:Data were analyzed using SPSS 17.0 software. The measurement data were expressed as the mean X±S. Comparison among groups adopted t tests, statistical analysis expressed as ratio. Comparison among groups adopted ?² tests. Statistical differences were established at p ? 0.05.Results:1 Expression of MEG3 in esophageal cancer tissues and cells1.1 The expression of Inc RNA MEG3 was significantly lower in esophageal cancer tissues than in adjacent normal ones.1.2 The expression of Inc RNA MEG3 was significantly lower in esophageal cancer cell lines Eca109 than in normal HEEC.2 Effect of MEG3 on esophageal cancer tissue proliferation, migration and invasion2.1 Cell proliferation assay in vitro showed up-regulation of MEG3 expression level could reduce proliferation of Eca109.2.2 Cell invasion assay in vitro showed that the number of trans-membrane Eca109 cell was smaller in MEG3 over-expression group than that of the control group.2.3 Cell migration assay showed the cell space was broader after MEG3 over-expression in the assay than that of the control group.2.4 Western blot assay showed the expression level of P53 protein in over-expression MEG3 Eca109 was higher than that of the control group.Conclusion:1 Inc RNA MEG3 is significantly down-regulated in esophageal cancer tissues and cells, indicating correlation with tumor development, and could exert tumor-suppressive functions in the genesis and progression of the cancer.2 Over-expression of MEG3 could suppress proliferation, invasion and migration of esophageal cancer cells, and would perform the above role through affecting expression of p53 protein.