The Study On Functions And Mechanisms Of MiRNA-214 In Theperitoneal Dissemination Of Gastric Cancer

BackgroundGastric cancer(GC) is one of the main reason of death from cancer. Studies have shown that a percentage of patients ranging from 15% to 50% or more have a peritoneal disease at the surgical exploration, becoming the leading cause of death. Peritoneal metastasis once established is associated with a poor survival with median values ranging from 1-1.6 months to 3.1-9 months. Various treatments, including intraperitoneal chemotherapy 、 hyperthermia intraperitoneal chemotherapy、 immunotherapy、high intensity focused ultrasound(HIFU), all of the treatment have been tested for effectiveness in peritoneal metastasis from gastric carcinoma, however the effects have been found to be unsatisfactory. Therefore, clarify the mechanism of peritoneal metastasis of gastric cancer, explore a novel means to inhibit peritoneal dissemination is not only significant for improving its treatment, but enhancing the patients’ survival rate.With the discovery of first micro RNA(mi RNA) in 1993, scholars to the investigation is more and more in-depth.Mi RNAs has been verified to be regulated in many types of diseases, such as Diabetes, Hypertension, AIDS, of course a variety of cancers. Many studies have reported that mi RNAs play a critical role in various aspects of GC such as tumor occurrence 、 development 、 diagnosis 、 treatment and prognostic evaluation. Mi RNAs can exerts its biological function as the form of tumor suppressors or oncogenes,influencing cell invasion 、migration、 proliferation 、 apoptosis and differentiation, mi RNAs as a new type of gene regulator that entirely or partially base-pairing to the 3’-untranslated regions(UTRs) of target m RNA, and influencing the m RNA translation in post-transcriptional level by formation the RNA-induced silencing complexus. Although many studies have shown that micro RNA has a close correlation with gastric cancer, yet the expression and function in peritoneal dissemination of GC were not known. Based on literatures domestic and overseas and high-profile micro RNA chip analyses, we initially predicted that the differential expression of micro RNA-214-3p( mi RNA-214、mi R-214) in gastric cancer cell line GC9811 and highly peritoneal dissemination of gastric cancer cell line GC9811-P, and may exert a special biological function. In this paper, we try to initially unravel the expression, function and possible mechanism of mi RNA-214 in gastric cancer cell lines with highly peritoneal dissemination.Objects1. To investigate the levels of mi RNA-214 in gastric cancer cell lines.2. To study the biological function of mi RNA-214 in gastric cancer cell lines.3. To explore the mechanism of mi RNA-214 in gastric cancer cell lines.Methods1. We revive and foster the cells GC9811 and GC9811-P at the same condition, then picked up the small RNA after two or three generation in logarithmic phase, determing small RNA’s OD values between 1.8-2.0; using q RT-PCR to detect mi R-214 differential expression situation in human gastric cancer cell line GC9811 and GC9811-P, count relative value by 2-ΔΔCt, selected U6 as internal reference contrast.2. Ordering mi RNA-214 lentiviral vector,then using the negative control virus and mi RNA-214 up-regulated virus to infect the cells GC9811,marked GC9811+LV-NC and GC9811+LV-mi RNA-214 respectively;using the negative control virus and mi RNA-214 reduced virus to infect the cells GC9811-P, marked to GC9811-P+LV-NC and GC9811-P+LV-mi RNA-214 IH respectively and used q RT- PCR method to verify the transfection effect.3. Using stably transfected cell lines to process transwell migration, invasion assay, MTT proliferation assay, flat cloning experiments, apoptosis experiments et al, and investigating its biological function on gastric cancer cell lines GC9811 and GC9811-P after ectopic expression of mi RNA-214.4. Bioinformatics analysis software Target Scan and Mi Randa were used to predict target genes of mi RNA-214, then we select the cancer-promoting genes that related to facilitate cell invasion and migration. Using q RT-PCR 、western-blot to verify predicted target genes encoding proteins.Result1. The mi RNA-214 expression was increased in peritoneal dissemination cell lines GC9811-P, compared to their corresponding gastric cancer cell lines GC9811.2. We successfully constructed a stably transfected cell lines, and using q RT-PCR to identify the effect of that expression. It revealed that these cell lines can significantly up and down-regulated mi RNA-214 expression levels in cells.3. Up-regulated the expression of mi RNA-214 can obvious enhanced the ability of cell invasion 、 migration 、 proliferation, and the ability of apoptosis was significantly reduced in gastric cancer cells GC9811;while down-regulated of mi RNA-214 can significantly reduced the ability of cell invasion、 migration、proliferation,but significantly enhanced the ability of cells apoptosis in GC9811-P cell lines,with high peritoneal dissemination potential.4. Bioinformatics predicted that PTEN may be one of target genes for mi RNA-214, q RT-PCR revealed that mi RNA-214 expression have no effect in PTEN m RNA level;The result of Western-blot demonstrated that up-regulation mi RNA-214can decreased PTEN protein in GC9811 cells while PTEN protein was increased by down-regulation mi RNA-214 in GC9811-P cells.Conclusion1. Micro RNA-214-3P were significantly up-regulated in highly peritoneal dissemination of gastric cancer cell lines GC9811-P. Suggesting that mi RNA-214 may be a oncogenes promotes gastric cancer metastasis to peritoneum.2. Micro RNA-214-3P in vitro can significantly increase the ability of cell invasion、migration、proliferation and can reduce the ability of cell apoptosis.3. PTEN may be one of the target genes for micro RNA-214-3P, micro RNA-214-3P negatively regulated PTEN expression at the posttranscriptional level,not m RNA level.