The Role Of Artemin Gene In Proliferation And Invasiveness Of Human Endometrial Carcinoma Cells

Endometrial carcinoma(EC)is one of the most common genital tract malignancy in women.The morbidity of endometrial carcinoma tends to increase in recent years.The majority of EC cases that in early stage are cured with surgery and show a favorable prognosis.However,someone reported median survival times in patients with late stage or recurrent disease treated by chemotherapy is hardly more than 1 year.EC can be divided into estrogen dependent(type ?)and non-estrogen dependent(type ?).The former has the most cases and good prognosis.The latter,considering it highly aggressive biological nature and early distant metastases tendency,has the poor prognosis.Artemin(ARTN)is a neurotrophic factor,belonging to the glial cell-derived neurotrophic factor(GDNF)family.ARTN is widely distributing in many tissues,it is of great significance for the development of the nervous system and the non-nervous system.ARTN also maintains the physiological function.ARTN is closely related to the genesis and progression of pancreatic,esophageal,stomach,breast cancer etc.In the subject,through interferring and forcing expression of ARTN,investigates the effects of proliferation,migration and invasion of ARTN in EC cells.Objective:To investigate the effects of proliferation,migration and invasion on EC in cell line by functional inhibition and forced expression of ARTN.Inhibition of ARTN may be considered as a potential therapeutic strategy to EC.Methods:1 Endometrial carcinoma cell lines(Ishikawa and HEC-1-A)were cultured in votro.Western Blot analysis was used to detect the expression of ARTN protein in two EC cell lines.2 The eukaryotic expression plasmid and short hairpin RNA for ARTN were constructed.3 The pGPU6/GFP/Neo-ARTN shRNA and pcDNA3.1-ARTN recombinant plasmid were transfected into Ishikawa and HEC-1-A cell lines by Lipofectamine TM 2000.The expression of mRNA and protein in ARTN were detected by qRT-PCR analysis and Western Blot analysis.4 CCK growth curve and Transwell chamber assays were used to detect the effect of ARTN on EC cells proliferation,migration and invasion.Results:1 ARTN protein was detected in two kinds of EC cell lines at different levels.The relative expression of ARTN protein were 0.808±0.015 and 0.895±0.008.2 The relative expression of ARTN mRNA in two cell lines decreased after being transfected by pGPU6/GFP/Neo-ARTN shRNA(F=47.882,F=394.326,P<0.05).While being transfected by pcDNA3.1-ARTN,the relative expression of ARTN mRNA was detected significantly increasing(F=276.007,F=861.159,P<0.05).The relative expression of ARTN protein in two cell lines reduced by inhibition of ARTN(F=726.323,F=1780.714,P<0.05).Forced expression of ARTN in two cell lines significantly increased the relative expression of protein(F=1145.127,F=519.357,P<0.05).3 After being transfected by pGPU6/GFP/Neo-ARTN shRNA,the proliferation of two cell lines had no difference between each groups in 24h(P>0.05),while decreasing significantly in 48 h,72h,96h(P<0.05).Forced expression of ARTN by pcDNA3.1-ARTN enhanced the proliferation in 48 h,72h and 96h(P<0.05).4 The invasion experiment showed that the number of membrane penetrating cells in interference group were less than the negative control group and control group(F=55.609,F=108.865,P<0.05).Contrary to it,the number of membrane penetrating cells in overexpression group were much more than the negative control group and control group(F=42.880,F=32.004,P<0.05).In the migration experiment,the number of membrane penetrating cells in interference group were less comparing to the negative control group and control group(F=52.938,F=90.224,P<0.05).In the contrary,the number of membrane penetrating cells in overexpression group were much more comparing to the negative control group and control group(F=196.899,F=100.167,P<0.05).In the invasion and migration experiment,the number of membrane penetrating cells of HEC-1-A cell line in each group increased significantly,comparing to the Ishikawa cell line(P<0.05).Conclusion:1 ARTN protein was detected in two kinds of EC cell lines at different levels.2 RNAi could inhibit the expression of ARTN mRNA and protein.The expression of ARTN mRNA and protein was increased by forcing expression of ARTN.3 The proliferation,migration and invasion of EC cells were significantly reduced by functional inhibition of ARTN.Concordantly,forced expression of ARTN in EC cells increased these effects.The migration and invasion ability of HEC-1-A cell line was more stronger than Ishikawa cell line.