The Effects Of MicroRNA-153-3p On The Migration,Proliferation And Chemosensitivity In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma?ESCC?is the major histopathological subtype of esophageal cancer in China,where esophageal cancer ranks eight among all neoplasm.Even though management of ESCC patients at the level of surgery,chemotherapy or targeted therapy,and radiation therapy has improved over the years,ESCC shows a poor prognosis due to high lymphatic metastatic recurrence rates after Ivor Lewis esophagectomy as well as chemotherapy resistance.In fact the five-year survival rate for patients with resectable disease is<40%.Thus,it is very important to identifying new biological markers in the carcinogenesis and progression of ESCC and improve ability to predict the response to chemotherapy of ESCC.MicroRNAs?microRNAs?are endogenous,small non-coding RNAs that play essential roles in the regulation of gene expression,and which have been linked to chemotherapy resistance.Recently,microRNA-153-3p has been shown to target SNAIL or Nrf-2 in regulating proliferaton,migration and EMT in hepatocellular carcinoma and melanoma.However,whether microRNA-153-3p plays a critical role in regulating proliferaton,migration and EMT as well as chemotherapy resistance in ESCC is still unknown.In this study,we will explore the effect of microRNA-153-3p involved in proliferaton,migration and EMT as well as chemotherapy resistance in ESCC through Snail and Nrf-2.Part one:micro RNA-153-3p inhibits cell proliferation and chemot-herapy resistance in esophageal squamous cell carcinoma by targeting Nrf-2Objective:To explore whether microRNA-153-3p inhibits cell proliferation and chemotherapy resistance in esophageal squamous cell carcinoma by targeting Nrf-2.Method:Eca109 cells were transfected with control or microRNA-153-3p mimics,and the cell proliferation and Snail,CyclinD1 and Survivin expression was detected.A dual-luciferase reporter system analysis was performed on Eca109 cells co-transfected with the wild-type/mutant 3'UTR sequences of Nrf-2 and control or microRNA-153-3p mimics.The Eca109cells transfected with microRNA-153-3p mimics or Nrf-2 were treated DDP,and IC₅₀,cell viability,and apoptosis were measured.We also applied 60cases of human ESCC samples as well as the Eca109 cells transfected Nrf-2to explore the effect of Nrf-2 on SOD-2 expression.Result:1.Overexpression of micro RNA-153-3p in Eca109 cells inhibited cells viability and colonies formation?P<0.05?.The expression of CyclinD1 and Survivin in cells transfected with microRNA-153-3p mimics was lower than that in the control?P<0.05?.2.Different concentration of DDP cuased cell death and apoptosis in Eca109 cells transfected with microRNA-153-3p mimics compared with that in the control?P<0.05?.IC₅₀ of DDP in microRNA-153-3p-overexpressed cells was lower than that in the control?P<0.05?.Thus,overexpression of microRNA-153-3p significantly increased the chemosensitivity of ESCC to cisplatin.3.Real-time PCR results of twenty-five ESCC samples and matched para-carcinoma showed an inverse relationship between the expression of microRNA-153-3p and Nrf-2 mRNA.Overexpression of microRNA-153-3p inhibited Nrf-2 expression in Eca109 cells,and a dual-luciferase reporter system analysis confirmed that microRNA-153-3p targeted 3'UTR of Nrf-2.Blocking Nrf-2 inhibited cells viability and colonies formation in Eca109cells?P<0.05?.4.Different concentration of DDP cuased cell death and apoptosis in Eca109 cells transfected with Nrf-2 siRNA compared with that in the control?P<0.05?.IC₅₀ of DDP in Nrf-2 si RNA cells was lower than that in the control?P<0.05?.Thus,inhibition of Nrf-2 significantly increased the chemosensitivity of ESCC to cisplatin.5.Increased expression of Nrf-2?CyclinD1?Survivin and SOD-2 was shown by IHC in 60 cases of human ESCC.Correlation between Nrf-2 and CyclinD1,Survivin and SOD-2 was shown.Kaplan-Meier analysis indicated higher expression of Nrf-2,CyclinD1,and Survivin associated with a poor overall survival in ESCC patients?P<0.05?.6.Real-time PCR results of twenty-five ESCC samples and matched para-carcinoma showed no relationship between the expression of microRNA-153-3p and SOD-2 m RNA.Overexpression of microRNA-153-3p or inhibition of Nrf-2 did not affect SOD-2 expression in Eca109 cells.Part two:TNF-?upregulating SOD-2 contributes to cell proliferation and chemotherapy resistance in esophageal squamous cell carcinomaObjective:Though we found overexpression of micro RNA-153-3p or inhibition of Nrf-2 did not affect SOD-2 expression in Eca109 cells in the part I,we still wanted to explore whether SOD-2 involvoed in cell proliferation and chemotherapy resistance as well as whether/how TNF-?regulates SOD-2 expression in esophageal squamous cell carcinoma.Method:We also applied 60 cases of human ESCC samples to measure the expression of SOD-2 as well as its role in overall survival in ESCC patients.The Eca109 cells transfected with SOD-2 si RNA were treated with DDP,and IC₅₀,cell viability,and apoptosis were measured.Finally,Eca109cells were transfected with SOD-2 si RNA or NF-?B si RNA,and then treated with TNF-?.The cell proliferation as well as CyclinD1 and Survivin expression was detected.Results:1.Increased expression of SOD-2 was observed in 60 cases of human ESCC.Correlation between SOD-2 and Cyclin D1,Survivin was shown.Kaplan-Meier analysis indicated higher expression of SOD-2 associated with a poor overall survival in ESCC patients?P<0.05?.2.TNF-?increased cells viability and colonies formation in Eca109cells,and upregulated SOD-2 as well as Cyclin D1,Survivin expression?P<0.05?.3.We blocked SOD-2 expression in Eca109 cells transfected with siRNA,and found that blocking SOD-2 expression significantly inhibited TNF-?-increased cells viability and colonies formation.Increased CyclinD1 and Survivin induced by TNF-?was inhibited in Eca109 cells transfected with siRNA.TNF-?induced cell proliferation through SOD-2in Eca109 cells.4.We found that blocking NF-?B expression by siRNA significantly inhibited TNF-?-increased cells viability and colonies formation in Eca109 cells.Increased Cyclin D1 and Survivin induced by TNF-?was inhibited in Eca109 cells transfected with siRNA.Thus,TNF-?upregulate SOD-2 through NF-?B pathway to contribute to cell proliferation through in Eca109 cells.5.Different concentration of DDP cuased cell death and apoptosis in Eca109 cells transfected with SOD-2 si RNA compared with that in the control?P<0.05?.IC₅₀ of DDP in SOD-2 si RNA cells was lower than that in the control?P<0.05?.Thus,inhibition of SOD-2 significantly increased the chemosensitivity of ESCC to cisplatin.Part three:microRNA-153-3p inhibits tumor cell migration in esophageal squamous cell carcinoma by targeting SNAILObjective:To explore whether microRNA-153-3p inhibits tumor cell migration in esophageal squamous cell carcinoma by targeting Snail.Method:Metagenomic analysis of 115 TCGA patients and twenty-five ESCC samples and matched para-carcinoma were applied to explore the relationship between Snail and microRNA-153-3p in esophageal cancer patients.The expression of microRNA-153-3p and Snail as well as cell migration were determined in HET-1,OE21 as well as OE21 cell transfected with microRNA-153-3p mimics or NC mimics.Finally,firefly luciferase expressing OE21 cells,stably transfected with either NC mimics or microRNA-153-3p mimics were intravenously injected into tail vein of athymic nude mice.Mice were imaged by in vivo luciferase imaging up to 8weeks to detect lung colonization and metastasis.Results:1.Metagenomic analysis of The Cancer Genome Atlas?TCGA?data identified an inverse correlation between microRNA-153-3p and Snail expression in ESCC.Real-time PCR results of twenty-five ESCC samples and matched para-carcinoma showed an inverse relationship between the expression of micro RNA-153-3p and Nrf-2 mRNA?P<0.05,Pearson correlation r=-0.663?.2.Complementary 7mer-m8 seed match between microRNA-153-3p and the 3?UTR of Snail was predicted by TargetScan software.Overexpression of microRNA-153-3p inhibited Snail expression in OE21 cells,and a dual-luciferase reporter system analysis confirmed that micro RNA-153-3p targeted 3'UTR of Snail.3.Blocking of Snail inhibited cells migration in OE21 cells?P<0.05?.The expression of Snail,Vinmentin,and MMP-9 was lower in OE21 cells transfected with Snail siRNA compared with that in the control?P<0.05?.4.Whereas,OE21 control mimics cells showed robust metastasis in 3 out of 5 animals after 8 weeks,while OE21/microRNA-153-3p mimics significantly attenuated lung colonization in the ESCC cells.Cumulatively,this indicated that loss of microRNA-153-3p expression function as a suppressor of metastasis in the context of ESCC.Conclusion:1.Micro RNA-153-3p targeted 3'UTR of Nrf-2 to downregulating Nrf-2expression in ESCC,which significantly inhibited cells proliferation and increased the chemosensitivity to cisplatin in Eca109 cells.2.Higher expression of SOD-2 is associated with a poor overall survival in ESCC patients.TNF-?through NF-?B pathway could upregulate SOD-2expression,which promoted cell proliferation as well as increased chemotherapy resistance to cisplatin in Eca109 cells.3.microRNA-153-3p targeted 3'UTR of Snail to downregulating Snail expression in ESCC,which significantly inhibited cell migration and invasion in ESCC.