| Background Lung cancer,a leading cause of mortality among the cancer patients,is divided into two categories,non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),which account for about 85% and 15% of the total lung cancers respectively,according to different histomorphological features,and lung adenocarcinoma(LUAD)accounts for about 50% of NSCLC.Various diagnostic techniques like CT-imaging,bronchoscopy and sputum cytology and main treatments like surgical resection,radiotherapy and chemotherapy,have been modified recently years for better sensitivity and accuracy,but the effects are still not ideal,one of the primary causes for poor effect is the metastasis of tumor.Therefore,it is necessary to explore the key factors and the molecular mechanisms involved in lung cancer migration and invasion for the development of diagnostic and treatment strategies.Micro RNA(mi RNA)are non-coding single-stranded small RNA molecules(19-25 nt)that regulate the expression of target genes at the post-transcriptional level by suppressing translation or inducing degradation of target genes m RNA.Abnormal expression of mi RNAs has been widely recognized during the development and progression of cancers.Therefore,mi RNAs are expected to be markers for clinical diagnosis and prognosis as well as potential therapeutic targets of cancer.It has been reported that mi R-198-5p is down-regulated in a variety of malignancies,mi R-198-5p is also reported to inhibit proliferation and promoted apoptosis of NSCLC cells by targeting SHMT1 or FGFR1.Nevertheless,the role of mi R-198-5p in the migration and invasion of LUAD and the potential molecular mechanisms involved have not been clear.Fucosylation involves a variety of physiological and pathological processes such as cell growth,adhesion,and tumor metastasis.FUT8 shows stable expression under normal state,however,studies have found that FUT8 is up-regulated in some malignant tumor tissues,and knocking down FUT8 significantly inhibits malignant behaviors including invasion and proliferation of lung cancer cells.And high expression of FUT8 is associated with an unfavorable clinical outcome in LUAD patients.Objective 1.To investigate the expression level of mi R-198-5p in LUAD tissues and its relationship with clinical pathological types of LUAD patients.2.To investigate the effect of mi R-198-5p on invasion and metastasis of LUAD cells and the EMT.3.To clarify that mi R-198-5p is involved in the molecular mechanisms that influence the invasion and metastasis of LUAD cells and the EMT.Method Part 1 Expression of mi R-198-5p and FUT8 in LUAD tissues and their correlation 1.Relative expressions of mi R-198-5p in LUAD tissue specimens and the corresponding adjacent non-cancerous specimens were detected by q RT-PCR 2.To investigated the correlation of mi R-198-5p expression with clinicopathologic characteristics of LUAD patients.3.Predicted potential target genes for mi R-198-5p by target gene prediction software.4.The expressions of FUT8 in LUAD tissues and adjacent non-cancerous specimens were detected by q RT-PCR,and its correlation with mi R-198-5p was analyzed.Part 2 The role of mi R-198-5p in the invasion,migration and EMT in LUAD CELLS 1.According to the expression of mi R-198-5p in four LUAD cell lines,we chose NCI-H1650 and A549 cells for next assays 2.After transfection of mi R-198-5p mimic and mi R-198-5p inhibitor into NCI-H1650 cells and A549 cells for 48 hours,q RT-PCR was used to detect the m RNA expression of mi R-198-5p in the two cells.3.Detect the migration ability of cells by using wound healing assay.4.Detect the invasion ability of cells by using Transwell invation assay.5.Stable NCI-H1650 cells transfected with mi R-198-5p overpression plasmids were injected into the tail vein of the nude mice,then analyzed the effect of mi R-198-5p on the metastasis of LUAD cells.6.Western blot was used to detect the expression of E-cadherin,N-cadherin and Vimentin in each group after transfection,and immunofluorescence was used to detect the expression of Vimentin.Part 3 mi R-198-5p inhibits the invasion,migration and EMT of LUAD Cells by targeting FUT8 1.The dual luciferase reporter assay confirmed whether FUT8 is a target gene for mi R-198-5p.2.q RT-PCR and western blot were used to detect the change of FUT8 expression after transfection with mi R-198-5p mimic or inhibitor.3.Detect the effects of mi R-198-5p and/or FUT8 overexpression on cell invasion,migration,and EMT.Results Part 1 Expression of mi R-198-5p and FUT8 in LUAD tissues and their correlation 1.The results demonstrated that mi R-198-5p expression in LUAD tissues was significantly down-regulated than that in adjacent non-cancerous tissues(p<0.05).Just the opposite,the FUT8 expression in LUAD tissues was significantly up-regulated than that in adjacent non-cancerous tissues 2.lower expression of mi R-198-5p was closely correlated with more lymph node metastasis,and higher grade TNM stage of LUAD patients.3.The expression of FUT8 was negatively correlated with mi R-198-5p in LUAD tissues.Part 2 The role of mi R-198-5p in the invasion,migration and EMT in LUAD Cells 1.The efficiencies of mimic and inhibitor in modulating mi R-198-5p expression were confirmed by q RT-PCR.The results showed that the expression of mi R-198-5p in NCI-H1650 cells transfected with mi R-198-5p mimic was increased(p<0.01).And the expression of mi R-198-5p in A549 cells transfected with mi R-198-5p inhibitor was significantly lower than that in NC group(p<0.01).2.The wound healing assay demonstrated that transfection with mi R-198-5p mimic markedly decreased the cell migration in NCI-H1650 cells,compared with the NC.By contrast,transfection with mi R-198-5p inhibitor markedly increased the cell migration in A549 cells,compared with the NC.3.After NCI-H1650 transfected with mimic,the number of transmembrane cells was significantly decreased(p<0.01).While the number of transmembrane cells of A549 transfected with inhibitor was significantly increased(p< 0.01).4.Overexpression of mi R-198-5p can inhibite the metastasis of LUAD in nude mice.5.Key marker proteins E-cadherin,N-cadherin and Vimentin were detected using western blot,and Vimentin was also detected using immunofluorescence method.The results showed that mi R-198-5p upregulation by mi R-198-5p mimic could increase E-cadherin expression and decrease N-cadherin and Vimentin expressions in NCI-H1650.mi R-198-5p downregulation induced by mi R-198-5p inhibitor could decrease E-cadherin expression and increase N-cadherin and Vimentin expressions in A549 cells.Part 3 mi R-198-5p inhibits the invasion,migration and EMT of LUAD cells by targeting FUT8 1.Dual-luciferase reporter assay revealed that the luciferase activity was significantly decreased in HEK293 T cells transfected with FUT8-3'-UTR-wt and mi R-198-5p mimic.2.q RT-PCR and western blot analyses further confirmed that the mi R-198-5p overexpression significantly reduced FUT8 m RNA and protein expressions in NCI-H1650 cells,whereas,the suppression of mi R-198-5p enhanced the expression of FUT8 m RNA and protein in A549 cells.3.Wound healing and transwell assays were used to evaluate the effect of FUT8 on the inhibition of migration and invasion by mi R-198-5p in NCI-H1650 cells.The results showed that mi R-198-5p overexpression resulted in slower wound closure,whereas,FUT8 overexpression counteracted this inhibited migration of NCI-H1650 cells.Analogously,mi R-198-5p reduced invasion ability in NCI-H1650 cells,whereas,FUT8 overexpression dramatically impaired this reducing invasion ability.mi R-198-5p upregulated E-cadherin and downregulated N-cadherin and Vimentin expressions in NCI-H1650 cells,whereas FUT8 reversed the change in these proteins,and the immunofluorescence of Vimentin showed a consistent change.4.Overexpression of FUT8 reversed the inhibitory effect of mi R-198-5p on PI3K/AKT pathway.Conclusions 1.The expression of mi R-198-5p was decreased in LUAD tissues,and its expression level was correlated with lymph node metastasis and TNM staging.The expression of FUT8 in tumor tissues was higher than that in adjacent normal tissues.FUT8 and mi R-198-5p was negatively correlated.2.mi R-198-5p blocks the PI3K/AKT pathway by targeting FUT8,and inhibits the expression of EMT in lung adenocarcinoma cells. |
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