The Effect METTL14 On Prolifetation And Metastasis Of Pancreatic Cancer Cells And Its Mechanisms

Part 1.Detection of N6-methyladenosine(m6A)in pancreatic cancer cell lines and pancreatic cancer tissuesObjective:To detect the expression levels of m6A in pancreatic cancer tissues and corresponding peritumorial tissues,then compare the expression differences.Methods:We detected 21 cases of pancreatic cancer tissues and their adjacent none-tomor tissues by colorimetric method,6 pancreatic cancer cell lines and 8 normal pancreas tissues,then we analysed the expression difference between pancreatic cancer tissues and their adjacent none-tomor tissues.We detected 21 cases of pancreatic cancer tissues and their adjacent none-tomor tissues by colorimetric method,7 pancreatic cancer cell lines and the immortalized human pancreatic ductal epithelial cell line,then we analysed the expression difference between pancreatic cancer tissues and their adjacent none-tomor tissues.Results:Compared with normal pancreas tissues and the immortalized human pancreatic ductal epithelial cell line,m6A levels were upregulated.Conclusion:Expression level of m6A in pancreatic cancer is increased.Part 2.The expression level and prognosis of m6A methyltransferase METTL14 in pancreatic cancerObjective:Detection the expression levels of METTL14 in 120 cases of pancreatic cancer tissues,combining the clinicopathology characteristics,we researched the expression difference of METTL14 in pancreatic cancer and adjacent none-tumor tissues,then we studied the relation of METTL14 expression with clinicopathology characteristics and prognosis.Methods:Expression of METTL14 was detected by immunohistochemistry in 120 cases of pancreatic cancer tissues,analyzing the clinicopathology characteristics,we studied the relation of METTL14 expression with clinical stages,pathological grading,lymphatic metastasis and prognosis.Then 17 cases pancreatic cancer tissues were detected by real-time PCR and 20 cases of pancreatic cancer tissues were detected by western blot to further assess and qualify the expression of METTL14.Results:1.Compared with adjacent none-tumor tissues,METTL14 was overexpressed in pancreatic cancer tissues,the difference is significant(P<0.001).Analysing clinicopathology characteristics of pancreatic cancer patients,the expression level of METTL14 was related with lymphatic metastasis(P<0.05),and patients with the high expression level of METTL14 had poor prognosis(P<0.05).2.mRNA levels of METTL14 increased in pancreatic cancer tissues compared with adjacent none-tumor tissues.3.Protein levels of METTL 14 increased in pancreatic cancer tissues compared with adjacent norne-tumor tissues.Conclusion:The expression of METTL 14 is up-regulated in pancreatic cancer and overexpressed METTL 14 predicts lymphatic metastasis and a poor prognosis.Part 3.The effect of METTL14 on the growth and metastasis ofpancreatic cancer in vivo and vitroObjective:Three pancreatic cancer cell lines were used as tools,we constructed stable cell lines with METTL14 knocked down or overexpressed to study the effect of METTL14 intervention on the growth ability of pancreatic cancer cells.Methods:We constructed the stable PANC-1,Mia and BxPC-3 cell lines overexpressing or knocking-down METTL14 using a lentiviral delivery system.After METTL14 intervention,we detected the contents of m6A.CCK8 and clone formation assay were used to evaluate the growth ability of pancreatic cancer cells after METTL14 intervention in vitro.Overexpressing or knocking-down METTL14 in PANC-1 cell were generated and injected in a subcutaneous transplant model.Then we evaluated the growth ability of pancreatic cancer cells after METTL14 intervention in vivo.Overexpressing or knocking-down METTL14 in PANC-1 cell were generated and injected into the spleen to get a liver metastasis model,then we evaluated the invasion ability of pancreatic cancer cells after METTL14 intervention in vivo.Results:1.METTL14 could modulate the content of m6A in pancreatic cancer.2.Of all the three pancreatic cancer cell lines,down-regulation of METTL14 could inhibit the growth and metastasis abilities of pancreatic cancer cells.3.Overexpression of METTL14 in pancreatic cancer cell line could promote the growth and metastasis abilities of pancreatic cancer cells.4.For the subcutaneous transplant model of pancreatic cancer cells,down-regulation of METTL14 could inhibit the tumorigenicity of pancreatic cancer cells,while over-regulation of METTL14 could promote the tumorigenicity of pancreatic cancer cells,compared with the negative control group,the difference is significant.5.For the liver metastasis model by spleen injection of pancreatic cancer cells,down-regulation of METTL14 could inhibit the micro metastases of pancreatic cancer cells in the liver,while over-regulation of METTL14 could promote the micro metastases of pancreatic cancer cells,compared with the negative control group,the difference is significant.Conclusion:METTL14 promoted the growth and metastasis of pancreatic cancer cells.Part 4.The molecular mechanisms for the promotion of prolifatation and metastasis of pancreatic cancer cells by METTL14Objective:To research the mechanism underlying METTL14 promoting malignant biological behavior of pancreatic cancer.Methods:Using the second generation of sequencing technology to screen changes of signaling pathways and microRNAs after METTL14 konocking down,with bioinformatics method,to predict the microRNAs that targeted the changes of MAPK signaling,which METTL14 depended on.Results:1.Activation of MAPK signaling by METTL14 was denpendent on Rap 1B.2.METTL14 could regulate the expression of miR-1-3p,and Rap1B was the target gene of miR-1-3p.3.Blocking Rap1B could inhibit the activation of MAPK signaling by METTL14 and inhibit the promoting effect of growth and metastasis of pancreatic cancer cells by METTL14.Conclusion:METTL14 regulate growth and metastasis of pancreatic cancer cells through MAPK signaling by inhibiting the expression of miR-1-3p.