| BackgroundPancreatic cancer is one of the leading causes of the death in cancer worldwide,with the main characteristics of difficult early diagnosis,high recurrence rate and low survival rate.Even patients have postoperative adjuvant chemotherapy,the survival rate of the patients was not significantly improved.At present,studies have reported that autophagy is closely related to cancer.The best combination of autophagy inhibition and other traditional therapies(chemotherapy or radiotherapy)in different tumor types and stages can be a successful way to improve the anti-cancer treatment.MiRNAs may play a role in the process of autophagy.However,the complex relationship and mechanism between them are still unclear.In the preliminary experiments of this project,we found that the expression of miR-154-3p was down-regulated and the expression of ERBB4 was up-regulated after the treatment of pancreatic cancer with chloroquine.It was also found there was a potential targeting relationship between miR-154-3p and ERBB4.The purpose of this study was to explore the effects of chloroquine and miR-154-3p on the growth of pancreatic cancer,and to provide new ideas for exploring the therapeutic method in pancreatic cancer.Methods1.Seventy-six paraffin-embedded tissues of pancreatic cancer and 39 adjacent tissues were collected from the department of pathology,the First Affiliated Hospital of Guangxi Medical University.Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the expressions of miR-154-3p,ERBB4 and autophagy-related genes ATG5,LC3 B and p62 in paraffin-embedded tissues.The data from public databases including GEO,TCGA,ArrayExpress and Oncomine were screened for integrated analysis of miR-154-3p,ERBB4 and autophagy-related genes expression in pancreatic cancer tissue.Immunohistochemical staining was conducted to detect the protein expression level of ERBB4 in pancreatic cancer and adjacent tissues.2.RT-PCR was used to detect the expressions of miR-154-3p,ERBB4 and autophagy-related genes in the pancreatic cancer cell lines of Panc-1 and SW1990.The expressions of miR-154-3p,ERBB4 and autophagy-related genes were detected again after chloroquine(CQ)treatment in pancreatic cancer cells to verify the chip results of previous experiments.Western blot(WB)was used to detect the expression of ERBB4 and autophagy-related genes in pancreatic cancer cells treated with CQ.At the same time,the proliferation,migration and apoptosis of pancreatic cancer cells were conducted after CQ treatment.3.The effect of miR-154-3p on the migration and invasion of pancreatic cancer cells was detected by wound healing assay and transwell assay.Double luciferase reporter assay verified the targeting relationship between miR-154-3p and ERBB4.Changes in ERBB4 expression after inhibition and overexpression of miR-154-3p were detected by RT-qPCR.Results1.Expression and significance of miR-154-3p/ERBB4 and autophagyrelated genes in pancreatic cancer.The average expression level of miR-154-3p in paraffin-embedded tissue of pancreatic cancer was higher than that in para-carcinoma tissue,while the average expression level of ERBB4 in paraffin-embedded tissue of pancreatic cancer was lower than that in para-carcinoma tissue.Autophagy related gene ATG5,LC3 B in paraffin-embedded tissue of pancreatic cancer have a high expression trend,while p62 has low expression trend.However,their differences were not statistically significant.The data of GEO,TCGA,ArrayExpress,Oncomine and RT-qPCR in this study were integrated for a meta-analysis,and it was found that the expression difference of miR-154-3p was not statistically significant.Meanwhile,the expression of ERBB4 was down-regulated,and the expression of autophagy related genes ATG5,LC3 B and p62 were up-regulated,with statistically significant differences.It is worth noting that in the GSE60980 gene chip,the expression of miR-154-3p was high in pancreatic cancer while ERBB4 was low expression,and the area under the ROC curve of both miR-154-3p and ERBB4 is more than 70%.Immunohistochemical results showed that ERBB4 protein was both negative and positive in pancreatic cancer tissues,and the difference was not statistically significant.2.Effects of chloroquine on miR-154-3p /ERBB4 and autophagy-related proteins and on proliferation and migration of pancreatic cancer cellsMiR-154-3p was high expression,while ERBB4 was low expression in Panc-1 cell lines.ATG5 and LC3 B were both high expression,and p62 was low expression in Panc-1 cell lines.The differences were all statistically significant.The expression changes of miR-154-3p and ERBB4 after CQ treatment of pancreatic cancer cells were consistent with the results of previous microarray,that is,miR-154-3p expression was down-regulated,and ERBB4 expression was up-regulated.We were also found ATG5,LC3 B and p62 expression were all down-regulated.In the detection of WB,ERBB4 protein level was also increased after CQ treatment.At the same time,the protein expression levels of LC3 B were decreased,while the protein expression level of p62 was increased.In addition,after the addition of CQ autophagy inhibitors to pancreatic cancer cells,the cell proliferation and migration ability were significantly reduced,while the apoptosis rate was increased.3.The effect of miR-154-3p on the migration and invasion of pancreatic cancer cells and the verification of the targeting relationship between miR-154-3p and ERBB4.After inhibiting the expression of miR-154-3p,the results of wound healing assay and transwell assay showed that the migration and invasion ability of pancreatic cancer cells was inhibited.The dual-luciferase report showed that miR-154-3p had a direct targeting relationship with ERBB4.ERBB4 expression was increased while miR-154-3p expression was inhibited in pancreatic cancer cells.ERBB4 expression was decreased after overexpression of miR-154-3p.ConclusionCQ may play an inhibitory role in pancreatic cancer.MiR-154-3p may play a role in promoting cancer in pancreatic cancer.CQ may function as cancer inhibitor through miR-154-3p targeting ERBB4. |
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