| Purpose Pancreatic cancer(PC)is an extremely invasive malignant tumor with hidden onset and rapid development.The 5-year overall survival rate is less than 10%.Pancreatic ductal adenocarcinoma(PDAC)is the most common histologic type of PC,accounting for more than 85%.The pathogenesis of PDAC is not clear,and the lack of specific tumor marker has brought great difficulties for the diagnosis and treatment of the disease.Therefore,it is of great significance for PDAC patients to explore the occurrence and development mechanism of PDAC and seek new tumor markers and targets.Autophagy is an important mechanism of cell quality control,which plays an important role in maintaining tissue homeostasis and resisting the occurrence and development of foreign pathogens and tumors.Autophagy has a dual effect on cells: On one hand,autophagy can remove damaged proteins and organelles then provide nutrients for cells;On the other hand,autophagy overactivated may cause the degradation of intracellular substances,which will lead to cell death.At present,more and more researches were focusing on the effect and mechanism of autophagy in tumor.Non-coding RNA(nc RNA)refers to the RNA that does not code protein.It has been confirmed that nc RNA can affect the occurrence and development of tumor by regulating the expression of some proteins andregulating different signal pathways.Micro RNA(mi RNA)is a conserved non-coding RNA withabout 22 nucleotides length.Mi RNA can regulate the expression of target genes through RNA-induced silencing complex(RISC),then participate in post-transcriptional regulation,mi RNA splicing and translation.Mi R-663a(also named mi R-663)had been reported in many malignant tumors.For example,mi R-663 a were reported as a potential tumor inhibitor in acute lymphoblastic leukemia,glioblastomas,colorectal cancer and thyroid cancer,while it played a role as a carcinogenic gene in nasopharyngeal carcinoma and breast cancer.In recent years,more and more mi RNAs have been found to be involved in the process of autophagy regulation.Because of the close relationship between autophagy and tumor,researchers began to pay attention to the role of mi RNA in autophagy.Mi RNA can inhibit tumor by influencing autophagy.Our previous microarray chips data showed that inhibiting autophagy can significantly down-regulated the expression level of mi R-663 a in PDAC.In the chip results,we selected the genes that were significantly upregulated after inhibiting autophagy,then intersected with the target genes of mi R-663 a predicted by the network bioinformatics database.Finally,IL2 RB were selected as the experimental target.We hypothesis that inhibiting mi R-663 a can inhibit autophagy and hinder the proliferation and migration of PDAC cells by targeting IL2 RB.It is hoped that this study can enrich the study of the relevant mechanism of PDAC and provide theoretical and experimental basis for finding better diagnosis and treatment methods.The experiment was divided into two parts:PartⅠ.Expression of mi R-663a/IL2 RB in PDAC and inhibitory effect of mi R-663 a on Proliferation and Migration of PDAC cellsMaterials and methods 1.Materials 1.1 Immunohistochemical experiment included PDAC paraffin wax samples diagnosed by the Department of Pathology,the first affiliated Hospital of Guangxi Medical University from 2012 to 2018,including 78 cases of cancer tissues and 36 cases of paracancerous tissues.Particularly,68 cases of cancer tissues and 36 cases of normal tissues from 2013 to 2018 were selected to evaluate the m RNA level of mi R-663 a and IL2 RB by RT-q PCR.1.2 PDAC cell lines: PANC-1,SW1990,MIA Pa Ca;human pancreatic ductal cell: Hped6-C7.2.Methods 2.1 Bioinformatics analysis: Chip data were downloaded from GEO,Array Express,TCGA and Oncomine databases,and the expression data of mi R-663 a and IL2 RB in PDAC were extrated.The expression mean and standard deviation of mi R-663 a and IL2 RB in PDAC and control group were calculated,meta-analysis were conducted,and funnel chart was used to evaluate publish bias.2.2 RT-q PCR was used to detect the expression of mi R-663 a and IL2 RB in cell lines and PDAC paraffin wax blocks,and the clinical parameters were analyzed.2.3 Immunohistochemical staining was used to detect the protein level of IL2 RB in PDAC and paracancerous pancreatic tissues.2.4 The targeting relationship between mi R-663 a and IL2 RB were verified by double luciferase experiment.The effects of inhibition of mi R-663 a on the proliferation and invasion of PDAC cells were detected by CCK-8 test,scratch test and transwell chamber migration test.Result 1.Expression of mi R-663 a in PDAC and its clinicopathological significance.Six tissue chips in GEO database were found to be involved in the expression of mi R-663 a,of which,4 chips suggested higher mi R-663 a expression level in PDAC,while the other 2 chips suggested lower expression level of mi R-638 in PDAC.The expression of mi R-663 a in cell lines was also detected by RT-q PCR.The results showed that mi R-663 a was highly expressed in PANC-1 and SW1990(P = 0.0379,P < 0.0251)but lowly expressed in MIA-PACA(P = 0.833).The expression of mi R-663 a in paraffin wax was detected by RT-q PCR.The results showed that mi R-663 a was highly expressed in PDAC(P =0.2895).The expression level of mi R-663 a was not significantly correlated with age,tumor growth site,tumor maximum diameter,history of smoking and drinking,TMN stage,lymph node metastasis and vascular infiltration,but correlated with sex(P= 0.038)and nerve infiltration(P = 0.028).2.Expression of IL2 RB in PDAC and its clinicopathological significance.Meta analysis showed that the expression of IL2 RB in PDAC was slightly lower than that in non-cancerous tissues(P=0.732).The integration of GEO,Array Express,TCGA and Oncomine microarray data showed that the expression of IL2 RB in PDAC was slightly higher than that in non-cancerous tissues.The expression of IL2 RB in cell lines was detected by RT-q PCR.The results showed that IL2 RB was low expressed in PANC-1 and SW1990(P = 0.0001,P = 0.0447),while high expressed in MIA-PACA(P = 0.0400).The expression of IL2 RB in paraffin samples detected by RT-q PCR showed that the expression of IL2 RB in PDAC tissues was lower than that in paracancerous tissues(P < 0.0788).The expression level of IL2 RB has no correlation with age,sex,maximum diameter of tumor,history of smoking and drinking,TMN stage,lymph node metastasis and neurovascular infiltration,but related to tumor growth site(P < 0.027).The results of immunohistochemistry showed that the expression of IL2 RB in PDAC was lower than that in paracancerous tissues(χ 2 = 27.40,P < 0.05).3.Prediction and verification of targeting relationship of mi R-663 a and IL2 RB.Target Scans Human 7.2 showed that there was binding sites between mi R-663 a and target gene IL2 RB,which was verified by double luciferase experiment.4.The effect of inhibiting mi R-663 a on the proliferation of PDAC cells.After the inhibition of mi R-663 a,the OD values were detected by cck8 proliferation assay.The results showed that the inhibition of mi R-663 a could inhibit the proliferation of PANC-1 cells,but there was no significantly difference between the experimental group and the blank group(P > 0.05).5.The effect of inhibiting mi R-663 a on migration ability of PDAC cells.The results of scratch test showed that the inhibition of mi R-663 a hindered the plane migration of PANC-1 cells(P < 0 05).The results of transwell migration test showed that the inhibition of mi R-663 a hindered the migration ability of PANC-1 cells(P < 0.05).Part II.The interaction between mi R-663 a and autophagy in PDAC cells.Materials and methods 1.Materials 1.1 PDAC cell lines: PANC-1,SW1990,MIA Pa Ca;human pancreatic ductal cell: Hped6-C7.1.2 Westtern Blot antibody: IL2RB;autophagy associated protein p62,ATG5,LC3 B.2.Methods 2.1 RT-QPCR was used to detect the expression of ATG5,LC3 B and p62 in cell lines and paraffin wax blocks.2.2 PANC-1 cells were treated with 100 μmol/L chloroquine to inhibit autophagy.After 24 hours later,the expressions of mi R-663 a and IL2 RB were detected by RT-q PCR.2.3 After the inhibition of mi R-663 a,the protein expression of ATG5,LC3 B and p62 was detected by Western Blot.The autophagy level was judged by the change of autophagy protein expression.Result.1.Expression of autophagy related genes in PDAC cell line.The results of RT-q PCR showed that the expression of ATG5 was up-regulated in PANC-1(P < 0.0330)and down-regulated in SW1990 and MIA-PACA(P < 0.0001).The expression of LC3 B was up-regulated in PANC-1 and MIA-PACA(P < 0.0001)and down-regulated in SW1990(P < 0.005).The expression of p62 was down-regulated in PANC-1,SW1990 and MIA-PACA(P =0.002,P = < 0.0001).RT-q PCR was also used to detect the expression of PDAC and autophagy-related genes in tissues.ATG5 and LC3 B were high expression in PDAC tissue,while p62 was low expression(P=>0.05).2.Effect of inhibiting autophagy on the expression of mi R-663 a.The results of RT-q PCR showed that the expression of mi R-663 in PDAC cells down-regulated after autophagy inhibition(P=<0.05).3.Effect of inhibition of autophagy on the expression of IL2 RB.The results of RT-q PCR showed that the expression of IL2 RB in PDAC cells up-regulated after autophagy inhibition(P=<0.05).The results of Western Blot showed that the expression of IL2 RB protein in PDAC cells was up-regulated after autophagy inhibition.4.Inhibitory effect of mi R-663 a on autophagy.The expression of autophagy associated protein ATG5,LC3 B and p62 was detected by Western Blot.The results showed that after inhibiting mi R-663 a,the expression of ATG protein was down-regulated,the expression of LC3 BI and p62 protein up-regulated,but there was no statistical significance(P > 0.05).Conclusion 1.After inhibition of autophagy,the expression of mi RNA-663 a in PDAC down-regulated and the expression of IL2 RB up-regulated,but there was no significantly change in autophagy protein after inhibition of mi R-663 a.Mi R-663 a did not regulate tumor by regulating autophagy in PDAC.2.Inhibition of mi RNA-663 a expression could inhibit the migration of PDAC cells.There is a direct regulatory targeting relationship between IL2 RB and mi RNA-663 a,and mi RNA-663 a binds to IL2 RB to inhibit its expression.Mi R-663 a may affect the occurrence and development of PDAC by regulating IL2 RB. |
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