| Objective:Pancreatic cancer?PDAC?is a malignant tumor of the pancreatic exocrine gland,with a poor prognosis and a five-year survival rate of less than 6%,earning the title"king of cancer"[1].The onset of PDAC is insidious,with local infiltration and metastasis occurring early in the disease.At present,clinical screening is mainly conducted by detecting the content of Carbohydrate antigen19-9?CA19-9?in the blood of patients[2].However,due to the relatively low sensitivity of CA19-9,early diagnosis is difficult.In addition,during the treatment of pancreatic cancer,patients showed insensitivity to radiotherapy and easy to develop resistance to existing chemotherapy drugs,which led to high mortality of pancreatic cancer.In the United States,PDAC has become the third deadliest cancer in the world and is likely to surpass breast cancer as the second most deadly cancer by 2030[3,4].Statistics in the past 20 years show that the incidence of pancreatic cancer in China has increased by about 6 times,ranking the 6th among all fatal cancers[5].It was previously thought that the development of pancreatic cancer was influenced by environmental factors,personal habits and other health diseases.However,the pathogenesis of pancreatic cancer is limited.Therefore,an in-depth understanding of the pathogenesis of early pancreatic cancer.Finding the relevant molecular markers of pancreatic cancer is of great significance for the timely diagnosis,control and treatment of pancreatic cancer.In recent years,more and more studies have pointed out that Long non-coding RNA?lncRNA?plays an important role in the progression of pancreatic cancer.LncRNA is a kind of non-coding RNA with a length of more than 200nucleotides[6],which is an important regulatory factor in vivo.LncRNA is widely involved in various life processes in vivo and plays an important role in epigenetic regulation,cell cycle regulation and cell differentiation regulation and other life activities[7-11].Proposed by Karreth FA[12]competitive endogenous RNA?competitive,endogenous RNA ceRNA?hypothesis,namely the lncRNA can be used as ceRNA and competitive combination of micrornas,the combination of interference of micrornas with downstream target genes,regulation of gene expression,which involved in cell proliferation,differentiation,apoptosis,and angiogenesis,and other biological processes[13,14].At the same time a number of studies have shown that lncRNA was associated with the incidence of pancreatic cancer.For example,Sun et al.[15]found increased expression levels of lncRNA H19 and PTK1 in pancreatic cancer tissues in their study.Further studies showed that lncRNA H19 combined with mir-194competitively could up-regulate the expression level of PTK1,thus promoting the proliferation and migration of pancreatic cancer cells.Gao et al.[16]pointed out that lncRNA DLEU1 could regulate the expression level of CXCR4 and promote the deterioration of pancreatic cancer by binding to mir-381.In the study of Wang et al.[17],merritine induction was found to increase the expression level of lncRNA NONHSAT105177 in pancreatic cancer cells and inhibit the proliferation of pancreatic cancer cells.OIP5 gene was first discovered in 1988[18],the coding of protein structure and function of centromere form the necessary components.Several studies have shown that OIP5 is involved in the development of human gastric cancer,colon cancer and female acute myeloid leukemia[19-21].LncRNA OIP5-AS1 is the antisense transcription product encoding OIP5 gene and has been shown to play a pro-tumor role in a number of diseases.In the study of the pathogenesis of glioma,Liu et al.[22]found that the high expression of lncRNA OIP5-AS1 can enhance the proliferation and diffusion ability of glioma cells.The main mechanism is to promote the expression of CEBPA through competitive binding with miR-367-3p,so as to play a positive regulatory role in the occurrence and development of cancer.Zhang et al.[23]found in their study that lncRNA OIP5-AS1 inhibited the interference of miR-186a-5p on ZEB1 by binding to miR-186a-5p,and upregulated ZEB1 gene expression level,thus promoting the proliferation and differentiation of hepatoblastoma cells.LncRNA OIP5-AS1 is highly expressed in cervical cancer tissues and cells,and can be competitively combined with miR-143-3p to up-regulate the expression of ITGA6 and SMAD3,so as to enhance the proliferation and migration ability of cervical cancer cells and play a pro-cancer role[24,25].However,no studies have been reported on its role in pancreatic cancer.In the early stage,we found binding sites between lncRNA OIP5-AS1 and miR-342-3p through online bioinformatics software analysis.At the same time,studies have found that miRNAs arranged in specific gene cluster regions tend to have similar expression patterns during the development of malignant tumors[26],and multiple studies have confirmed that mi RNAs are involved in the development of tumors.It has been reported that miR-342-3p plays an important regulatory role in the progression of colorectal cancer[27],gallbladder cancer[28]and prostate cancer[29].In addition,the study of Permuth-Wey J et al.[30]found that the expression level of miR-342-3p was decreased in papillary myxoma tissue,the predecessor of PDAC.Therefore,we speculated that lncRNA OIP5-AS1 might play a role in promoting the development of pancreatic cancer through the adsorption of miR-342-3p.However,the mechanism of mir-342-3p in PDAC remains to be further explored.Studies have shown that the activity of the intracellular Phosphoinositide3-kinase/Protein kinase B?PI3K/AKT?signaling pathway and the sudden change of its upper and lower molecular expression patterns are closely related to the process of tumor occurrence[31].The activation of PI3K/AKT pathway can activate the expression of proto-oncogenes and thus participate in the process of tumor occurrence.For example,Zhao et al.[32]found that the expression level of mir-21 was positively correlated with the activation state of MAPK/ERK and PI3K/AKT signaling,and the increased expression level of mir-21 could activate the PI3K/AKT pathway,thus enhancing the amplification capacity of PDAC cells.Xiong et al.[33]pointed out that down-regulation of mir-107 could inhibit the activity of PI3K/AKT pathway and the expression levels of caveolin-1 and PTEN proteins,and inhibit the proliferation and invasion ability of PDAC cells.It is suggested that miRNA is closely related to the PI3K/AKT pathway.It is worth noting that the Liu,etc.[34]in the process of liver cancer research found that miR-342-3p PI3K/AKT/GLUT1 regulation of signaling pathways activated state,after the activation of PI3K/AKT/GLUT1signaling pathways can activate the downstream target genes,the sugar intake of liver cancer cells,and lower levels of glycolysis and ATP production,extracellular acidification rate,increase the oxygen consumption rate of liver cells,inhibiting liver cancer cell proliferation.Therefore,we speculated that there was also a regulatory relationship between mir-342-3p and the PI3K/AKT pathway in the pathogenesis of pancreatic cancer.Studies have shown that the activity of the intracellular Phosphoinositide3-kinase/Protein kinase B?PI3K/AKT?signaling pathway and the sudden change of its upper and lower molecular expression patterns are closely related to the process of tumor occurrence[31].The activation of PI3K/AKT pathway can activate the expression of proto-oncogenes and thus participate in the process of tumor occurrence.For example,Zhao et al.[32]found that the expression level of mir-21 was positively correlated with the activation state of MAPK/ERK and PI3K/AKT signaling,and the increased expression level of mir-21 could activate the PI3K/AKT pathway,thus enhancing the amplification capacity of PDAC cells.Xiong et al.[33]pointed out that down-regulation of mir-107 could inhibit the activity of PI3K/AKT pathway and the expression levels of caveolin-1 and PTEN proteins,and inhibit the proliferation and invasion ability of PDAC cells.It is suggested that miRNA is closely related to the PI3K/AKT pathway.It is worth noting that Liu et al.[34]found in the study of liver cancer that mir-342-3p can regulate the activation state of the PI3K/AKT/GLUT1 signaling pathway.The activated PI3K/AKT/GLUT1 signaling pathway can activate downstream target genes and reduce the sugar intake of liver cancer cells.In turn,the glycolysis level,ATP production and extracellular acidification rate are reduced.Increase the oxygen consumption rate in hepatocellular carcinoma cells and inhibit the proliferation of hepatocellular carcinoma cells.Therefore,we speculated that there was also a regulatory relationship between mir-342-3p and the PI3K/AKT pathway in the pathogenesis of pancreatic cancer.To sum up,we propose a reasonable scientific hypothesis that lncRNA OIP5-AS1 may play a role of ceRNA in the development of pancreatic cancer,regulating the expression level of miR-342-3p and thus playing a pro-cancer role.This study will investigate the regulatory relationship between lncRNA OIP5-AS1 and miR-342-3p and whether lncRNA OIP5-AS1 can affect the proliferation of pancreatic cancer cells by regulating miR-342-3p and its downstream signaling pathway,and preliminarily elucidate the relevant mechanism.Methods:From September 2015 to May 2018,28 pairs of cancer and paracancer specimens were collected from patients undergoing surgery for pancreatic cancer in Shengjing hospital affiliated to China medical university.These patients did not undergo chemoradiotherapy before surgery.All collected specimens were immediately placed in liquid nitrogen and stored at-80 degree refrigerator before RNA extraction.The research was approved by the ethics committee.The expressions of lncRNA OIP5-AS1 and miR-342-3p in pancreatic and paracancer samples were detected by quantitative real-time PCR.We analyzed the correlation between OIP5-AS1 and mi R-342-3p expression in pancreatic cancer and paracancer.The survival of high expression and low expression OIP5-AS1 in pancreatic cancer was analyzed by GEPIA database.In vitro,the pancreatic cancer cell lines ASPC-1 and PANC-1 with higher expression of OIP5-AS1 were selected for inhibition test.ASPC-1 and BXPC-3 pancreatic cancer cell lines with lower miR-342-3p expression were selected for overexpression test.CCK-8 was used to detect the proliferation changes of pancreatic cancer cells.Immunofluorescence was used to detect the expression of Ki67.Flow cytometry was used to detect the cell cycle changes of pancreatic cancer cells,and Western blot was used to detect the cyclinD1,CDK4 and CDK6 cyclins,to identify the effects of lncRNA OIP5-AS1 and miR-342-3p on the proliferation of pancreatic cancer cells.Wild-type lncRNA OIP5-AS1 and AGR2 seed sequence vectors containing miR-342-3p binding sites?wt-OIP5-AS1,wt-AGR2?and mutant OIP5-AS1 and AGR2 seed sequence vectors?mut-OIP5-AS1,mut-AGR2?were constructed and transfected with mir-342-3p mimic or NC mimic,respectively.The binding of miR-342-3p with OIP5-AS1 and AGR2 was detected by double luciferase assay.LncRNA OIP5-AS1 siRNA or NC si RNA were co-transfected with miR-342-3p inhibitor or NC inhibitor,respectively.CCK-8 was used to detect the activity of the transfected cells,and immunofluorescence was used to detect the expression of Ki67 in the transfected cells.Western blot analysis was performed to detect the expression of cyclinD1,CDK4,CDK6,AGR2,AKT,p-AKT?Ser473?,ERK1/2,p-ERK1/2?Thr202/Tyr204?after transfection to determine whether lncRNA OIP5-AS1 affects the proliferation of pancreatic cancer cells through miR-342-3p and its downstream signaling pathway.In vitro nude mice were divided into the control group,the NC shRNA group and the OIP5-AS1 shRNA group.At the end of the experiment,tumor bodies were taken and weighed.Expression of lncRNA OIP5-AS1 and miR-342-3p in tumor tissues were detected by fluorescence quantitative PCR.Western blot was used to detect the expression of cyclinD1,CDK4,CDK6,AGR2,AKT,p-AKT?Ser473?,ERK1/2,p-ERK1/2?Thr202/Tyr204?in tumor tissues,and to determine the effect of lncRNA OIP5-AS1 on the growth of pancreatic cancer in vivo.Results:The results of qRT-PCR showed that,compared with adjacent tissues,the expression level of lncRNA OIP5-AS1 in pancreatic cancer tissues was significantly up-regulated,while the expression level of miR-342-3p was significantly down-regulated.Appropriate cell lines were selected in vitro,lncRNA OIP5-AS1 was interfered with by siRNA or miR-342-3p was overexpressed by mimic.Cell proliferation was detected by CCK-8,expression of Ki67 was detected by immunofluorescence,cell cycle was detected by flow cytometry,and expression of cycle-related indicators was detected by Western blot.The results showed that interference with lncRNA OIP5-AS1 or overexpression of miR-342-3p could inhibit cell proliferation,reduce Ki67 expression,induce cell cycle arrest,and down-regulate the expression of cyclinD1,CDK4,and CDK6.In addition,lncRNA OIP5-AS1 could act as an adsorption sponge for miR-342-3p to inhibit its expression and function.The results showed that miR-342-3p inhibitor could effectively reverse the effect of interfering lncRNA OIP5-AS1 on the occurrence and development of pancreatic cancer.In addition,we also established a nude mouse tumor-forming animal model,and found that lncRNA OIP5-AS1 significantly inhibited tumor volume in tumors silenced by shRNA,and the expression level of lncRNA OIP5-AS1 in tumor tissues was down-regulated,while the expression level of miR-342-3p was up-regulated.Subsequently,the expression levels of Ki67 in tumor tissues were detected by immunohistochemistry,and the expression levels of AKT/ERK signaling pathway related molecules in cell cycle were detected by Western blot.The results showed that silencing lncRNA OIP5-AS1 significantly inhibited the expression levels of cyclinD1,CDK4,CDK6,AGR2,p-AKT and p-ERK in tumor tissues.Conclusions:It was preliminarily confirmed that lncRNA OIP5-AS1 plays a pro-cancer role in pancreatic cancer.Moreover,interference with lncRNA OIP5-AS1can improve the inhibition of AGR2 by miR-342-3p,thus inhibiting the activation of AKT/ERK signaling pathway.Therefore,it can inhibit the occurrence and development of pancreatic cancer. |
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