The Function And Mechanism Of MiR-29c In The Development Of Pancreatic Cancer

Pancreatic cancer(PC)is a kind of digestive cancer with extremely high malignancy and poor prognosis,and about 90% of the cases of PC are pancreatic adenocarcinoma(PDAC).Of the 18 million cases diagnosed globally in 2018,about 500,000 were PC cases.In addition,all forms of PC had the lowest survival rate of all cancer types.While significant progress has been made in the development of new cancer therapies,survival rates for PC have not improved over the past 40 years.The root cause is the difficulty of early diagnosis of PC and the limitations of late treatment.In recent years,the emergence of drug resistance in advanced chemotherapy agents for PC has become another major difficulty in PC treatment.There is therefore an urgent need for early detection and treatment to improve survival and to better understand its pathogenesis to help create effective treatments.Micro RNA(mi RNA)is a tiny non-coding RNA that regulates the expression of a variety of proteins during post-translation.Recent studies have shown that abnormally expressed mirnas in PC are related to diagnosis,guidance of optimal treatment and prediction of therapeutic response,so they are expected to be used as diagnostic and prognostic markers and potential therapeutic targets for PC.Different mi RNA expression profiles can be correlated with the staging of PC and have potential as biomarkers,prognostic markers and clinical targets.However,limited understanding and validation of the specific role of this mi RNA has hindered clinical applications.Targeted gene prediction using algorithmic tools provides a wide range of possible targets,but these mirnas still need to be validated through preclinical studies to determine the linkage genetic effect.Therefore,understanding the main effector genes and downstream pathways in PC can identify possible therapeutic candidates for mirnas and find more effective and safe therapeutic strategies for PC.In this study,bioinformatics techniques were used to screen out key mirnas involved in the occurrence and development of PC,and mi R-29 c was identified as the key mi RNA.The influence of mi R-29 c on the development of PC was further studied.The bioinformatics tool was then used to predict possible downstream targets and pathways for mi R-29c: the MAPK pathway regulated by MAPK1.QRT-PCR,Western Blot,double luciferase reporter gene assay,and immunohistochemical staining were used to determine whether the MAPK signaling pathway promotes the occurrence and development of PC by mediating the mi R-29c-MAPK1 axis.Exploring the role of mi R-29c/MAPK1 in PC proliferation could help shed light on the pathogenesis of PC and provide a basis and direction for the search for new biomarkers and therapeutic targets for PC.Part One The key mi RNAs involved in the occurrence and development of pancreatic cancer were screened based on GEO and TCGA databasesObjective: Objectives: PC is a digestive tumor with an extremely high degree of malignancy and a poor prognosis,and the pathological type in nearly 90% of PC cases is PDAC.Related genes are involved in the occurrence and development of PC,and the specific regulatory mechanisms are still unclear.Studies have shown that mi RNA plays an important role in tumor development and regulation.In this study,the GEO and TCGA databases were used to search for key mi RNAs involved in PC development.Methods:1.The GEO database was used to retrieve the pancreatic cancer expression profile data set,and differential analysis was performed to screen pancreatic cancer-related mi RNAs.2.Receiver operating characteristic(ROC)curve analysis was performed on the selected pancreatic cancer related mi RNAs to further screen the mi RNAs that can better distinguish the normal control and pancreatic cancer.3.The expression and prognosis of pancreatic cancer-related mi RNAs screened by the GEO database in the TCGA database were analyzed using the UALCAN website.Results:1.The pancreatic cancer expression profile dataset and differential analysis results were searched through the GEO databaseThree pancreatic cancer expression profiling datasets(GSE24279,GSE28955 and GSE53325)were retrieved.Seven mi RNAs(hsa-mi R-29 c,hsa-mi R-107,hsa-mi R-324-3p,hsa-mi R-375,hsa-mi R-210,hsa-mi R-623 and hsa-mi R-765)were obtained by intersection analysis of three pancreatic cancer expression datasets.2.The receiver operating characteristic(ROC)curve of the screened mi RNAs related to pancreatic cancer was analyzed.Among the 7 mi RNAs,hsa-mi R-29 c,hsa-mi R-107,hsa-mi R-324-3p,hsa-mi R-375 and hsa-mi R-210 were able to distinguish between normal control and pancreatic cancer(AUC > 0.80).3.The expression and prognosis of these 5 mi RNAs in TCGA database were analyzed by UALCAN website.hsa-mi R-29 c and hsa-mi R-324-3p were lowly expressed,while hsa-mi R-375,hsa-mi R-107 and hsa-mi R-210 were extremely expressed in pancreatic cancer tissues.Combined with the results of GEO database microarray,only hsa-mi R-29 c,hsa-mi R-107 and hsa-mi R-210 showed the same trend in pancreatic cancer tissues.Only mi R-29 c,mi R-375 and mi R-324-3p were associated with the prognosis of pancreatic cancer patients.Furthermore,combining the expression levels of these five mi RNAs in pancreatic cancer tissue,we found that only mi R-29 c was down-regulated in pancreatic cancer tissue,and that pancreatic cancer patients with excessive mi R-29 c expression had a relatively better prognosis.Therefore,mi R-29 c was selected as the target gene for subsequent studies.Summary:1.7mi RNAs(hsa-mi R-29 c,hsa-mi R-107,hsa-mi R-324-3p,hsa-mi R-375,hsa-mi R-210,hsa-mi R-623 and hsa-mi R-765)were differentially expressed in pancreatic cancer.2.5mi RNAs(hsa-mi R-29 c,hsa-mi R-107,hsa-mi R-324-3p,hsa-mi R-375 and hsa-mi R-210)could distinguish pancreatic cancer from normal control.3.1 mi RNA: mi R-29 c was low expressed in pancreatic cancer tissues,and the prognosis of pancreatic cancer patients with high expression of mi R-29 c was relatively good.Part two Effects of mi R-29 c on proliferation,migration,invasion,apoptosis and tumorigenic ability of pancreatic cancer cells in nude miceObjective: q RT-PCR was employed to assess the expression levels of mi R-29 c in human pancreatic cancer tissues and corresponding adjacent normal tissues,as well as in human pancreatic cancer cell lines(As PC-1,PANC-1,Bx PC-3,Hs766 T and CFPAC-1)and human normal pancreatic ductal epithelial cell lines.This approach confirmed mi R-29 c as a differentially expressed gene in pancreatic cancer.The effect of mi R-29 c on the occurrence and development of pancreatic cancer was verified by constructing transfected cell lines and tumor formation experiment in nude mice.Methods:1.q RT-PCR was employed to quantify the expression of mi R-29 c in human pancreatic cancer tissues and their corresponding adjacent normal tissues.2.Kaplan-Meier method was used to analyze the correlation between mi R-29 c expression and overall survival(OS)of pancreatic cancer patients.3.The expression levels of mi R-29 c were quantified in five human pancreatic cancer cell lines(As PC-1,PANC-1,Bx PC-3,Hs766 T and CFPAC-1)as well as a normal human pancreatic ductal epithelial cell line HPDE using q RT-PCR.The two cell lines with the highest and lowest expression levels were selected for mi R-29 c inhibition and overexpression experiments.4.Following cell transfection,the expression levels of mi R-29 c were assessed in two pancreatic cancer cell lines using q RT-PCR.5.MTS assay was used to detect the proliferation ability of As PC-1 and PANC-1 before and after mi R-29 c was interfered.6.Transwell assay was used to detect the invasion and migration ability of pancreatic cancer As PC-1 and PANC-1 cells before and after mi R-29 c expression was interfered.Flow cytometry was used to analyze the effect of mi R-29 c expression on cell cycle and apoptosis of pancreatic cancer cells.7.The mi R-29 c overexpression As PC-1 cell line was constructed by mi R-29 c Agomir,and the expression level of mi R-29 c in normal and mi R-29 c Agomir transfected As PC-1 cell lines was detected by q RT-PCR.8.The impact of mi R-29 c on the tumorigenic potential of pancreatic cancer cells in nude mice was evaluated.Results:1.Expression level of mi R-29 c in human pancreatic cancer tissues and corresponding adjacent normal tissuesThe expression level of mi R-29 c in pancreatic cancer tissues was significantly lower than that in adjacent normal tissues(P<0.001).2.Correlation between mi R-29 c expression and survival of pancreatic cancer patientsThe OS of pancreatic cancer patients in mi R-29 c high expression group was significantly higher than that in mi R-29 c low expression group(P=0.0143).3.To assess the expression levels of mi R-29 c in five human pancreatic cancer cell lines(As PC-1,PANC-1,Bx PC-3,Hs766 T and CFPAC-1)as well as a normal human pancreatic ductal epithelial cell line HPDE.Compared to the human normal pancreatic ductal epithelial cell line HPDE,the expression of mi R-29 c was significantly lower in five pancreatic cancer cell lines(P<0.001).Among them,As PC-1 cells exhibited the lowest level of mi R-29 c expression while PANC1 cells showed the highest.Therefore,As PC-1 and PANC1 were selected for mi R-29 c inhibition and overexpression experiments.4.Expression levels of mi R-29 c in two pancreatic cancer cell lines transfected with mi R-29 c inhibition and overexpressionCompared with mimic-NC group,the expression of mi R-29 c was significantly increased after transfection with mi R-29 c mimic.Compared with inhibitor-NC,mi R-29 c expression was significantly decreased after transfection with mi R-29 c inhibitor.5.mi R-29 c is involved in regulating the proliferation of As PC-1 and PANC1 cell linesThe mi R-29c-inhibited and overexpressed As PC-1 and PANC1 cell lines were successfully established.Compared with the transfected inhibitor-NC group,the OD value of the mi R-29 c inhibitor group began to increase at 24 h,and statistically significant differences were observed at 72 h and 96 h.Compared with the mimic-NC group,the OD value of the mi R-29 c mimic group began to decrease at 48 hours,and the difference was statistically significant at 72 hours and 96 hours.6.mi R-29 c is involved in regulating the migration and invasion of As PC-1 and PANC1 cell linesCompared to the inhibitor-NC group,down-regulation of mi R-29 c expression in the mi R-29 c inhibitor group significantly enhanced the migration and invasion abilities of pancreatic cancer cells.Conversely,up-regulation of mi R-29 c expression in the mi R-29 c mimic group significantly inhibited these abilities compared to the mimic-NC group.Compared with mimic-NC group,up-regulation of mi R-29 c expression in mi R-29 c mimic group significantly inhibited the invasion ability of pancreatic cancer cells.7.mi R-29 c is involved in the regulation of cell cycle and apoptosis in As PC-1 and PANC1 cell linesCompared to the inhibitor-NC group,the mi R-29 c inhibitor group exhibited a significant decrease in the proportion of cells in G0/G1 phase and an increase in S phase for both As PC-1 and PANC-1 cell lines.Conversely,compared to mimic-NC,the mi R-29 c mimic group showed a significant increase in G0/G1 phase cells and a decrease in S phase cells.These results suggest that mi R-29 c may slow down the cell cycle by inhibiting DNA replication in S phase.Compared with mimic-NC cells,the proportion of apoptotic cells was significantly increased after overexpressing mi R-29 c in As PC-1 and PANC-1 cells.Compared with NC cells,the proportion of apoptotic cells was significantly decreased after the reduction of mi R-29 c by As PC-1 and PANC-1.These results suggest that mi R-29 c can promote the apoptosis of pancreatic cancer cells.8.Overexpression of mi R-29 c inhibited the tumorigenic ability of pancreatic cancer cells in nude miceCompared to the Agomir NC group,the transplanted tumor in the mi R-29 c Agomir group exhibited slower growth and significantly smaller volume than that of the control group.After 30 days,mice were euthanized to extract tumors and their weights were measured.Results showed that tumors in the mi R-29 c Agomir group were significantly smaller than those in the Agomir NC group(P<0.05).In addition,the expression level of proliferation index Ki-67 was detected by immunohistochemical staining.Our experimental results showed that the expression of Ki-67 in the mi R-29 c Agomir group was lower than that in the Agomir NC group to varying degrees(P<0.05).Summary:1.The expression level of mi R-29 c was significantly lower in pancreatic cancer tissues and cell lines compared to corresponding adjacent normal tissues and cell lines.Patients with high mi R-29 c expression had a significantly higher overall survival rate than those with low mi R-29 c expression.Low mi R-29 c expression is associated with poor prognosis in patients with pancreatic cancer,suggesting that it may be a key differential gene affecting patient outcomes.2.The overexpression of mi R-29 c impedes the proliferation,migration,and invasion while promoting apoptosis in pancreatic cancer cells.3.Overexpression of mi R-29 c inhibited the tumorigenic ability of pancreatic cancer cells in nude mice.Part Three mi R-29 c inhibits the activation of ERK/MAPK signaling pathway by targeting MAPK1 to prevent the growth and metastasis of pancreatic cancerObjective: Bioinformatics tools and in vitro and in vivo experiments were used to further clarify the molecular mechanism of mi R-29 c affecting the biological characteristics of pancreatic cancer cells.Methods:1.Star Base was used to find the downstream genes of mi R-29 c,and GEPIA website was used to obtain the highly expressed genes and prognosis related genes in TCGA database.Intersection genes were obtained through the jvenn website.2.Functional analysis of intersection genes was performed through KEGG website to predict key downstream genes and signaling pathways.The Star Base database was used to predict the possible targets.3.The expression of key downstream genes in pancreatic cancer was analyzed by UALCAN website.4.Total RNA was extracted from 42 pairs of pancreatic cancer tissues and corresponding adjacent normal tissues,and pancreatic cancer cell lines were cultured.q RT-PCR was used to detect the expression of key downstream genes in pancreatic cancer tissues and corresponding adjacent normal tissues,and Western blot was used to detect the expression of MAPK1 in pancreatic cancer cell lines.5.Spearman correlation analysis was used to verify the correlation between mi R-29 c expression and MAPK1 expression in pancreatic cancer cells.6.q RT-PCR and Western blot were used to detect the effect of mi R-29 c on MAPK1 m RNA and protein expression.7.Plasmid of MAPK1 3’UTR-wild type and MAPK1 3’UTR-mutant type were constructed.Dual luciferase reporter assay was used to verify that mi R-29 c regulated MAPK1 by targeting the 3’UTR region in As PC-1 and PANC-1 cells.8.MTS assay,Transwell assay and flow cytometry were used to detect the effect of MAPK1 knockdown on the proliferation,migration,invasion,cell cycle and apoptosis of pancreatic cancer cells.9.As PC-1 and PANC-1 cells were transfected with MAPK1 interfering lentivirus,and Western blot was used to observe the effect of MAPK1 knockdown on the tumorigenic ability of pancreatic cancer cells.10.The effect of MAPK1 knockdown on the tumorigenic ability of pancreatic cancer cells in vivo was further analyzed by knocking down MAPK1 in tumor-bearing nude mice.11.Western blot was used to further investigate whether MAPK signaling pathway was involved in mi R-29c/ MAPK1-mediated growth and apoptosis of pancreatic cancer cells.The effect of MAPK1-RNAi on the biological characteristics of pancreatic cancer cells was investigated by in vitro cell function experiments.Immunohistochemical staining of MAPK1,ERK,Bcl-2,Caspase-3 and Ki-67 in human pancreatic cancer tissues was performed to further determine whether MAPK signaling pathway promotes the occurrence and development of pancreatic cancer by mediating mi R-29c-MAPK1 axis.Results:1.MAPK1 may be a downstream gene of mi R-29 c and has a targetFourteen intersection genes(KCNN4,S100A16,MYEOV,ANXA2,PLCD3,EFNB2,TPX2,PYGB,ARNTL2,HTATIP2,TMOD3,VASH1,RAPH1 and MAPK1)were obtained.Functional analysis showed that MAPK signaling pathway was dominant in pancreatic cancer,and only MAPK1 among these 14 genes regulated MAPK signaling pathway.Star Base database predicted that there was a 3’UTR region of MAPK1 that was the target of mi R-29 c.UALCAN analysis showed that MAPK1 was highly expressed in pancreatic cancer tissues,and high expression of MAPK1 was associated with poor prognosis of pancreatic cancer patients.2.q RT-PCR was used to detect MAPK1 expression in pancreatic cancer tissues and corresponding adjacent normal tissues,and Western blot was used to detect MAPK1 expression in pancreatic cancer cell linesThe expression level of MAPK1 in pancreatic cancer tissues was significantly higher than that in the corresponding adjacent normal tissues(P<0.05),and MAPK1 was highly expressed in As PC-1 cells(P<0.001).3.Correlation between mi R-29 c expression and MAPK1 expression in pancreatic cancer cellsThere was a significant negative correlation between mi R-29 c and MAPK1 expression in pancreatic cancer cells(r=-0.5765,P<0.0001).4.q RT-PCR and Western blot were used to detect the effect of mi R-29 c on the m RNA and protein expression of MAPK1Overexpression of mi R-29 c significantly decreased the m RNA and protein levels of MAPK1,while knockdown of mi R-29 c significantly increased the m RNA and protein levels of MAPK1(P<0.05).5.Dual luciferase reporter assay was used to test the effect of mi R-29 c on MAPK1 by targeting the 3’UTR region of MAPK1Co-transfection of MAPK1 3’UTR-WT and mi R-29 c mimic significantly reduced the luciferase activity compared with co-transfection of mimi-NC(P< 0.05),while co-transfection of MAPK1 3’UTR-MUT did not change the expression of Ki67 protein in the xenograft in nude mice.6.Knockdown of MAPK1 inhibited the tumorigenicity,proliferation,migration,cell cycle,and increased apoptosis of pancreatic cancer cells.MTS assay was used to detect the effect of MAPK1 on the proliferation of pancreatic cancer cells.Compared with the transfected NC group,the OD value of si-MAPK1-1 group began to decrease at 48 h,and the difference was statistically significant at 48 h,72h and 96h(P<0.05).Transwell assay was used to detect the effect of MAPK1 on the migration and invasion of pancreatic cancer cells.Compared with the NC group,the down-regulation of MAPK1 expression in the si-MAPK1-1 group significantly inhibited the migration of pancreatic cancer cells(P<0.05).Compared with the NC group,the down-regulation of MAPK1 expression in the si-MAPK1-1 group significantly inhibited the invasion ability of pancreatic cancer cells(P<0.05).Flow cytometry was used to analyze the effect of MAPK1 on cell cycle and apoptosis of pancreatic cancer cells.Compared with NC,the proportion of cells in G0/G1 phase in si-MAPK1-1 group was significantly increased,while the proportion of S phase cells was decreased(P<0.05).These results suggest that MAPK1 may accelerate the cell cycle by promoting DNA replication in S phase.Compared with NC group,the apoptotic cells in As PC-1 and PANC-1were significantly increased after MAPK1 expression was reduced,suggesting that MAPK1 could inhibit the apoptosis of PC cells(P<0.05).7.Effect of MAPK1 knockdown on the tumorigenic ability of pancreatic cancer cells xenografts and pancreatic cancer cells in nude miceCompared with the control vector group,MAPK1 protein expression was significantly down-regulated in the MAPK1 interference lentivirus transfection group(Fig.6-G).The results of in vitro cell proliferation assay showed that the proliferation ability of As PC-1 and PANC-1 cells was significantly inhibited in MAPK1-Rnai group,suggesting that MAPK1 knockdown can inhibit the proliferation of pancreatic cancer cells in vitro.The tumor growth rate and volume of MAPK1-RNAi group were significantly lower than those of the control group.After 30 days,the nude mice were sacrificed,and the tumors were dissected out and weighed.The results showed that the tumors in the MAPK1-RNAi group were significantly smaller than those in the control group(P<0.05).8.mi R-29 c inhibits the growth of pancreatic cancer cells by inhibiting the ERK/MAPK signaling pathwayThe Western blot results demonstrated that inhibition of mi R-29 c activated the MAPK signaling pathway,resulting in an up-regulation of p-ERK,p-MEK and Bcl-2 expression.Furthermore,the promotion effect of mi R-29 c inhibitor on G1/S phase transition was found to be inhibited by MAPK1-RNAi(P<0.05).In vitro cell function experiment showed that the proliferation ability of cells with mi R-29 c inhibitor expression was enhanced,but MAPK1-RNAi alleviated the enhancement of cell proliferation ability induced by mi R-29 c inhibitor expression to a certain extent(P<0.05).These results indicated that MAPK1-RNAi could reverse the growth promotion effect of mi R-29 c inhibitor on pancreatic cancer cells.The results of immunohistochemical staining for MAPK1,ERK,Bcl-2,Caspase-3 and Ki-67 in human pancreatic cancer tissues showed that the expression of mi R-29 c was negatively correlated with the expression of MAPK1,ERK,Ki67 and Bcl-2,and positively correlated with the expression of Caspase3 protein.The findings suggest that mi R-29 c suppresses the proliferation of pancreatic cancer cells by inhibiting the ERK/MAPK signaling pathway.Summary:1.The expression of MAPK1 was significantly upregulated in pancreatic cancer tissues compared to adjacent normal tissues,and this upregulation was found to be negatively associated with the expression of mi R-29 c.Furthermore,we identified a 3’UTR region on MAPK1 that serves as a target for mi R-29 c.2.MAPK1 3’-UTR-wild type and mutant plasmids were constructed,and it was successfully verified that mi R-29 c regulated MAPK1 by targeting the3’-UTR region of MAPK1.3.The stable inhibition of MAPK1 was successfully achieved in the pancreatic cancer cell lines As PC-1 and PANC-1,resulting in a significant reduction of their tumorigenic potential in nude mice.4.mi R-29 c suppresses the proliferation of pancreatic cancer cells by impeding the ERK/MAPK signaling pathway.Conclusions:1.mi R-29 c may be a key differential gene affecting the prognosis of pancreatic cancer patients.2.Overexpression of mi R-29 c or knockdown of MAPK1 suppressed the proliferation,migration and invasion while promoting apoptosis in pancreatic cancer cells.3.Overexpression of mi R-29 c or MAPK1 knockdown inhibited the tumorigenic ability of pancreatic cancer cells in nude mice.4.mi R-29 c can target MAPK1 to inhibit the activation of ERK/MAPK signaling pathway,and ultimately prevent the occurrence and development of pancreatic cancer.