Study On The Mechanism Of LINC01133 Promoting The Development Of Pancreatic Cancer By Targeting MiR-126a-3p/ITGA6 Axis

Background:Pancreatic cancer is a highly malignant tumor of digestive system,and and one of the most poorly prognosed solid malignancies.Its morbidity and mortality are increasing year by year in China and worldwide,posing a serious risk to human health.Currently,the main treatment for pancreatic cancer is still surgery.Radical surgical resection combined with postoperative adjuvant therapy has prolonged the survival of pancreatic cancer patients to some extent.Unfortunately,it is the majority of patients who have lost the chance of surgery at the time of diagnosis,and only a minority(about 15-20%)of patients can undergo radical surgery.For unresectable pancreatic cancer,systemic chemotherapy is the main treatment,but the results are not satisfactory.Immunotherapy may be the future hope for pancreatic cancer treatment,and numerous research efforts are focusing on modulating the pancreatic tumor microenvironment to improve the efficacy of immunotherapeutic strategies.Long non coding RNAs(Lnc RNAs)are a special class of non coding RNAs(nc RNAs)that regulate gene expression through various mechanisms.Lnc RNAs contain micro RNA(miRNA)reaction elements that can act as miRNA "sponges" to competitively bind specific miRNAs to target m RNA.Lnc RNA-miRNA-m RNA competitive endogenous RNA(ce RNA)networks are a hot topic in oncology research today.In the early stage,the research team screened Lnc RNAs differentially expressed in pancreatic cancer through bioinformatics analysis and high-throughput sequencing,then found that the expression level of LINC01133 was significantly up-regulated in pancreatic cancer based on cell line research.Through bioinformatics prediction,it was found that there might be an interaction between LINC01133 and miR-126a-3p,and there was a binding site between them.There is no report on the interaction between LINC01133 and miR-126a-3p in pancreatic cancer in the literature.ITGA6 is an integrin subunit.Previous studies have shown that ITGA6 participates in various physiological and pathological processes in the human body,and plays an important regulatory role in various tumors.However,the regulatory mechanism of ITGA6 in pancreatic cancer is still unclear.ITGA6 is an integrin subunit,which has been shown to be involved in various physiopathological processes in humans and plays an important regulatory role in various tumors.However,the regulatory mechanism of ITGA6 in pancreatic cancer is still unclear.We have predicted and screened the possible target genes of miR-126a-3p by prebiotic analysis,the results showed that ITGA6 has a binding site with miR-126a-3p,and the expression of both is negatively correlated,suggesting that ITGA6 is a potential target gene of miR-126a-3p.Based on the above understanding,our research team proposed a hypothesis:LINC01133 targets miR-126a-3p and upregulates the expression of ITGA6 in tumor cells,thus promoting the occurrence and development of pancreatic cancer.This study intends to experimentally validate this hypothesis through a series of in vivo and ex vivo experiments at three levels: tissue,cellular and animal.Objective:To verify the existence of LINC01133-miR-126a-3p-ITGA6 regulatory axis,and explore the role of this regulatory axis in the occurrence and development of pancreatic cancer.Methods:(1)The expression of LINC01133 in clinical pancreatic cancer tissues(50 pairs)and pancreatic cancer cells(Panc-1,Bx PC-3,Mia Ca Pa,SW1990 and Pa Tu8988)was detected by quantitative real-time polymerase chain reaction(qRT-PCR).The relationship between LINC01133 expression and clinicopathological parameters of pancreatic cancer patients was also analyzed.In addition,the relationship between the expression of LINC01133 and the prognosis of pancreatic cancer patients was confirmed by Kaplan-Meier curve.(2)Based on the expression level of LINC01133 in the above five cell lines,we selected intermediate expressing Panc-1 cells and SW1990 cells as experimental objects,and used lentivirus transfection to overexpress LINC01133 in Panc-1 cells and knock down LINC01133 expression in SW1990 cells,respectively,to construct stable transfected cell lines.CCK8,scratch test and Transwell test were used to observe the effects of LINC01133 on the proliferation,migration and invasion of pancreatic cancer cells.Cell cycle and apoptosis were detected by flow cytometry.Western blot(WB)was used to detect the expression of apoptosis related proteins(Bax,Bcl-2,Caspase-3).(3)To explore the upstream and downstream regulatory relationship between LINC01133 and miR-126a-3p.Firstly,qRT-PCR was used to detect the expression of miR-126a-3p in the above pancreatic cancer tissues and cell lines,and Spearman method was used to analyze the correlation between LINC01133 and miR-126a-3p in pancreatic cancer.Then we explored the role of miR-126a-3p in pancreatic cancer,and observed the changes of biological behavior of pancreatic cancer cells by up regulating and down regulating the expression level of miR-126a-3p in pancreatic cancer cell lines(up regulating the use of Panc-1 cells,down regulating the use of SW1990 cells).Bioinformatics tools include Targetscan,NCORI database and Snapgene software were used to predict the binding site of LINC01133 and miR-126a-3p.The dual luciferase experiment verified the regulatory relationship between LINC01133 and miR-126a-3p.The expression of LINC01133 in pancreatic cancer cells was up-regulated and down-regulated,and the expression of miR-126a-3p was observed.Then,a recovery experiment was designed to downregulate the expression of miR-126a-3p while knocking down LINC01133;Overexpression of LINC01133 and up regulation of miR-126a-3p expression to observe the effect on proliferation,migration and invasion of pancreatic cancer cells.(4)To explore the molecular biological mechanism of LINC01133-miR-126a-3p axis inducing malignant biological behavior of pancreatic cancer,and to clarify the existence of LINC01133-miR-126a-3p-ITGA6 molecular regulatory axis in pancreatic cancer.GEPIA database was used to analyze the expression of ITGA6 in pancreatic cancer tissues.qRT-PCR was used to detect the expression of ITGA6 in the above pancreatic cancer cell lines and 50 pairs of pancreatic cancer and corresponding adjacent tissues,and Spearman was used to analyze the correlation between ITGA6 and the expression levels of LINC01133 and miR-126a-3p in pancreatic cancer.Bioinformatics was used to predict the binding targets of miR-126a-3p and ITGA6,and the regulatory relationship between miR-126a-3p and ITGA6 was verified by double luciferase experiment.The expression of ITGA6 in pancreatic cancer cells was detected after up regulating and down regulating the expression of LINC01133.The expression level of ITGA6 was detected after overexpression and inhibition of miR-126a-3p expression in pancreatic cancer.Subsequently,the expression of LINC01133 was upregulated or co transfected with miR-126a-3p mimic,while the expression of LINC01133 was downregulated or co transfected with miR-126a-3p inhibitor.The expression level of ITGA6 in each group of cells was detected.Finally,a nude mouse model of subcutaneously transplanted pancreatic cancer was constructed.The expression of LINC01133 was up-regulated and knocked down in vivo(Panc-1 cells were up-regulated and SW1990 cells were knocked down).The growth curve of transplanted tumors in each group was calculated and weighed,and the tumor volume was calculated.Routine pathological examination was performed on the tumor tissue.TUNEL was used to detect tumor cell apoptosis.qRT-PCR was used to detect the expression level of each molecule in the regulatory axis of LINC01133-miR-126a-3p-ITGA6.Detection of the expression of Bax,Bcl-2,Caspase-3,and Ki67 in transplanted tumors in each group by IHC.Results:(1)Compared with normal pancreatic duct epithelial cells(h TERT-HPNE),the expression level of LINC01133 in pancreatic cancer cell lines was significantly higher(P<0.05).The expression level of LINC01133 in pancreatic cancer tissue was also significantly higher than that in normal adjacent pancreatic tissue(P<0.05).In addition,the expression level of LINC01133 in tumor tissue was related to tumor differentiation(P=0.037),T stage(P=0.041),lymph node metastasis(P=0.021),and TNM stage(P=0.047)in pancreatic cancer patients,but not to gender(P=0.777),age(P=0.395),and neural invasion(P=0.390).Kaplan Meier survival analysis curve confirmed that the overall survival rate of pancreatic cancer patients in the LINC01133 overexpression group was significantly lower than that in the low expression group(P=0.022)..(2)Upregulation of LINC01133 significantly promoted the proliferation,migration,and invasive ability of Panc-1 cells(P<0.05),induced an increase in S phase cells,a decrease in G0/G1 phase cells,and a decrease in apoptosis;However,knockdown of LINC01133 significantly inhibited the proliferation,migration,and invasion of SW1990 cells,resulting in a decrease in S phase cells,an increase in G0/G1 phase cells,and an increase in apoptosis.In addition,after upregulation of LINC01133,the expression of Bcl-2 increased,while the expression of Bax and cleaved Caspase-3 decreased,while the expression of pro Caspase-3 protein did not change significantly;After down-regulation of LINC01133,the expression of Bcl-2decreased,and the expression of Bax and cleaved Caspase-3 increased,while the expression of pro Caspase-3 protein did not change significantly.(3)miR-126a-3p was underexpressed in pancreatic cancer cell lines and tissues,and its expression level was negatively correlated with LINC01133(P<0.05).Upregulation of the expression of miR-126a-3p in Panc-1 will reduce the proliferation,migration and invasion of Panc-1,increase apoptosis,reduce the proportion of S phase cells,and increase the number of G0/G1 phase cells.Downregulation of miR-126a-3p resulted in increased proliferation,migration,and invasion of SW1990 cells,decreased apoptosis,increased proportion of S phase cells,and decreased G0/G1 phase cells.The opposite effect.Double luciferase gene reporting experiments confirmed that LINC01133 can target and regulate miR-126a-3p.The results of the recovery experiment showed that the effects of overexpression of LINC01133 on the proliferation,migration,invasion,apoptosis and cell cycle of pancreatic cancer cells were reversed by the upregulation of miR-126a-3p(P<0.05).Similarly,down-regulation of miR-126a-3p can also reverse the change of malignant phenotype of pancreatic cancer caused by LINC01133 knockout.(4)GEPIA database showed that ITGA6 was highly expressed in pancreatic cancer;qRT-PCR also confirmed that the expression level of ITGA6 in pancreatic cancer cell lines and pancreatic cancer tissues was significantly higher than that in normal pancreatic duct cells and normal adjacent tissues,respectively.The expression level of ITGA6 in pancreatic cancer tissues was negatively correlated with miR-126a-3p,while positively correlated with LINC01133(P<0.05).After upregulating the expression of miR-126a-3p in Panc-1 cells,the expression level of ITGA6 significantly decreased,while after inhibiting the expression of miR-126a-3p in SW1990 cells,the expression level of ITGA6 significantly increased.The double luciferase reporter gene experiment confirmed that ITGA6 is the target gene for miR-126a-3p.Overexpression of LINC01133 in Panc-1 inhibited miR-126a-3p and upregulated the expression of ITGA6(P<0.05);Knocking down the expression of LINC01133 in SW1990 has the opposite effect.Upregulation of miR-126a-3p expression in pancreatic cancer cells can offset the promotion of overexpression of LINC01133 on ITGA6,while downregulation of miR-126a-3p expression in pancreatic cancer cells can offset the inhibition of LINC01133 on ITGA6(P<0.05).In addition,in vitro blocking experiments showed that blocking ITGA6 could partially reverse the effects of LINC01133 overexpression on the proliferation,migration and invasion of pancreatic cancer cells.The experimental results of nude mice transplanted tumors showed that compared with the control group,the subcutaneous transplanted tumors in the overexpression LINC01133 group grew faster,resulting in a larger tumor volume and increased mass(P<0.05).The results of qRT-PCR and WB showed that the expression level of ITGA6 in the transplanted tumor of the overexpression LINC01133 group was significantly higher than that of the control group(P<0.05),while the expression level of miR-126a-3p was significantly lower than that of the control group(P<0.05).TUNEL test results showed that the level of apoptosis of tumor cells in the overexpression LINC01133 group was significantly lower than that in the control group.Immunohistochemical(IHC)results showed that compared with the control group,the overexpression of LINC01133 significantly decreased the expression of Bax and Caspase-3 proteins in tumor tissue,while the expression levels of Bcl-2 and Ki67 proteins were significantly increased.Correspondingly,compared with the control group,the growth rate of subcutaneously implanted tumors in the knockdown LINC01133 group was slower,resulting in a smaller tumor volume and lower weight(P<0.05).The results of qRT-PCR and immunohistochemistry showed that the expression of ITGA6 in the transplanted tumor of the knockdown LINC01133 group was lower than that of the control group(P<0.05),and the expression level of miR-126a-3p was higher(P<0.05).TUNEL detection showed that the level of apoptosis of tumor cells increased,the expression of Bax and Caspase-3 proteins increased,and the expression of Bcl-2 and Ki67 proteins significantly decreased in nude mice tumor tissue.Conclusions:LINC01133 was highly expressed in pancreatic cancer tissues and cells,and its expression level was negatively correlated with the expression of miR-126a-3p,while positively correlated with the expression of ITGA6;LINC01133 expression is closely related to T stage,lymph node metastasis,TNM stage and prognosis of pancreatic cancer patients;LINC01133 can promote the proliferation,migration and invasion of pancreatic cancer cells,induce cell cycle arrest and inhibit apoptosis.In mechanism,LINC01133 upregulates the expression of ITGA6 in pancreatic cancer cells by targeting miR-126a-3p,thus participating in the regulation of the genesis and development of pancreatic cancer.