LncRNA FAM83H-AS1 Promotes The Proliferation,Invasion And Metastasis Of Pancreatic Cancer By Stabilizing FAM83H MRNA To Protect ?-catenin From Degradation

Part 1:Screening of the long non-coding RNAs related to lymph node metastasis and distant metastasis in pancreatic cancerResearch objective:As a highly malignant and refractory gastrointestinal tumor,pancreatic cancer has brought us huge challenges in the aspects of early diagnosis,effective treatment,improving prognosis and better improvement of the quality of life of patients.Long noncoding RNA(lncRNA)plays a vital regulatory role in human diseases,especially in human cancers.This part of study aims to screen abnormally expressed lncRNAs in pancreatic cancer in the development of tumors,especially in the pancreatic cancer with lymphatic metastasis and distant metastasis.Experimental Methods:The abnormal expression of lncRNAs in pancreatic cancer are screened through TCGA database.According to the comparison result of T2N0M0 vs.T2N1M0,T2N0M1 and T2N1M1,and T3N0M0 vs.T3N1M0,T3N0M1 and T3N1M1,the differentially expressed lncRNAs are enriched via the seventh edition of the TNM staging standard of the American Joint Committee on Cancer(AJCC)system.Take the intersection of the lncRNAs selected in the above three models,and then find 6 lncRNAs.Then the formation state of microfilament and microtubules is evaluated by immunofluorescence.Wound healing assays and Transwell assays are conducted.Through Kaplan-Meier analysis,the OS times that are affected by the 6 lncRNAs were analyzed,and a representative lncRNA was selected for further study.Results:We analyzed the transcriptome results of 178 cases of pancreatic cancer tissues and 4 cases of adjacent tissues in the TCGA database to enrich all abnormally expressed lncRNAs.Comparing the lncRNA expression profiles of T2N0M0 with T2N1M0,T2N0M1 and T2N1M1 in the same cohort,275 abnormally expressed lncRNAs were entrenched.The same strategy was applied in the comparison of T3N0M0 with T3N1M0,T3N0M1 and T3N1M1 and another 169 aberrantly expressed lncRNAs were found out.Take the intersection of the lncRNAs selected in the above three models,and then find 6 lncRNAs,which represent abnormally expressed lncRNAs in pancreatic cancer tissues that are prone to metastasis.Then through survival analysis,it was found that the expression of LINC00365,LINC00628,AFAP1-AS1 and HNF1A-AS1 did not affect the survival and prognosis of patients.FAM83H-AS1 and LINC00261 had an impact on the survival of patients with pancreatic cancer,while only the high expression groups of FAM83H-AS1 can shorten the overall survival time of patients.In vitro experiments,we found that FAM83H-AS1 has the strongest ability to promote the metastasis of pancreatic cancer cells.Conclusion:The database screened the 6 abnormally expressed lncRNAs that are most relevant to the metastatic pancreatic cancer.In vitro experiments we found that FAM83HAS1 has the strongest ability to promote the metastasis of pancreatic cancer cells.Therefore,as a potential oncogene,FAM83H-AS1 becomes the object of our next in-depth study.Part 2:The expression of long non-coding RNA FAM83H-AS1 in pancreatic cancer and its relationship with the malignant biological behavior of pancreatic cancerResearch objective:In the process of tumorigenesis,the characteristics of malignant biological of tumors are empowered by the interactional networks from variant molecules in both transcriptional level and post-transcriptional level.The activation/inactivation of a variety of signals enables tumors to maintain the malignant activity of tumors,such as unlimited reproduction,invasion,migration,angiogenesis,immune escape,and so on.LncRNA is also a class of molecules with the above-mentioned complex biological characteristics.At the same time,because of its diversity in the types of intermolecular interactions,lncRNA plays a vital role in the evolution of tumors.This part of study explored the relationship between the long non-coding RNA FAM83H-AS1 and the biological behavior of pancreatic cancer.Experimental Methods:Through the first part of the screening,we focus on the lncRNA FAM83H-AS1 that most significantly affects the invasion and metastasis of pancreatic cancer.Real-time fluorescence quantitative PCR was used to detect its expression in pancreatic cancer adjacent tissues/pancreatic cancer tissues,pancreatic normal ductal epithelial cells/pancreatic cancer cell lines.The relationship between the expression of FAM83H-AS1 and patient survival was analyzed by Kaplan-Meier method.The distribution of FAM83HAS1 in pancreatic cancer cells was detected by real-time fluorescence PCR of nuclear/cytoplasmic fractionation and fluorescence in situ hybridization.Lentivirus was used to construct stably expressed FAM83H-AS1 overexpression and knockdown pancreatic cancer cell lines,and to construct models of proliferation,invasion and metastasis in vivo and in vitro to detect the relationship between FAM83H-AS1 and the malignant biological behavior of pancreatic cancer.Results:It was found that the expression of FAM83H-AS1 in pancreatic cancer tissues was significantly higher than that in adj acent pancreatic cancer tissues.Compared with the normal pancreatic ductal epithelial cells,the expression of FAM83H-AS1 in pancreatic cancer cell lines was increased to varying degrees.Through Kaplan-Meier analysis,it was found that patients in the FAM83H-AS1 high expression group had shorter overall survival time and worse prognosis.Through nuclear/cytoplasmic fractionation real-time fluorescent PCR and fluorescence in situ hybridization,it was found that FAM83H-AS1 was mainly located in the cytoplasm of pancreatic cancer cells.In vitro,through CCK8,clone formation and EdU experiments,it was found that overexpression of FAM83H-AS1 can promote the proliferation of pancreatic cancer cells,while knocking down FAM83H-AS1 has the opposite effect.In vivo,by constructing a subcutaneous tumorigenesis model of immunodeficiency nude mice,it was found that FAM83H-AS1 can promote the tumorigenesis of pancreatic cancer cells.By constructing tail-vein systemic metastasis model of immunodeficiency nude mice,it was found that FAM83H-AS1 can promote pancreatic cancer cells metastasis from tail-vein to other organs.Conclusion:FAM83H-AS1 is highly expressed in pancreatic cancer,and its abnormally high expression is positively correlated with the poor prognosis of patients.Inhibiting the expression of FAM83H-AS1 can suppress the proliferation,invasion and metastasis of pancreatic cancer cells and other malignant biological behaviors.Part 3:Study on the mechanism of long non-coding RNA FAM83H-AS1 in regulating the proliferation,invasion and metastasis of pancreatic cancer Research objective:The diversity of lncRNA regulatory functions is often reflected in the fact that lncRNA can not only interact with proteins,but also with DNA/RNA.In this part of study,the downstream molecules that interact with FAM83H-AS1 were screened to explore the mechanism of the influence of FAM83H-AS1 on the malignant biological behavior of pancreatic cancer.Experimental Methods:Through bioinformatics analysis and an overview of the reported regulation and activation pathways in which FAM83H-AS1 was involved in tumors,we are looking for the key molecules of the signaling pathways that FAM83H-AS1 may potentially regulate.Then it was verified by Western blot.The key molecule ?-catenin in the signaling pathway is used to find the bridge molecule FAM83H that connects FAM83H-AS1 and the pathway.The regulation mode of FAM83H-AS1 and FAM83H was clarified through the experiments of cycloheximide and actinomycin.Co-immunoprecipitation technology(Co-IP)was used to verify the direct binding of FAM83H to ?-catenin and the ubiquitination level of?-catenin regulated by FAM83H.Through real-time fluorescent quantitative PCR technology and protein immunoblotting technology,the expression of the downstream effector molecules of the canonical Wnt/?-catenin pathway regulated by FAM83H under the condition of FAM83H-AS1 knockdown was detected.The rescue experiment was used to verify the regulatory relationship between FAM83H-AS 1,FAM83H and ?-catenin.Results:Through screening and analysis,WB assays verified that FAM83H-AS1 regulates?-catenin,a key molecule in the canonical Wnt/?-catenin signaling pathway in pancreatic cancer.Under the condition of overexpression/knockdown of FAM83H-AS1,FAM83H protein level will change accordingly.After the intervention of cycloheximide,overexpression of FAM83H-AS1 did not affect the degradation of FAM83H protein.After actinomycin treatment,overexpression of FAM83H-AS1 can delay the degradation of FAM83H mRNA,thereby significantly increasing the expression of FAM83H at the protein level.Through co-immunoprecipitation(Co-IP),it was found that FAM83H can directly bind to ?-catenin,and knocking down FAM83H can increase the ubiquitination level of ?-catenin.Overexpression of FAM83H-AS1 also reduces the ubiquitination level of ?-catenin.However,knocking down FAM83H at the same time,the ?-catenin ubiquitination level will increase significantly.Overexpression of FAM83H-AS1 increased the expression of MYC,MMP2,and CD44,the downstream effector molecules of the canonical Wnt/?-catenin signaling pathway.At the same time,knocking down FAM83H,the expression of MYC,MMP2,and CD44 were decreased.Conclusion:FAM83H-AS1 increases the expression level of FAM83H by stabilizing the mRNA of FAM83H.FAM83H directly interacts with ?-catenin in the canonical Wnt/?catenin signaling pathway to reduce the ubiquitination level of ?-catenin.Thereby,increasing the expression of ?-catenin allows more ?-catenin to translocate into the nucleus for transcriptional regulating the expression of effector molecules MYC,MMP2 and CD44.It shows that FAM83H-AS1 promotes the malignant biological behavior of pancreatic cancer through the activation of FAM83H-AS1/FAM83H/Wnt/?-catenin signal axis.