Long Noncoding RNA 00976 Promotes Pancreatic Cancer Proliferation And Matastasis Via The Axis Of MiR-137/OTUD7B/EGFR

Objective: Identification of key Lnc RNAs involved in the development of pancreatic cancer using bioinformation method and verified the expression and its relationship with patient outcome of one key Lnc RNA(Long noncoding RNA 00976;Linc00976).Methods: Using the TCGA public database to screen the lnc RNA expressed differently by pancreatic cancer tissue and peripheral tissue.Using real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)to verify the expression in 52 cases of pancreatic cancer tissue and pericancerous tissue and its expression in human pancreatic duct epithelial cells HPDE and 7 human pancreatic cancer cells.The location of Linc00976 in PANC-1 human pancreatic adenocarcinoma cells was detected by nuclear plasma separation and fluorescence in situ hybridization(FISH).Results:q RT-PCR shows that Linc00976 has a relatively high expression in pancreatic cancer tissue(P<0.05),and the expression in 7 human pancreatic adenocarcinoma cells was also relatively higher than the human pancreatic duct epithelial cell HPDE(P<0.05).The expression of Linc00976 in pancreatic cancer tissue was negatively correlated with the patient’s lifetime(P<0.05).Nuclear plasma separation technology and fluorescence in situ hybridization(FISH)all show that Linc00976 is mostly located in cytoplasm.Conclusion: Linc00976 is upregulated in pancreatic cancer tissues,mainly located in the cytoplasm.Objective: To investigate the effects of Linc00976 on the proliferation,invasion and migration of pancreatic cancer cells.Methods: A stable expression of Linc00976 negative control of the virus carrier,over expression and inhibition expression were constructed,and were was infected to pancreatic cancer cell lines PANC-1 and MIA Pa Ca-2 respectively.The effects of Linc00976 on the proliferation ability of pancreatic cancer cells were detected by CCK-8 and Colony formation assay.Flow cytometry was used to detect cell cycle;The effects of Linc00976 on the invasive and migration ability of pancreatic cancer cells were detected by Transwell.A hypodermic tumor model was established in vivo to simulate the effect of Linc00976 on the hypodermic tumor ability of pancreatic carcinoma cells.The immunohistochemistry experiment detected the effect of Linc00976 on the expression of Ki67 and PCNA in the tumor.In vivo,a liver and lung metastasis model was established to detect the effects of Linc00976 on the invasive metastasis of pancreatic cancer cells.Western bolt detects the expression of cyclin D,CDK2,vimentin and N-cadherin.Results: Successful construction of a slow virus carrier,CCK-8 and Colony formation assay showed that increasing the expression of Linc00976 can significantly promote the proliferation ability of pancreatic cancer cells(P<0.05);Flow cytometry showed that inhibition of Linc00976 expression caused pancreatic cancer cell cycle block in the G1 phase(P<0.05);Transwell invasion,Matrigel-transwell migration experiment showed that the increase of Linc00976 expression,compared with the negative control,can significantly promote the invasion and metastasis of pancreatic cancer cells,and the reduction of Linc00976 expression,compared to the negative control,significantly inhibits the invasion and metastasis of pancreatic cancer cells ability,The differences are statistically significant(P<0.05).The naked mouse subcutaneous tumor experiment showed that the reduction of the expression of Linc00976 significantly inhibited the ability of pancreatic cancer cells to subcutaneous tumor(P<0.05);The immunohistochemistry showed that lowering the expression of Linc00976 inhibited the expression of Ki67 and PCNA.The liver metastasis model of naked rat spleen showed that over-expression of Linc00976 could lead to significantly more liver metastases than negative control groups(P<0.05),and the life cycle was significantly shorter than that of the negative control group(P<0.05)The weight of nude mice was also significantly reduced in the control group(P<0.05),low expression of Linc00976,can inhibit the metastasis of pancreatic cancer cells to the liver,extend the life cycle of naked mice,and increase the weight of naked mice.Western bolt results showed that Linc00976 can promote the expression of cycllin D1、CDK2、 vimentin and N-cadherin,promote the proliferation of pancreatic cancer cells and metastasis.Conclusion: Linc00976 can reduce G0 stasis of pancreatic cancer cells promote proliferation、 migration and invasion of pancreatic cancer cells,and promote subcutaneous tumorigenesis and in vivo metastasis of pancreatic cancer cells.Objective: To explore the related molecular mechanism of Linc00976 involved in the proliferation and metastasis of pancreatic cancer cells.Methods:The Linc00976-interacting micro RNA was predicted by mi Rcode.The dual luciferase reporter assay was used to verify the relationship between Linc00976 and one key targeted micro RNA,mi R-137.The inhibitor of mi R-137 was used to treated normal control and Linc-00976 knock down pancreatic cancer cells.CCK8,transwell assays and hypodermic tumor model were used to detect the proliferation,migration and invasion of pancreatic cancer cells in these four groups in vitro and in vivo.ITRAQ protein profiling was performed to detect the overall protein change after Linc00976 knockdown.Differently expressed protein was analysed and regarded as the candidate protein directly or indirectly regulated by Linc00976.The target gene of mi R-137 was predicted using a online database.The predicted target genes and the deferentially expressed proteins analysed from mass spectrometry were used to perform intersected analysis,and the candidate proteins of the intersection were screened out.OTUD7 B was selected from the candidate protein for dual luciferase reporter assay to verify mi R-137.Interaction with OTUD7 B,mi R-137 inhibitor and OTUD7 B inhibitor were used to detect the effect of mi R-137 and OTUD7 B on the proliferation and invasion of pancreatic cancer cells in vitro.The OTUD7 B protein was co-immunoprecipitated and the precipitate was used for LC-MS.Proteomics was used to detect the ubiquitination interaction protein profile of OTUD7 B protein.The protein interaction network map was analyzed by String online software,and the interaction network diagram was visualized by Cytoscape online software.Results: Mi Rcode predicted that mi R-137 interacts with Linc00976.Linc00976 can interact with mi R-137 using dual luciferase reporter assay,and Linc00976 can inhibit mi R-137 mimic luciferase activity.The vector binding sites of Linc00976 and OTUD7 B were mutated separately,the inhibition of mi R-137 luciferase activity disappeared or the inhibition of OTUD7 B luciferase activity disappeared,demonstrating that mi R-137 is a downstream direct target micro RNA of Linc00976,and OTUD7 B is direct target gene of mi R-137.CCK8 and Transwell assay showed that mi R-137 can inhibit the proliferation and infiltration of pancreatic cancer cells,and OTUD7 B can promote the proliferation and infiltration ability of pancreatic cancer cells.Co-immunoprecipitation and Western blot showed that OTUD7 B and EGFR bind to each other,resulting in reduced ubiquitination and degradation of EGFR.Conclusion:Eubiquitination enzyme OTUD7 B is the target gene of mir-137,which can be complementary and paired with the m RNA of OTUD7 B,thus inhibiting the transcription of OTUD7 B.Linc00976 is the upstream regulatory molecule of mi R-137,which can be complementary and paired with mi R-137,making mi R-137 unable to bind and inhibit its downstream target genes.In pancreatic cancer,the increased level of Linc00976 and the increased binding with mi R-137 attenuated the transcriptional inhibition effect of OTUD7 B,and thus up-regulated the expression of OTUD7 B.OTUD7B binds to EGFR and protects it from ubiquitination,maintaining high expression of EGFR in pancreatic cancer,thereby promoting the development and metastasis of pancreatic cancer.