| Pancreatic cancer is one of the leading causes of malignant tumor-related death worldwide.Compared with other digestive tract malignancies,although the incidence is low,due to its rapid development and early metastasis,only 15%-20%of patients can undergo surgery,and postoperative tumor recurrence and lack of effective assistance Treatment resulted in a five-year survival rate of less than<9.3%.As the study progressed,long-chain non-coding RNA(LncRNA)was thought to be involved in the development of various cancers,playing a role in cancer promotion and tumor suppression,respectively.LncRNA CASC2 has been found to be involved in the development of a variety of cancers through competitive endogenous binding microRNAs(miRNAs),and there is increasing evidence that LncRNA CASC2 is a tumor suppressor in a variety of cancers.Studies have shown that LncRNA CASC2 is lowly expressed in pancreatic cancer and may inhibit tumor progression.The non-coding RNA miR-24 has been found in other studies to show increased expression in pancreatic cancer cells and may be associated with poor prognosis.At the same time,in a study of liver cancer,it was found that LncRNA CASC2 can bind to miR-24 to regulate tumorigenesis.The binding and interaction of LncRNA CASC2 and miR-24 in pancreatic cancer and its molecular mechanism are still unclear.Mucins are a group of highly glycosylated proteins that have been shown to have a regulatory role in the proliferation,migration and invasion of pancreatic cancer.MUC6 is a large-molecular-weight secreted poly-mucin in the mucin family,which is expressed in pancreatic cancer and inhibits cell-cell adhesion in the local microenvironment of the tumor.Through bioinformatics prediction,MUC6 can directly bind to miR-24,and the relationship between miR-24 and MUC6 in pancreatic cancer has not been studied.Integrin ?4(ITGB4),integrin ?4 is a major regulator of epithelial cell and interstitial adhesion.In the study of pancreatic cancer,it can be combined with focal adhesion kinase(FAK)signaling to regulate tumor progression.In addition to one of the subtypes of mucin,MUC5AC has been shown to be mediated by the ITGB4/FAK signaling pathway in lung cancer cells.In the study of pancreatic cancer,whether there is a similar regulatory mechanism between MUC6 and ITGB4/FAK pathway is still unknown.In view of the above unknown effects and relationships,we have designed corresponding experiments to verify and regulate.First,qPCR was used to detect the expression of CASC2,miR-24 and MUC6 in pancreatic cancer specimens and cell lines.Western blot analysis was used to determine the protein levels of MUC6,ITGB4,p-FAK and several epithelial-mesenchymal transition(EMT)markers in pancreatic cancer cells.MTT,clone colony formation assay,scratch wound healing assay,transwell invasion assay and flow cytometry were performed to detect in vitro cell proliferation,colony formation,migration,invasion and apoptosis.Morphological changes of pancreatic cancer cells were assessed by light microscopy.The interaction between CASC2,miR-24 and MUC6 was assessed by dual luciferase reporter assay.Tumor xenograft models were established to study tumor growth in vivo.The results showed that CASC2 and MUC6 were down-regulated and miR-24 was up-regulated in pancreatic cancer specimens and cell lines.Functionally,CASC2 overexpression or miR-24 knockdown suppressed pancreatic cancer cell proliferation,colony formation,migration,and invasion,and promoted apoptosis,along with altered cell-cell adhesion as demonstrated by the attenuated ITGB4,p-FAK,N-cadherin proteins as well as the morphological changes.Mechanistically,CASC2 overexpression inhibited miR-24 and activated its downstream target MUC6 to suppress pancreatic cancer growth and progression.CASC2 exerted tumor-suppressive functions in pancreatic cancer through the miR-24/MUC6 axis that might be a promising target for pancreatic cancer therapy.Part ? Expression of LncRNA CASC2/miR-24/MUC6 in pancreatic cancer tissues and cell linesOBJECTIVE:To determine the difference in expression levels of LncRNA CASC2,miR-24,and MUC6 in pancreatic cancer tissues and pancreatic cancer cells,and to establish a basis for subsequent functional mechanism experiments.METHODS:Pancreatic cancer and adjacent normal tissues were collected and cultured pancreatic HPNE normal cells and cancer cell lines PANC-1,AsPC-1,PATU8988T.RT-PCR was used to detect the differential expression of RNA levels of LncRNA CASC2,miR-24 and MUC6 in tissues and cells.WB and IHC-P were used to detect differences in protein expression of MUC6 in tissues and cells.RESULTS:The expression of LncRNA CASC2 and MUC6 in pancreatic cancer tissues and cancer cells was lower than that in adjacent normal tissues and normal epithelial cells,while the expression of miR-24 was increased.CONCLUSION:The expression of each index in the functional axis was preliminarily verified,which is in line with the expected experimental results.Part ? LncRNA CASC2 inhibits the growth and progression of pancreatic cancer cells,and the expression of related proteinsOBJECTIVE:To detect the effects on cell proliferation,migration,invasion and apoptosis by overexpressing CASC2 in PANC-1 and AsPC-1 pancreatic cancer cell lines.As well as the detection of ITGB4/FAK signaling pathway-related proteins,and EMT-related proteins and matrix metal-related proteins,to verify the changes and regulatory mechanisms of cell function.METHODS:Overexpression plasmids were constructed by pIRES2 overexpression plasmid vector,and overexpression plasmids were transfected in both cells.After exogenous overexpression of LncRNA CASC2,colony colony formation,scratch assay,transwell and flow cytometry were used to detect changes in cell proliferation,migration,invasion and apoptosis.Western blot was used to detect the changes of ITGB4/FAK signaling pathway-related proteins and EMT-related proteins.RESULTS:After overexpressing CASC2 in the two cells,MTT and clone formation experiments showed that the cell proliferation and colony formation ability was significantly reduced,while the scratch test and Transwell invasion suggested that the cell migration and invasion ability was inhibited,and the number of apoptosis increased.The fusiform structure,shrinking to a circular shape,and reduced synapses indicate a decrease in cell adhesion.Through WB detection of related proteins,we found that ITGB4 and p-FAK,Fibronectin expression decreased significantly.There was no significant difference in total FAK protein.Among proteins associated with EMT,the expression of E-cadherin,an epithelial cell marker,increased after overexpression of CASC2,while the expression of proteins with interstitial properties,N-cadherin and Vimentin,decreased,and Snail,MMP-2 and MMP-9 decline.CONCLUSION:Increased expression of CASC2 can inhibit pancreatic cancer cell proliferation,migration,invasion,and promote cell apoptosis,reduce cell adhesion,and has a significant tumor suppressive effect.It is a tumor suppressor gene for pancreatic cancer.By inhibiting the ITGB4/FAK signaling pathway and the EMT process,it may be its mechanism of tumor suppression.Part ? miR-24 promotes the growth and progression of pancreatic cancer cells,and the expression of related proteinsOBJECTIVE:To inhibit the expression of miR-24 in PANC-1,AsPC-1 pancreatic cancer cell lines,and to examine the biological effects of cell proliferation,migration,invasion and apoptosis.Changes in ITGB4/FAK signaling pathway-associated proteins and EMT-related proteins were examined.METHODS:We constructed miR-24 inhibitor analogs to inhibit miR-24 expression in both PANC-1 and AsPC-1 cells.Through MTT assay,cell cloning,scratching,transwell and flow cytometry to detect the effects of miR-24 overexpression on cell proliferation,migration,invasion and apoptosis,cell morphology changes,and verify its role in promoting cancer.Western blotting experiments were performed to detect changes in ITGB4/p-FAK pathway-associated proteins,as well as changes in EMT-related proteins such as E-cadherin,N-cadherin,Vimentin,Snail,and MMP-2,MMP-9.RESULTS:After inhibiting the expression of miR-24 in the two cell lines,MTT and clone formation experiments showed that the proliferation and colony formation ability of the cells was inhibited,while the scratch experiment and Transwell invasion indicated that the cell migration and invasion ability decreased,and the number of apoptosis was relatively increase.Compared with the control group,the tight structure between cells became loose,similar to the shuttle.The shape of the structure shrinks toward a circular shape,and the synapse decreases,indicating that the adhesion ability of the cells is decreased.After PANC-1 and AsPC-1 cells were transfected with miR-24 inhibitor,WB detection showed that ITGB4 and p-FAK,Fibronectin protein expression decreased significantly.FAK total protein did not change significantly.Among the proteins associated with EMT,the expression of E-cadherin,an epithelial cell marker,is increased,while the expression of proteins with interstitial properties,N-cadherin and Vimentin,is reduced,and the expression of EMT transcription factors Snail,MMP-2,and MMP-9 Expression is reduced.CONCLUSION:Inhibition of miR-24 expression can inhibit pancreatic cancer cell proliferation,migration,invasion,and promote cell apoptosis,reducing intercellular adhesion,indicating that miR-24 has a significant cancer-promoting effect and is a cancer-promoting gene for pancreatic cancer.Its role in promoting pancreatic cancer progression is partly related to the ITGB4/FAK signaling pathway and EMT processes.Part ? Verify the targeting relationship and functional regulation of LncRNA CASC2/miR-24/MUC6 axisOBJECTIVE:To verify the relationship between LncRNA CASC2/miR-24/MUC6 and to determine the regulatory relationship of functional axes.Recovery experiments were performed in AsPC-1 cells to verify the binding relationship between miR-24 and LncRNA CASC2,as well as the effects on cell function.At the same time,the protein changes in the above mechanism were verified.METHODS:PANC-1 and AsPC-1 cells were transfected by constructing CASC2,MUC6 dual luciferase reporter plasmid.By exogenously interfering with miR-24 expression,changes in fluorescence are detected to determine if there is a direct targeted binding relationship.At the same time,the changes in RNA levels or protein levels of the three genes are detected by corresponding molecular biology detection methods.The regulatory relationship between the three was examined by cell function experiments.Recovery experiments were performed in AsPC-1 cells to verify the binding relationship between miR-24 and LncRNA CASC2,as well as the effects on cell function.RESULTS:According to the prediction of bioinformatics,there is a binding sit e between CASC2 and miR-24,and there is also a binding site between miR-24 and MUC6.Luciferase report test shows that there is a binding relationship between the two.In PANC-1 and AsPC-1 cells were transfected with pIRES2-CA SC2 overexpression plasmid and miR-24 inhibitor respectively.After transfectio n with CASC2 overexpression plasmid,miR-24 expression decreased and MUC 6 expression increased.In the transfected miR-24 inhibitor group,MUC6 expre ssion also increased relatively,while CASC2 expression did not change signific antly.Recovery experiments were performed on AsPC-1 cells.When MTT and colony formation experiments were used to detect cell proliferation and colony formation ability,the CASC2 overexpression group was the same as before,a nd cell proliferation and colony formation were inhibited.In the miR-24 mimic s group,cell proliferation and colony formation ability was significantly increas ed.The overexpression of CASC2+miR-24 mimics group increased cell prolifer ation and colony formation ability,but lower than the miR-24 mimics group,i ndicating that miR-24 overexpression partially reversed the ability of CASC2 to inhibit proliferation and colony formation.The scratch experiment and Transw ell invasion experiment also found that compared with the CASC2 overexpressi on group,the migration and invasion ability of the CASC2 overexpression plas mid+miR-24 mimics group increased,but it was significantly lower than the mi R-24 mimics group.The ratio of apoptosis in the CASC2 overexpression plasm id+miR-24 mimics group was reduced compared with the CASC2 overexpressio n group,but significantly lower than the miR-24 mimics group.In the recover y experiment,it was shown that in the CASC2 overexpression+miR-24 mimics group,compared with the CASC2 overexpression plasmid group,ITGB4 and p-FAK,Fibronectin protein increased,E-cadherin protein expression decreased,Vimentin,Snail,MMP-2 and MMP-9 protein increased.Compared with miR-24 mimics group,ITGB4 and p-FAK,Fibronectin protein expression decreased,E-cadherin protein expression increased,Vimentin,Snail,MMP-2 and MMP-9 pr otein expression decreased.Conclusion:From this part of the experimental study,we initially found that the LncRNA CASC2/miR-24/MUC6 function axis exists in pancreatic cancer and plays a role in inhibiting the progression of pancreatic cancer.At least part of the mechanism is achieved by inhibiting the ITGB4/FAK signaling pathway and inhibiting the EMT process.Part V The validation of LncRNA CASC2 tumor suppressor and miR-24 cancer promotion in vivoOBJECTIVE:To verify the anti-cancer effect of LncRNA CASC2 and miR-24 by in vivo experiments,and to verify the relationship between the two genes.METHODS:By transfecting LncRNA CASC2 and miR-24 overexpressing lentivirus in AsPC-1 cells,nude mice were subcutaneously implanted with corresponding cells to form tumors,and tumor growth was continuously monitored.Tumors were collected 30 days later,volume and tumor were measured,and The expression of CASC2,miR-24,MUC6 was detected in each group.RESULTS:Tumors were collected after 30 days.Compared with the subcutane ous tumors of mice in the control group,tumors in the overexpressed CASC2 group were significantly smaller in volume and weight than the control group.However,in the subcutaneous tumors of miR-24 overexpression group,the tum or volume and weight were significantly larger than those in the control group.In the CASC2 overexpression group,the expression of CASC2 and MUC6 in tumor tissues was significantly higher than that in the control group,and miR-24 was significantly lower than that in the control group.In the miR-24 over expression group,MUC6 expression was lower than that in the control group.CONCLUSION:In vivo experiments show that LncRNA CASC2 can inhibit the growth of pancreatic cancer in vivo and is a tumor suppressor gene.miR-24 promotes the growth of pancreatic cancer in vivo,is a cancer-promoting gene,and is consistent with the results of in vivo experiments.CONCLUSION1.LncRNA CASC2 and MUC6 are low expressed in pancreatic cancer tissues and cancer cells,while miR-24 is highly expressed.2.LncRNA CASC2 plays a tumor suppressor role in pancreatic cancer.It binds miR-24 through endogenous competition,resulting in the increase of downstream protein MUC6,thereby regulating the changes of ITGB4/FAK pathway and EMT process,inhibiting the proliferation and migration of tumor cells Invasion,cell adhesion and apoptosis.3.In vivo experiments verified that LncRNA CASC2 can inhibit tumor growth,and miR-24 promotes tumor growth.4.LncRNA CASC2/miR-24/MUC6 axis inhibits cancer in pancreatic cancer. |
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