The Functional Elucidation And Mechanism Study Of LncRNA-MIR99AHG In Regulation Of Invadopodia Mediated Metastasis In Colorectal Cancer

【Background】Colorectal carcinoma remains the most malignant gastrointestinal tumor throughout China and the world,with high morbidity and mortality.The five-year survival rate in patients with metastatic colorectal cancer is only 12.5%.Cancer metastasis is the major cause of colorectal cancer-related deaths.Thus,clarification of the crucial molecular events during colorectal cancer progression and elucidation of the underlying mechanisms will provide new strategies for colorectal cancer management.Invadopodia,a subset of invadosomes,have been described tumor cell surface protrusions with proteolytic activity to breach the cross-linked networks of extracellular matrix.Hence,the invadopodia are hallmarks of tumor cells that undergo systemic dissemination and metastasis.Long non-coding RNAs are a group of recently identified non-coding RNAs of more than 200 nucleotides in length.Lnc RNAs function in the epigenetic regulation of gene expression through a variety of mechanisms.Emerging evidences have demonstrated that a number of lncRNAs are aberrant expressed in numerous tumors and play a significant role in regulating cancer initiation and progression.However,the roles and underlying regulatory mechanisms of lncRNAs in invadopodia formation remain elusive.In this study,we compare the lncRNA expression profiles between a pair of colorectal cancer cell lines with different metastatic potential,to elucidate the roles and underlying regulatory mechanisms of lncRNAs in colorectal cancer metastasis.【Objectives】1.To screen and identify differentially expressed lncRNAs in the pair of colorectal cancer cell lines.2.To explore the roles of MIR99AHG in regulation of malignant biological characteristics of colorectal cancer.3.To determine whether MIR99AHG regulate the formation of invadopodia in colorectal cancer cells.4.To screen and identify the proteins that interact with MIR99AHG.5.To elucidate the underlying molecular mechanisms of MIR99AHG in regulating colorectal cancer metastasis.【Methods】1.Short tandem repeat（STR）authentication was utilized to analysis homogeneity of KM12C and KM12SM.Cell counting kit-8（CCK-8）,flow cytometry,colony formation and nude mice xenograft assays were performed to detect the growth and proliferation of KM12C and KM12SM in vitro and in vivo.Transwell migration and invasion,wound healing and nude mice tail vein metastatic assays were used to detect the metastatic potential of KM12C and KM12SM in vitro and in vivo.Morphological changes of KM12C and KM12SM were studied by two-dimensional and three-dimensional culturing system.Using whole-exome sequencing and RNA sequencing（RNA-Seq）and small RNA sequencing（small RNA-Seq）to screen and identify differentially expressed RNAs between KM12C and KM12SM.Gene ontology（GO）enrichment analysis,KEGG pathway analysis and gene set enrichment analysis（GSEA）were further used to analyze the differentially expressed genes between KM12C and KM12SM.The expression of MIR99AHG was verified by in situ hybridization（ISH）and quantitative polymerase chain reaction（q PCR）in various colorectal cancer cell lines and tissues.Bioinformatic analyses were performed to compare the correlation between MIR99AHG and prognosis of colon cancer patients in the cancer genome atlas（TCGA）database.2.MIR99AHG plasmids and antisense oligonucleotides（ASO）were synthesized,and MIR99AHG overexpression and small guide RNA（sg RNA）lentiviral vectors were constructed.MIR99AHG gain-and loss-of-function models were established via transient transfection and stable infection.Cell counting kit-8（CCK-8）,flow cytometry,colony formation and nude mice xenograft assays were performed to determine the role of MIR99AHG in colorectal cancer cells proliferation.The influence of MIR99AHG on colorectal cancer metastasis was studied using transwell migration and invasion assays,wound healing assays and nude mice tail vein metastatic assays.3.The invadopodia in colorectal cancer cells were observed by transmission electron microscope（TEM）.Western blot analysis confirmed the expression level of specific and associated molecular markers of invadopodia.Immunofluorescence was performed to determine the sub-cellular localization of TKS5.The small interfering RNA（si RNA）of TKS5 and Coractin were synthesized,and loss-of-function models of TKS5 and Coractin were established via transient transfection.Transwell assays were conducted to determine colorectal cancer cells migration and invasion after down-regulation of TKS5 and Coractin expression.4.Five MIR99AHG truncated plasmids were constructed,and the functional region of MIR99AHG was determined by transient transfection and transwell migration and invasion assays.The cytoplasmic and nuclear RNA isolation assays and fluorescence in situ hybridization（FISH）were performed to verify the sub-cellular localization of MIR99AHG.Biotinylated lncRNA pull down assays and mass spectrometry（MS）were used to screen and identify the proteins that can interact with the functional region of MIR99AHG in the nucleus.RNA immunoprecipitation（RIP）was used to verify the candidate proteins that can interact with MIR99AHG.5.Western blot and q PCR analysis confirmed the expression level of PTBP1 after up-or down-regulation of MIR99AHG expression.Proteasome inhibitor MG132 and protein synthesis inhibitor Cycloheximide（CHX）were used to determine the expression level of PTBP1 after up-regulation of MIR99AHG expression.The role of PTBP1 in colorectal cancer metastasis was studied using transwell migration and invasion assays.Western blot and q PCR analysis confirmed the expression level of specific and associated molecular markers of invadopodia after down-regulation of MIR99AHG or PTBP1 expression.Bioinformatic analyses were performed to determine the relationship among MIR99AHG,PTBP1 and TKS5 in TCGA database.【Results】1.STR authentication results demonstrated that both KM12C and KM12SM originate from KM12 cells.CCK-8,flow cytometry,colony formation and nude mice xenograft assays revealed that the growth and proliferation of KM12C and KM12SM is no difference in vitro and in vivo.Transwell migration and invasion,wound healing and nude mice tail vein metastatic assays revealed that KM12SM has higher metastatic potential than KM12C in vitro and in vivo.RNA-Seq and small RNA-Seq results revealed that lncRNA-MIR99AHG and three embedded micro RNAs,mi R-99a,mi R-125b-2 and let-7c,are upregulated in KM12SM that processes higher metastatic potential than KM12C.GO enrichment,KEGG pathway and gene set enrichment analysis results demonstrated that a set of genes associated with cell migration,invasion and cell membrane structure are upregulated in KM12SM.QPCR results revealed that colorectal cancer cell lines express higher MIR99AHG compared with immortalizing human normal colon mucosa cells.ISH in tissue arrays demonstrated that MIR99AHG expression is greatly upregulated in the colorectal cancer tissues compared with adjacent normal tissues and further increased in the metastatic sites compared with primary tumors.Furthermore,MIR99AHG expression is negatively correlated with the overall survival（OS）and disease free survival（DFS）of colon cancer patients in TCGA database.2.MIR99AHG expression is successfully modulated either by transient transfection of MIR99AHG plasmids and ASO or by stable transfection of overexpression and sg RNA lentiviral vectors.CCK-8,flow cytometry,colony formation and nude mice xenograft assays revealed that MIR99AHG has no effects on the proliferation and growth of colorectal cancer cells.Transwell migration and invasion assays and wound healing assays indicated that MIR99AHG facilitates the migration and invasion of colorectal cancer cells.Furthermore,nude mice tail vein metastatic assays revealed that MIR99AHG has the capacity to promote colorectal cancer cells forming pulmonary metastatic sites in nude mice.3.TEM analysis revealed that the width and length of cell protrusions in KM12SM are greater than KM12C.Compared with negative control,the width and length of cell protrusions are greater in KM12C after up-regulation of MIR99AHG expression.Conversely,compared with negative control,the width and length of cell protrusions is less in KM12SM after knockout of MIR99AHG expression.Western blot analysis revealed that the expression of specific and associated molecular markers of invadopodia is higher in KM12SM compared with KM12C.After up-regulation of MIR99AHG expression,the expression of specific and associated molecular markers of invadopodia is higher in KM12C and DLD-1 compared with negative control.Conversely,after knockout of MIR99AHG expression,the expression of specific and associated molecular markers of invadopodia is lower in KM12SM compared with negative control.Immunofluorescence analysis demonstrated that KM12SM could form as individual puncta compared with KM12C.After up-regulation of MIR99AHG expression,KM12C can also form as individual puncta.Conversely,after knockout of MIR99AHG expression,the number of individual puncta in KM12SM is significant deceased.Transwell assays indicated that the migration and invasion capacity of KM12SM and MIR99AHG gain-of-function models is reduced after down-regulation of TKS5 and Coractin expression.4.MIR99AHG truncated expression is successfully modulated by transient transfection of MIR99AHG truncated plasmids.Furthermore,transwell migration and invasion assays demonstrated that the functional region of MIR99AHG is located in 1262 bases of 5’end.Both cytoplasmic and nuclear RNA isolation assays and FISH indicated that MIR99AHG is mainly located in the nucleus.Biotinylated lncRNA pull down assays and MS results screen and identify PTBP1 can interact with the functional region of MIR99AHG in the nucleus.Subsequently,RIP results verify PTBP1 can interact with MIR99AHG.5.Western blot analysis results indicated that MIR99AHG could influence the expression of PTBP1 in protein level.After up-regulation of MIR99AHG,the expression of PTBP1 in protein level is upregulated.Conversely,after down-regulation of MIR99AHG,the expression of PTBP1 in protein level is downregulated.Furthermore,MIR99AHG also sustains PTBP1 stability by acting as an RNA scaffold.Transwell assays revealed that PTBP1 facilitates the migration and invasion of colorectal cancer cells.Both PTBP1 and MIR99AHG upregulate the expression of TKS5_long in m RNA and protein levels.Bioinformatic analyses showed that the expression of MIR99AHG is positively correlated with TKS5 and PTBP1 is positively correlated with TKS5 in TCGA database.【Conclusions】1.The current study screen and identify differentially expressed MIR99AHG in the pair of colorectal cancer cell lines with different metastatic potential and invadosomes.MIR99AHG expression is greatly upregulated in colorectal cancer cell lines and metastatic tissues.2.MIR99AHG do not influence the proliferation of colorectal cancer cells,but it could facilitate colorectal cancer metastasis.3.MIR99AHG could regulate the formation of invadopodia in colorectal cancer cells.4.PTBP1,an important regulator of alternative splicing,interacts with the functional region of MIR99AHG in the nucleus.5.MIR99AHG sustains PTBP1 stability by acting as an RNA scaffold and then changes the isoform expression of the invadopodia key effector TKS5,regulating the formation of invadopodia in colorectal cancer cells and facilitating colorectal cancer metastasis.