Transgelin-2 Interacts With CD44 To Regulate The Notch-1 Signaling Pathway And Participates In Colorectal Cancer Proliferation And Migration

Objective(s):To explore the role of transgelin-2(TAGLN2)in the proliferation,invasion and migration of colorectal cancer,and to clarify the mechanism of its regulation of Notch-1 signaling pathway and interaction with CD44 to promote the development of colorectal cancer,so as to further enrich the theoretical basis for molecular therapy of colorectal cancer.Methods:1.Target genes were screened by whole-transcriptome sequencing and bioinformatics analysis73 cases of colorectal cancer tissues,73 cases of para-carcinoma tissues,and 13 cases of liver metastasis were collected from the Third affiliated Hospital of Kunming Medical University from March 2018 to July 2020.Whole transcriptome sequencing was performed in 13 colorectal cancer tissues,13 para-carcinoma tissues,and 13 liver metastases.HE staining and IHC were performed in 60 cancer tissues and 60 paracarcinoma tissues.Difference analysis was conducted on transcriptome sequencing data,and protein-protein interaction network(PPI)was constructed for differentially expressed genes.Top 10 hub genes were screened by sequencing of genes with high expression according to Degree.Target genes were screened out according to sequence.The expression level of target genes in colorectal cancer and its relationship with prognosis of colorectal cancer were further analyzed by bioinformatics.qRT-PCR,WB,IHC and other experiments were used to verify the expression level of the screened target genes in colorectal cancer,and finally the target gene TAGLN2 was selected.2.Explore the effect of target gene TAGLN2 on proliferation,migration and invasion of colorectal cancer cells①qRT-PCR and WB assay were used to detect the difference of TAGLN2 expression between NCM-460 and colorectal cancer cell lines(HCT-116,HT-29,RKO).②TAGLN2-siRNA was constructed and transfected into colorectal cancer cells,and its transfection effect was verified by qRT-PCR and WB experiments.③After the transfection,CCK8,EdU and plate colony assay were used to detect the proliferation of cells.The migration and invasion ability of cells were detected by wound healing and Transwell assay.④The recombinant lentivirus TAGLN2 overexpressed plasmid was constructed to infect three colorectal cancer cells,and the proliferation ability of the cells was detected by plate colony assay.In addition,subcutaneous tumor-bearing experiment was performed on HCT-116 cell line with TAGLN2 overexpression and negative control to observe the effect of TAGLN2 overexpression on tumor-bearing growth in nude mice,and HE staining was performed on tumor-bearing tissues.3.Explore the mechanism of target gene TAGLN2 promoting the development of colorectal cancer①Bioinformatics was used to explore the mechanism of TAGLN2 promoting CRC cell proliferation and found that TAGLN2 was related to Notch-1 signaling pathway.After silencing TAGLN2 expression,the expression changes of Notch-1 signaling pathway related proteins were detected by WB and immunofluorescence assay.IHC experiments were performed on tumor-bearing tissues to observe the effects of overexpression of TAGLN2 on the expression of interacting proteins and Notch-1 signaling pathway-related proteins in vivo.②WB assay was used to detect the changes in TAGLN2 and Notch-1 signal pathway related protein expression after the addition of TAGLN2-siRNA after the Notch-1 receptor agonist Jagged-1 activated the Notch-1 signaling pathway.③According to the results of mass spectrometry and Top 10 Hub gene sequencing,the proteins that may interact with TAGLN2 were analyzed,and the combination of TAGLN2 was verified by immunoprecipitation assay.Then,the expression of TAGLN2 was silenced to observe the changes of possible interacting proteins.The expression of TAGLN2 was observed after silencing the interaction protein.The expression of Notch-1 signaling pathway related proteins was observed by silencing TAGLN2 and interacting proteins.After overexpression of TAGLN2,theexpression changes of possible interacting proteins were observed.Immunofluorescence assay was performed to explore their common location in cell membrane.④The changes in the expression of interactive proteins and Notch-1 signaling pathway related proteins in TAGLN2-overexpressing colorectal cancer cells were detected by WB assay.⑤Bioassay was used to explore the expression of interactive protein CD44 in colorectal cancer.The expression of CD44 in fresh colorectal cancer tissues was verified by IHC.The expression of CD44 in colorectal cancer cells was detected by WB assay.⑥CD44-siRNA was constructed and transfected into colorectal cancer cells,and its transfection effect was verified by qRTPCR experiment.After the transfection,CCK8,EdU and plate colony assay were used to detect the proliferation of cells.The migration and invasion ability of cells were detected by wound healing assay.Results:1.TAGLN2 was selected as the target geneAnalysis of total transcription sequencing data showed that there were 858 differentially expressed overlapping genes among colorectal cancer tissues,liver metastatic cancer tissues and para-carcinoma tissues.After constructing PPI network,Degree sequencing was used to find that TAGLN2 ranked first.Further retrieval of TCGA and GTEx databases showed that TAGLN2 expression was significantly upregulated in colorectal cancer tissues compared with normal tissues,and patients with high TAGLN2 expression tended to have lower overall survival.qRT-PCR and WB assay results showed that TAGLN2 expression was significantly up-regulated in colorectal cancer tissues compared with para-carcinoma tissues(p<0.01).HE staining showed that colorectal cancer tissues were disordered,and the nucleo-cytoplasmic ratio was significantly higher than that of para-carcinoma tissues.IHC showed that TAGLN2 expression was significantly increased in colorectal cancer tissues.Therefore,TAGLN2 was selected as the target gene in this study.2.TAGLN2 can promote the proliferation,migration and invasion of colorectal cancer cells①Compared with NCM-460,the expression of TAGLN2 was significantly increased in HCT-116 and RKO(p<0.001),and in HT-29(p<0.01).②After transfection of HCT-116,HT-29 and RKO colorectal cancer cells with TAGLN2-siRNA,qRT-PCR and WB results showed that the expression level of TAGLN2 was significantly decreased after transfection compared with that without transfection(p<0.01).③The results of CCK8 assay showed that the proliferation ability of TAGLN2 gene silenced colorectal cancer cells decreased(p<0.01).Colony assay showed that after TAGLN2 expression was silenced,the colorectal cancer cell colonies and volume decreased(p<0.01),while the colorectal cancer cell lines overexpressing TAGLN2 increased(p<0.001).EdU assay showed that the proportion of EdU positive cells decreased in TAGLN2 silenced colorectal cancer cells(p<0.001).Wound healing showed that the migration ability of colorectal cancer cells was significantly decreased after TAGLN2 gene silencing(p<0.001).Transwell results showed that downregulation of TAGLN2 expression reduced the number of cells crossing the polycarbonate membrane and inhibited the invasion ability of colorectal cells(p<0.001).④Overexpression of TAGLN2 significantly promoted the growth of tumor-bearing tissues,and the tumor volume and weight were significantly different from those of NC group(p<0.01).HE staining showed that compared with NC group,tissue cells in TAGLN2 overexpression group were disordered and nucleo-plasmic ratio was increased.3.TAGLN2 interacts with CD44 to regulate Notch-1 signaling pathway and promote the development of colorectal cancer①Based on transcriptome sequencing data,KEGG analysis showed that Notch-1 signaling pathway plays an important role in the development of colorectal cancer and is related to TAGLN2.Further analysis of mass spectrometry and cell function enrichment showed that TAGLN2 had the functions of targeting cell cycle and adhesion,which were closely related to Notch-1 signaling pathway.WB assay showed that Notch1 expression was down-regulated in colorectal cancer cells after TAGLN2 expression was silenced,and downstream genes Hes-1 and RBPSUH were down-regulated with the down-regulation of Notch-1 expression(p<0.001).Immunofluorescence assay showed that the expression of TAGLN2 was inhibited and the fluorescence intensity of Notch-1 was decreased.The expression of Notch-1 and Hes-1 in tumor-bearing tissues of nude mice increased after TAGLN2 overexpression.Compared with NC group,there were statistical differences in positive cell ratio,positive cell density,H-Score and positive score(p<0.05).②WB results showed that Notch-1 signaling pathway was activated after the addition of Notch-1 receptor agonist Jagged-1,but the expression of related proteins in Notch-1 signaling pathway was down-regulated after the addition of TAGLN2-siRNA(p<0.001).Thus,TAGLN2 can regulate Notch-1 signaling pathway.③According to Degree,we sorted the Top10 Hub genes in the constructed PPI network,and found that TAGLN2 and CD44 ranked first and second respectively.Further retrieval of TCGA database showed that TAGLN2 and CD44 were enriched in CRC tissues,and the expression was moderately correlated(Cor=0.62).The results of TAGLN2 immunoprecipitation showed that TAGLN2 binds to CD44,and the two proteins may interact.WB results showed that CD44 expression was down-regulated after TAGLN2 silencing(p<0.001),and TAGLN2 expression was down-regulated after CD44 silencing(p<0.001).Meanwhile,HT-29 cells were silenced TAGLN2 and CD44,WB results showed that Notch-1,Hes-1,and RBPSUH expression were decreased,and Cleaved Notch-1 expression was significantly decreased(p<0.001).CD44 expression was up-regulated in TAGLN2 overexpressed colorectal cancer cells(p<0.001).Immunofluorescence co-localization showed that TAGLN2 and CD44 were coexpressed in cell membrane.Immunohistochemical staining in tumor-bearing tissues of nude mice showed that the expression of CD44 increased after overexpression of TAGLN2.Compared with NC group,there were statistical differences in positive cell ratio,positive cell density,H-score and positive score(p<0.05).④After overexpression of TAGLN2 gene,CD44 expression and Notch-1 expression were up-regulated in colorectal cancer cells,and downstream genes Hes-1 and RBPSUH were also upregulated with the increase of Notch-1 expression(p<0.001).⑤By searching TCGA and GTEx data,CD44 expression was significantly up-regulated in colorectal cancer tissues compared with normal tissues;IHC staining showed that CD44 was highly expressed in colorectal cancer tissues.WB assay showed that CD44 was highly expressed in colorectal cancer cells(p<0.01).⑥qRT-PCR showed that siRNA could inhibit the expression of CD44(p<0.001).CCK8,colony and EdU assay showed that silencing CD44 expression could inhibit the proliferation of colorectal cells(p<0.001).Wound healing showed that silencing CD44 expression inhibited the migration ability of colorectal cancer cells(p<0.001).Conclusion(s):1.TAGLN2 and CD44 promote proliferation,invasion and migration of colorectal cancer cells.2.TAGLN2 may regulate notch-1 signaling pathway to promote the proliferation,invasion and migration of colorectal cancer.3.TAGLN2 interacts with CD44,which may promote the progression of colorectal cancer.