| Background:Cancer is a global public health problem,which has the characteristics of long incubation period,difficult early diagnosis and high mortality.It has become the main cause of death in China and brings a great burden on our social economy and population health.In recent decades,with the continuous improvement of diagnosis and treatment methods of cancer,the 5-year survival rate of patients with cancer has been improved,but the overall prognosis is still not optimistic.It is an important way to reduce the mortality of cancer by studying the mechanism of cancer occurrence and development,finding valid early diagnostic markers,and screening new therapeutic targets,and carrying out accurate diagnosis and individual treatment.Tandem C2 domains nuclear protein(TC2N)gene is a gene involved in tumorigenesis.The protein encoded by TC2 N contains C2 domain,which is not only binds to calcium-dependent phospholipids,but also participates in cell signal transduction and protein-protein interactions.Therefore,it may play a vital role in normal physiological processes and tumorigenesis.Previously,We found that the expression of TC2 N was significantly upregulated in Hepatocellular carcinoma(HCC)and Skin cutaneous melanoma(SKCM),indicating that TC2 N may regulate the development of HCC and SKCM.However,The specific role and mechanism of TC2 N in HCC and SKCM are still unclear.Therefore,this study intends to analyze the function and molecular mechanism of TC2 N gene in tumorigenesis and development of HCC and SKCM,and explore the relationship between the expression of TC2 N and the clinical characteristics of tumors,so as to evaluate the clinical significance of TC2 N in HCC and SKCM.Contents:1.Expression,clinical characteristics and significance of TC2 N gene in tumorsThe cancer genome atlas(TCGA)and Gene expression omnibus(GEO)database were used to analyze the Messenger RNA(m RNA)expression level of TC2 N gene in cancer tissues and normal tissues,and Real time quantitative PCR(RT-q PCR)was used to detect the m RNA expression of TC2 N in cell lines.TC2 N protein expression levels in cancer tissues and normal tissues were analyzed by Immunohistochemistry(IHC).The IHC and TCGA cohort were used to further evaluate the relationship between TC2 N gene expression and clinicopathological features.2.The role of TC2 N gene in HCC and its molecular mechanismBioinformatics was used to predict the biological function of TC2 N gene in HCC.A differentially expressed cell model was constructed in vitro.The cell proliferation was evaluated by Cell counting kit-8(CCK-8)assay.The cell colony formation was detected by colony formation assay.The cell cycle was analyzed by flow cytometry.Transwell assay was used to analyze the cell migration and invasion ability.The downstream signaling pathways and target molecules of TC2 N in regulating the proliferation and metastasis of HCC were predicted by bioinformatics,and verified by RT-q PCR,Western blot(WB)in differentially expressed cell model and knockout mouse model,so as to clarify the main molecular mechanism of TC2 N in HCC.3.The role of TC2 N gene in SKCM and its molecular mechanismThe biological functions of TC2 N in SKCM were evaluate by bioinformatics.CCK-8assay and Transwell assay were used to measure cell proliferation,migration and invasion in vitro.In vivo,knockout mouse tumor model was constructed to analyze the effect of TC2 N on tumor formation and growth.The downstream signaling pathways and target genes of TC2 N regulating SKCM growth were predicted by the gene expression matrix,and the main molecular mechanism of TC2 N gene in SKCM was analyzed by RT-q PCR and WB experiments in vitro and in vivo,.Results:1.TC2 N gene expression is associated with tumorigenesis and clinical significance.(1)TCGA database and GEO data set were used to analyze the expression of TC2 N m RNA in HCC tissues and normal liver tissues,the results showed that comparing with normal liver tissues,the expression of TC2 N m RNA was significantly higher than that in normal liver tissues.In normal hepatocytes and HCC cell lines,the expression of TC2 N was significantly higher than that in normal hepatocytes(P < 0.05).The human protein atlas(HPA)database analysis showed that the protein expression levels of TC2 N in HCC tissues was significantly higher than that in normal liver tissues.TC2 N protein expression in patients with HCC was detected by tissue microarray analysis,which revealed that the high expression of TC2 N was significantly correlated with the clinical stage and tumor size of HCC patients(P < 0.05).(2)TC2N m RNA expression levels in SKCM tissues and normal skin tissues were analyzed using the TCGA cohort,which showed that TC2 N expression was significantly higher in metastatic SKCM tumors than that in primary SKCM tumors(P < 0.01).Immunohistochemical staining of TC2 N in SKCM and normal skin tissues were explored using the HPA database,which showed that the protein expression levels of TC2 N in SKCM tissues was higher than that in normal skin tissues.When analyzing the relationship between TC2 N expression and clinicopathological parameters of cancer patients by TCGA cohort,the results showed that TC2 N expression was significantly associated with clinical stage and lymph node metastasis of SKCM patients(P < 0.05).2.TC2 N promotes the proliferation and metastasis of HCC cells by regulating Wnt/β-catenin signaling pathway(1)The list of co-expressed genes of TC2 N in HCC was obtained through Linked Omics website,and the Gene ontology(GO)analysis of biological process showed that TC2 N co-expressed genes were significantly enriched in biological processes such as cell proliferation,cell migration and cell adhesion.(2)The results of CCK-8 assay,colony formation assay and flow cytometry analysis showed that overexpression of TC2 N gene could significantly promote the proliferation of HCC cells and accelerate the process of cell cycle(P < 0.05),while knockdown of TC2 N expression could significantly inhibite the proliferation of HCC cells and induce the cell cycle arrest(P < 0.01).(3)Transwell assay using overexpression of TC2 N gene could significantly promote the migration and invasion of HCC cells(P < 0.01);On the contrary,the migration and invasion of HCC cells were significantly inhibited in the cells with interference of TC2 N expression(P < 0.01).(4)Enrichment analysis of co-expressed genes by Kyoto encyclopedia of genes and genomes(KEGG)showed that TC2 N was enriched in the Wnt/β-catenin signaling pathway in HCC;The correlation between TC2 N and the downstream target genes of Wnt/β-catenin signaling pathway were analyzed by TIMER 2.0 database,and it was found that TC2 N was significant positively correlated with downstream target genes of Wnt/β-catenin signaling pathway(P < 0.05).The transcriptional activity of nuclear Wnt/β-catenin was detected by TOP flash/FOP flash luciferase reporter assay.The results showed that overexpression of TC2 N gene significantly enhanced the transcriptional activity of Wnt/β-catenin signaling pathway(P < 0.01).At the same time,Wnt/β-catenin signaling pathway and its downstream target genes were detected,and the results showed that TC2 N significantly promoted the expression of β-catenin,Cyclin D1,MMP7 and c-Myc(P < 0.05).(5)The changes of cell proliferation and cell cycle were verified by functional experiments.The results showed that the promotion of TC2 N on cell proliferation,migration and invasion was significantly reduced by β-catenin interference(P < 0.01).WB results showed that the activation effect of Wnt/β-catenin signaling pathway and its target molecules by TC2 N was significantly inhibited by interfering with β-catenin expression,which indicated that TC2 N promoted the growth and development of tumors through Wnt/β-catenin signaling pathway.3.TC2 N promotes the growth of SKCM by activating ERK/MAPK signaling pathway(1)The list of co-expressed genes of TC2 N in SKCM was obtained from Linked Omics website,and Go enrichment analysis was performed by co-expressed genes.The results showed that TC2 N was mainly involved in cell proliferation,apoptosis,cell adhesion and other biological processes.(2)The results of CCK-8 assay showed that overexpression of TC2 N gene could significantly promote the proliferation of SKCM cells(P < 0.01).Transwell assay results showed that TC2 N overexpression could significantly promote the migration and invasion of SKCM cells(P < 0.05).(3)TC2N knockdown significantly inhibited the growth of tumor in vivo,which was observed in the knockout mouse model(P < 0.05).The proliferation of SKCM cells were detected by Tumor protein Ki-67(Ki67)staining,and the results showed that TC2 N knockout significantly inhibited the proliferation of SKCM cells(P < 0.01).(4)Gene set enrichment analysis(GSEA)showed that Mitogen-activated protein(MAPK)signaling pathway was significantly enriched in patients with high TC2 N expression(P < 0.01).In addition,TIMER 2.0 database analysis showed that TC2 N was a significant positive correlation between TC2 N and the expression of downstream target molecules of ERK/MAPK signaling pathway(P < 0.01).The expression of ERK/MAPK signaling pathway target molecules in differentially expressed cell lines and tumor tissues of knockout mouse was detected by RT-q PCR and WB,and the results showed that TC2 N significantly promoted the m RNA and protein expression of ERK1/2,p-ERK1/2 and the target molecules of ERK/MAPK signaling pathway,such as Cyclin D1,c-Myc,CDK2 and Cyclin E1(P <0.01).Conclusion:(1)TC2N is highly expressed in HCC and SKCM and may play a role as an oncogene.(2)TC2N promotes the proliferation and metastasis of HCC cells by activating Wnt/β-catenin signaling pathway.(3)In SKCM tumors,TC2 N promotes tumor formation and growth through activating the ERK/MAPK signaling pathway. |
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