The Expression Profile Of CRIP1 In Gastric Cancer And Mechanism Of Its Role On Promoting Lymphangiogenesis Via CREB1 Mediated VEGFC Expression

Background: Gastric Cancer(GC)is among the most common cancers all over the world.Compared with the western country,our country is still with a high GC incidence rate,with about 400,000 new GC cases each year,accounting for about 40% of the global GC cases.It is a major burden affecting the health of Chinese residents.In recent years,with the continuous improvement of various comprehensive diagnosis and treatment technologies,the treatment effect of GC has improved,but the mortality rate of GC is still high,and the five-year survival rate is hovering around 30%.The main reason for the poor prognosis of advanced GC patients is regional invasion and lymph node metastasis.The occurrence of lymph node metastasis is also an important initial step for GC distant metastasis.Lymph node metastasis is one of the common ways of tumor metastasis.The lymph node metastasis is closely related to tumor staging,treatment options and patient prognosis.Emerging evidence shows that the presence of new lymphatic vessels in the primary tumor and local lymph nodes can promote tumor metastasis to a certain extent.Lymphangiogenesis is characterized as the process of generating new lymphatic vessels on the basis of the original lymphatic vessels in the primary cancer,adjacent tissues or distant metastasis,including the proliferation and enlargement of lymphatic vessels.Through the activation of VEGF/VEGFR,angiopoietin system(angiopoietin system),hepatocyte growth factor pathway(Hepatocyte growth factor,HGF)and other lymphatic vessel growth pathways.It can simultaneously produce intratumoral lymphatic vessels(ILVs),peritumoral lymphatic vessels(PLVs)and promote the increase of sentinel lymph node(SLN).CRIP1(cysteine-rich intestinal protein 1)is a member of the LIM domain protein family and is dysregulated in many tumors including cervical cancer,thyroid cancer,colorectal cancer,prostate cancer etc.CRIP1 can transport zinc ions into the cell to promote GSK-3?phosphorylation to activate GSK-3?/m TOR signaling pathway inducing EMT;promoting the ubiquitination and degradation of Fas protein to reduce the chemotherapy sensitivity of tumor cells to 5-FU.In addition,studies have also shown that CRIP1 plays a tumor suppressor role in breast cancer and esophageal squamous cell carcinoma.CRIP1 can reduce the phosphorylation of MAPK and Akt,increase the phosphorylation of cdc2,and thereby inhibit tumor cell proliferation.At the same time,several studies have found that the upregulation of CRIP1 in tumor tissues is closely associated with lymph node metastasis.In GC,the role of CRIP1 still remains unclear.In this study,we used gene microarray to analyze the m RNA expression profile in GC tissues.Combined bioinformatics analysis and experimental confirmation,we find the lymph node metastasis associated marker CRIP1.The effect of CRIP1 on GC cell proliferation,invasion,metastasis was investigated in vitro and in vivo.In view of the close relationship between CRIP1 and GC lymph node metastasis,we further used in vivo and in vitro experiments to clarify the role of CRIP1 on GC lymph node metastasis.We further performed a series of experiments to investigate the mechanism of CRIP1 on lymphangiogenesis.Methods:1.Quantitative PCR and immunohistochemistry were performed to detect the expression of CRIP1 m RNA and protein in GC tissues and adjacent tissues.2.Western blot: Use Key Gen's whole protein extraction kit to extract GC cell protein,and use BCA protein quantification method for protein quantification.Further using western blot to detect the differential expression of protein between the experimental groups.3.Proliferation-related detection methods: using CCK8,EDU proliferation detection,colony formation and other proliferation-related detection methods to explore the changes in proliferation capacity in vitro,and using nude mouse subcutaneous tumor formation experiments to detect the regulatory effect of genes on proliferation in vivo.4.Invasion and metastasis related experiments: Transwell experiment detects the regulatory effects of CRIP1 and other genes on the migration and invasion of GC cells.5.Inject tumor cells via tail vein to construct a lung metastasis model to detect the regulation of genes on the metastasis of GC cells in vivo;construct a popliteal lymph node metastasis model to detect the influence of each intervention group on lymph node metastasis.6.Statistical analysis: All statistical analysis is based on SPSS version 19 software and Graph Pad Prism version 8 software.The rank sum test was used to analyze the differential expression of CRIP1 in GC tissues and adjacent non-cancerous tissues.Chi-square test were used to compare the relationship between CRIP1 expression and clinicopathological characteristics.Results:1.The high-throughput microarray results showed CRIP1 is highly expressed in GC tissues compared with non-tumorous adjacent tissues,and in GC tissues with lymph node metastasis compared with GC tissues without lymph node metastasis(Fold change>2,p<0.05).Using a large scape sample from our institution,we further verified the high expression of CRIP1 m RNA in GC tissues(p=0.037),which is closely associated with lymph node metastasis(p=0.034).At the same time,using immunohistochemistry experiments to verify the expression of CRIP1 protein in large-scale GC tissue microarray specimens,the results showed that CRIP1 protein was significantly upregulated in GC tissues compared with non-tumorous tissues(p<0.0001)and the upregulation of CRIP1 was positively associated with lymph node metastasis(p<0.001),T staging(p<0.001),TNM Staging(p<0.001).These results suggest that CRIP1 may be an important biomarker in GC and can be used as a marker for lymph node metastasis.2.Using the lymphatic vessel formation experiment and the lymphatic vessel density of subcutaneous tumor tissue to detect the regulation of CRIP1 on lymphangiogenesis,it is confirmed that CRIP1 can promote lymphangiogenesis in vivo and in vitro.Constructing a popliteal lymph node metastasis model to observe the influence of CRIP1 on the formation of lymph node metastasis in vivo,and the results showed that CRIP1 can increase the lymph nodes volume and lymph node metastasis.Functional experiments confirmed that CRIP1 can promote the proliferation,invasion and migration of GC cells.In vivo subcutaneous tumor formation experiments and tail vein lung metastasis model experiments showed that CRIP1 can promote subcutaneous tumor formation and lung metastasis.3.ELISA results show that CRIP1 can increase the level of VEGFC in the supernatant of GC conditioned culture medium.PCR and western blot were further performed to confirm that CRIP1 can increase the expression level of VEGFC at both RNA and protein levels.Subsequently,immunoprecipitation combined with mass spectrometry was performed to investigate the proteins that CRIP1 may interact with.Mass spectrometry data shows that CRIP1 may interact with CREB1,and the results of Co-IP experiments confirm their interaction.Western blot results show that CRIP1 can increase the phosphorylation level of CREB1 and enhance the transcriptional activity of CREB1.We further knocked down the expression of CREB1 in CRIP1 overexpressing cells and found that it can reverse CRIP1's promotion of CREB1 phosphorylation and VEGFC levels.On the contrary,overexpression of CREB1 in CRIP1 knockdown cells can also restore CRIP1' s effects.These results show that CRIP1 may affect the expression of VEGFC through CREB1,thereby regulating lymphangiogenesis and lymph node metastasis.Conclusion: 1.CRIP1 is highly expressed in 10 pairs of GC tissue microarrays,with overexpression in GC tissues compared with non-tumorous tissues.We further confirmed CRIP1 upregulation in large scale of GC tissues and revealed high expression of CRIP1 was positively correlated with the positive lymph node metastasis of GC and poor prognosis.2.CRIP1 promotes lymphangiogenesis and lymph node metastasis in vivo and in vitro.CRIP1 can promote the proliferation,invasion and migration of GC cells,and promote tumor growth and metastasis.CRIP1 up-regulates VEGFC levels at protein and RNA levels and affects lymphangiogenesis.CRIP1 interacts with CREB1 to affect its phosphorylation level and transcription activity,thus regulate the expression of VEGFC.