Study On The Mechanism Of PGM-1 Affecting Colorectal Cancer Progression Through PI3K/AKT Signaling Pathway

Colorectal cancer is one of the most common malignancies in the world.According to the global cancer statistics in 2015,the incidence of colorectal cancer ranks the third and the fatality rate ranks the fourth among malignancies.Colorectal cancer can be treated in a variety of ways,such as surgery,chemotherapy and radiation,but none of these treatments work well for patients with advanced colorectal cancer.Therefore,to investigate the pathogenesis of colorectal cancer,to find effective therapeutic targets,and to improve the survival and quality of life of patients with colorectal cancer has become a hotspot of current research.At present,most studies have proved that the metabolism and survival of cancer cells need energy maintenance and a suitable environment,which can ensure the survival of cancer cells under harsh conditions such as nutritional deficiency and oxidative stress.Current background data suggest that the occurrence of cancer is usually accompanied by abnormal glycogen metabolism.Under glucose depletion conditions(nutritional deficiency,oxidative stress and other microenvironments),glycogen catabolism pathway is significantly activated,accompanied by up-regulation of PGM1 expression.PGM1 is highly expressed in lung cancer tissues,and its high expression is closely related to poor prognosis of lung cancer patients.At the same time,studies have shown that under the condition of sufficient oxygen and nutrition,the up-regulation of PGM1 can convert more extracellular glucose into glycogen,thus inhibiting cell proliferation and tumor growth.Therefore,PGM1 plays a role in promoting or inhibiting the growth of tumor cells depending on different environmental conditions.However,the specific pathway through which PGM1 regulates its anti-tumor effect and the mechanism of action have not yet been clarified.In this study,we determined the effects of PGM1,PGM2,PGM3 and PGM5 among PGM homologous enzymes on the survival of tumor patients by Meta-analysis.It is clear that PGM1 can inhibit tumor growth.Bioinformatics studies,clinical data analysis,animal experiments and cellular experiments were used to investigate the PGM1-mediated inhibition of tumors in vitro and in vivo.We found that the higher the expression of PGM1,the weaker the proliferation and invasion ability of colorectal tumor cells,and PGM1 could inhibit the growth of colorectal tumor cells.In this study,we found for the first time the downstream factor of PGM1,SRPK2,and PGM1 may affect PI3K/AKT signaling pathway by regulating its downstream factor SRPK2,thus inhibiting the growth of tumor cells.In this study,we investigated the specific molecular mechanisms affecting the proliferation and invasive ability of tumor cells.This paper will be elaborated through Four parts.Part Ⅰ Meta-analysis of the effect of PGM on the survival prognosis of tumor patientsObjective: A systematic evaluation of the impact of phosphoglucose translocase PGM on the survival prognosis of tumor patients was conducted to understand its impact on tumors,in anticipation of improving the quality of survival and finding effective therapeutic targets for tumor patients.Methods: "PGM1","PGM2","PGM3","PGM4" "PGM5" as Chinese keywords,"PGM1","PGM2","PGM3" PGM1 cancer "," PGM2 cancer "," PGM3 cancer "," PGM4 "," PGM5 "," PGM1 cancer "," PGM2 cancer "," PGM3 cancer "," PGM4 cancer "," PGM5 cancer "," phosphoglucomutase The keywords "phosphoglucomutase" were searched in the databases of China Knowledge Network(CNKI),Wanfang,Wipu,Pub Med,EMBASE,Science Direct,web of science and Cochrane Library.Relevant studies published from the database establishment cut-off to April 2022 were collected.Studies that met the inclusion criteria were extracted and evaluated for quality with reference to the Cochrane 5.1.0 systematic evaluation method,and the quality assessment was performed using Revman 5.3 software.Results: The final results of 9 articles and 10 studies with a total of 3806 patients were included,including 272 patients in the PGM1 group,541 patients in the PGM2 group,1775 patients in the PGM3 group,and 1585 patients in the PGM5 group.results of Metaanalysis: after counting the results of the 9 articles,the results showed that the difference was statistically significant with a P value <0.05(HR=0.89,95% CI 0.69 to 1.09,P=0.000).Looking for sources of heterogeneity,the remaining 8 papers were tested after removing the highly sensitive literature,and the results showed I~2=26.5%,P<0.001,a statistically significant difference.the risk ratio(HR)for high expression of PGM1 and PGM2 and PGM5 was <1,while the risk ratio(HR)for high expression of PGM3 was >1.Conclusion: Although PGM1,PGM2,PGM3 and PGM5 are enzymes of the same family,their effects on tumors are different.high expression of PGM1,PGM2 and PGM5 can effectively prolong the overall survival of patients.In contrast,high expression of PGM3 reduced the overall survival of patients.The study of PGM family enzymes can provide help for subsequent tumor diagnosis,treatment and prognostic assessment.Part Ⅱ Expression of PGM1 in colorectal cancerObjective: Colorectal cancer,as the third most common malignancy worldwide,is one of the leading causes associated with cancer deaths.And the development of cancer is often accompanied by abnormal glucose metabolism,in which PGM1 plays a key role.PGM1 affects tumor cell proliferation,metabolism and migration by influencing glucose metabolism.In this study,the expression of PGM1 in tumor cells and normal tissues was confirmed by studying the clinical specimens of PGM1,so as to clarify its influence on tumors.Through the analysis of clinical data,it is clear which influencing factors can directly lead to the occurrence and development of tumors,so as to provide help for new drug research,clinical examination and treatment in the future.Methods: PGM1 expression was compared between cancer tissues and normal tissues using Graph Pad 8.0.Since the paired sample size between the two groups was the same,the paired t-test was used to draw a scatter plot for analysis.SPSS statistical software(version 22.0,IBM SPSS,USA)was used for statistical analysis.Chi-square test was used to analyze the relationship between PGM1 expression and clinicopathological data of patients.Multivariate binary Logistic regression analysis was used to determine the independent factors affecting the clinical progress of patients.P value less than 0.05 was considered to be statistically significant.Results: The inhibition of PGM1 on tumor cells was confirmed by bioinformatics screening.The area under the curve(AUC)was 0.635 in the ROC curve.As can be seen from the survival curve,the survival time of the group with high PGM1 expression was longer than that of the group with low PGM1 expression.Paired t test was used to verify the situation in normal tissues and cancer tissues,and the scatter plot showed that the expression level of PGM1 in cancer tissues was less than in normal tissues.Chi-square test was conducted to analyze the relationship between PGM1 expression level and clinical related factors,the results showed that there was no statistical significance between age,gender,degree of tumor differentiation and PGM1 expression,the P value was greater than 0.05.Chi-square test was performed for tumor size,clinical stage,lymph node metastasis,and distant metastasis.The statistical results indicated that P value was less than 0.05,the difference was statistically significant.We performed multivariate binary Logistic regression analysis on the variables that were valid in univariate analysis: tumor size,clinical stage,lymph node metastasis,and distant metastasis.The results showed that clinical stage,lymph node metastasis and distant metastasis had significant effects on the expression level of PGM1,the P value was less than 0.05,the difference was statistically significant.The size of the tumor had a partial effect on the expression level of PGM1,but the effect was not obvious,the P value was greater than 0.05.Conclusion: PGM1 is a tumor suppressor gene,its expression level is related to the survival and prognosis of tumor patients.PGM1 significantly inhibits the growth and proliferation of tumor cells by regulating the expression of glucose metabolism pathway,which can provide a more powerful target for clinical treatment in the future.Part Ⅲ: Effect and specific mechanism of PGM1 on tumor proliferation,invasion and migration.Objective: The PI3K/AKT signaling pathway affects colorectal cancer through phosphorylation,which in turn contributes to the proliferation,migration,and metastasis of tumor cells,acting as a promoter of tumor growth.Whether the role of PGM1 in tumor cells and its effect on glycolysis is related to PI3K/AKT signaling pathway has not been reported so far.Therefore,we hope to confirm it by this study.Methods: After culture and screening of colorectal cancer cell lines,a series of cell biology experiments were performed to assess the status of PGM1 in colorectal cancer tumor cells and its effect on the proliferation,invasion,and migration ability of tumor cells.To clarify the PGM1-related protein pathway and reveal the underlying mechanisms involved.Results: By cultured colorectal cancer cell lines,the comparison was made between the two groups with the least expression of PGM1 and the highest expression of PGM1.The results showed that the expression of PGM1 was the highest in HT29 colorectal cancer cell lines,and the lowest in SW620 colorectal cancer cell lines.q PCR was performed for the two groups of cell lines.CCK8,TRANSWELL,EDU,TUNEL,flow apoptosis and clone formation were performed after knockdown or overexpression of PGM1.The results all suggested that the overexpression of PGM1 could promote tumor apoptosis and inhibit the growth of tumor cells.However,the low expression of PGM1 can promote tumor growth and increase tumor proliferation,invasion and migration.We added the inhibitor LY294002 and found that the PI3K/AKT pathway was inhibited and the expression of PGM1 was increased,which reduced tumor invasion and migration.Through the study of PGM1-related protein pathway,it was found that when PGM1 was highly expressed,the protein expression levels of Bax and p21 were also increased,while the protein levels of p-PI3 K,p-AKT,Cyclin D1 and Bcl-2 were decreased.Conclusion: PGM1 can inhibit the occurrence and development of tumors,which is related to the inhibition of PGM1 on PI3K/ Akt signaling pathway.By inhibiting the phosphorylation of PI3K/ Akt signaling pathway,PGM1 can inhibit the growth of tumors.Part Ⅳ: To verify the effect of PGM1 on the tumorigenesis ability of colorectal cancer in vivoObjective: Comparison of bioinformatics,analysis of clinical data,and cellular assays made it clear that PGM1 inhibits the proliferation of colorectal cancer tumor cells.Next,the effect of PGM1 on subcutaneous tumorigenesis of colorectal tumor cells was verified by subcutaneous tumorigenesis in nude mice in vivo.Methods: The subcutaneous tumorigenesis model of nude mice was used to further evaluate the influence of PGM1 on subcutaneous tumorigenesis ability of colorectal cancer by Western Blot,HE stain,Immuohistochemical stainin and Ki67.Results: Through the establishment of nude mouse model,it was found that the high expression of PGM1 could inhibit the growth of colorectal cancer tumor cells,thereby reducing the growth of subcutaneous tumorigenesis in nude mice.WB detected the association between PGM1 and Bax,p21,p-PI3K、p-AKT、Cyclin D1、Bcl-2 related protein pathways.The results showed that PGM1 was related to PI3K/ AKT signaling pathway and promoted cell apoptosis by inhibiting the PI3K/AKT signaling pathway.Immunohistochemistry and Ki67 both verified that the high expression of PGM1 could inhibit the proliferation of tumors.Conclusion: PGM1 can reduce the phosphorylation of AKT by regulating PI3K/AKT signaling pathway,thereby inhibiting the growth of tumor cells and the growth of subcutaneous tumor-formation in nude mice.Therefore,PGM1 has an inhibitory effect on tumor cells and can inhibit tumor proliferation.Part Ⅴ: PGM1 regulates SRPK2 to affect PI3K/AKT signaling pathwayObjective:Previous studies have demonstrated that PGM1 can inhibit tumor cell growth by suppressing the PI3K/AKT signaling pathway.However,exactly how PGM1 affects the PI3K/AKT signaling pathway and what is its mechanism of action has not been reported.Therefore,we hope to clarify the specific mechanism of action of PGM1 affecting PI3K/AKT signaling pathway through this study.Methods: A preliminary screening of related proteins with significantly altered expression in colorectal cancer HT29 cells was performed by using a combination of Kemas Brilliant Blue staining and mass spectrometry to clarify the downstream related factors of PGM1 and reveal the potential mechanism of action.Results: The downstream factor SRPK2 of PGM1 was successfully screened by protein profiling.Conclusion: By means of protein profiling,we found SRPK2,a protein related to PGM1 downstream,and suggested that PGM1 may influence PI3K/AKT signaling pathway through its downstream factor SRPK2,thus inhibiting the growth of colorectum.