The Role And Mechanism Of PSMD12 In The Progress Of Liver Cancer

Part 1 Prognostic value analysis of lymph node metastasis of anaplastic thyroid carcinoma and bioinformatics analysis related to cancer incidenceObjective The incidence of thyroid cancer is increasing year by year,which seriously affects human health and safety.Undifferentiated thyroid cancer(ATC)is the least common pathological type of thyroid cancer but the worst prognosis.The American Joint Committee on Cancer(AJCC)staging manual is a general tool for assessing the prognosis of patients with thyroid cancer.The eighth edition of the AJCC(AJCC-v8)grading system revised on the basis of the seventh edition of AJCC(AJCCv7)adds lymph node status as the staging standard for ATC.However,the correlation between lymph node metastasis and the prognosis of ATC patients is still controversial.In addition,the pathogenesis and specific biomarkers of ATC are still unclear.Therefore,the purpose of this study is to first explore the relationship between lymph node status and the prognosis of ATC patients and evaluate the prognostic predictive ability of the AJCC-v8 model.At the same time,the molecular mechanism related to the occurrence and development of ATC is explored through bioinformatics analysis to provide a basis for finding specific biomarkers and therapeutic targets.Methods Use SPSS statistical software(version 25)and R software(R Foundation;version 3.6.3)for statistical analysis.COX regression analysis was used to determine the association between lymph node status and survival in ATC patients.The consistency index,Akaike Information Criterion(AIC)and Bayesian Information Criterion(BIC)are used to reflect the prediction performance of the model.Based on the GEO database,the pathogenesis of ATC was explored,and the online website tool DAVID was used to perform functional enrichment analysis and signal pathway enrichment analysis of differentially expressed genes.At the same time,analyze the expression and prognosis of differential genes in other types of tumors in the TCGA database.Results The analysis based on the SEER database proved that lymph node metastasis is an independent prognostic factor for ATC patients(OS: HR 1.32,95% CI1.03-1.68,P = 0.028;CSS: HR 1.33,95% CI 1.03-1.71,P = 0.028).AJCC-v8,which includes lymph node status as the staging standard,has a more accurate prognostic prediction ability.Compared with AJCC-v7,AJCC-v8 has a higher consistency index(0.60 vs.0.59),AIC(2728 vs.2732)and BIC(2732 vs.2735)is lower.In addition,through bioinformatics methods,differential analysis of the expression profiles of cancer tissues and normal tissues of ATC patients in the GEO database was performed to screen 98 differential genes.A functional enrichment analysis of differential genes was carried out by DAVID,suggesting that cell division is the most critical biological process in ATC,cell adhesion molecule binding is the most critical cell component,secretory granule membrane is the most critical molecular function,EMC receptor Interaction with NF-kappa B is the most critical signaling pathway in ATC.In particular,we found that PSMD12,as a gene related to the occurrence of ATC,is abnormally expressed in a variety of malignant tumors except thyroid cancer and is closely related to the poor prognosis of liver cancer and lung cancer,suggesting that this gene may be a key gene in the pathogenesis of many cancers.,Has potential research value.Conclusion Lymph node metastasis is an independent risk factor for poor survival in ATC patients.Compared with AJCC-v7,AJCC-v8,which includes lymph node status as the grading standard,has better prognostic performance for ATC patients.PSMD12 gene may play an important role in the mechanism of ATC.At the same time,PSMD12 is related to the poor prognosis of patients with lung cancer,liver cancer and other tumors.Demonstrated the potential research value in the related tumor field.Part 2 The role and mechanism of PSMD12 in liver cancerObjective Hepatocellular carcinoma is currently the fifth largest cancer type worldwide,and its incidence is increasing by 3.1% every year.With the advancement of scientific research and medical methods,the prognosis of patients with liver cancer has improved.However,due to the lack of biomarkers with strong applicability,the detection of liver cancer is often late,and most patients have a higher tumor stage at the time of diagnosis.In addition,due to the lack of effective therapeutic targets,the overall treatment effect of liver cancer patients is still poor and the mortality rate is high.PSMD12 is a non-ATPase subunit on the 26 S ubiquitin proteasome,and its function has been studied in the mechanism of neurogenesis and development.However,we still know little about the function of PSMD12 in tumor-related progression.The latest research has found that PSMD12 has the effect of inhibiting tumor cell apoptosis in breast cancer,but there is no related research on PSMD12 in other types of tumors.Kinesin family member 15(kinesin family member 15,KIF15)is an N-terminal and forward-oriented motor,which plays a key role in the formation of bipolar spindles,and participates in mitosis,meiosis,and the transportation of macromolecules.Kind of cell biology process.KIF15 is highly expressed in a variety of malignant tumor tissues including breast cancer,pancreatic cancer and glioma.In addition,KIF15 has been shown to activate the MEK-ERK signaling pathway in a variety of tumor cells and promote the malignant progression of tumor cells.Our research intends to determine the expression of PSMD12 in liver cancer through public database analysis and immunohistochemistry,and analyze the relationship between its expression and prognosis,and determine whether it has the potential value as a prognostic marker of liver cancer.In addition,we hope to further study the possible functions and related molecular mechanisms of PSMD12 in liver cancer cells,fill the gaps in its research in the tumor neighborhood and provide a basis for the development of targeted drugs in the future.(8)In vivo,8 nude mice were selected to construct the subcutaneous xenograft tumor model.Four of them were injected with Hepg2 cells stably knocking down PSMD12,and the other four were injected with Hepg2 cells transfected with NC.After four weeks of culture,they were sacrificed and the nude mice were dissected.The mouse took out the tumor.We observed that compared with transplanted tumors transfected with NC,tumors with stable knockdown of PSMD12 were significantly smaller and lighter in weight.Immunohistochemical staining found that the expression of KIF15 and KI67 in transplanted tumors with PSMD12 knockdown was also lower than that in transplanted tumor tissues transfected with NC.Methods(1)Determine the expression of PSMD12 in liver cancer by analyzing the transcriptome data of liver cancer tissues and adjacent tissues in the TCGA database,and analyze the relationship between PSMD12 expression and prognosis in combination with patient survival information,and incorporate clinical factors for multivariate COX regression Analyze whether PSMD12 is an independent factor of patient prognosis.In addition,immunohistochemical staining was performed on liver cancer specimens collected in our hospital to determine the expression of PSMD12 in cancer tissues and adjacent tissues.(2)Use si RNA to knock down the PSMD12 of liver cancer cells Hepg2 and Huh7,and use the plasmid to overexpress PSMD12.CCK8 and clone formation experiments were used to detect tumor cell proliferation,scratch test and transwell were used to detect tumor cell migration and invasiveness,and the effect of PSMD12 protein expression on the biological functions of liver cancer cells was studied.(3)The enriched signaling pathway(MAPK)related to the expression of PSMD12 in liver cancer was screened by GSEA.And analyze the relationship between PSMD12 and key genes(MEK,ERK)expression in the pathway.Western blotting was used to detect the expression of related pathway genes after PSMD12 knockdown and overexpression.(4)In response to the experiment,cells were treated with ERK pathway inhibitor U0126,and the effect of ERK pathway inhibition on the malignant phenotype of cells in cells overexpressing PSMD12 was observed.(5)Analyze the correlation between the m RNA expression of PSMD12 and KIF15 in liver cancer in the TCGA database,and verify it by immunohistochemistry.In addition,the effect of knockdown and overexpression of PSMD12 on the expression of KIF15 was tested in liver cancer cells,and the effect of knockdown of KIF15 on the expression of PSMD12 protein was tested.(6)Analyze the expression of KIF15 in liver cancer in the TCGA database and its relationship with the prognosis,and verify it by immunohistochemistry.In addition,KIF15 was knocked down in hepatoma cells Hepg2 and Huh7 in vitro to detect the biological phenotype of related tumors and the activation of ERK pathway.(7)The response experiment verified that the function of PSMD12 to activate the ERK pathway and promote tumor progression is related to KIF15 by performing KIF15 knockdown on liver cancer cells overexpressing PSMD12.(8)Use lentivirus to perform stable knockdown of Hepg2 PSMD12,and use PSMD12 knockdown Hepg2 cells and normal Hepg2 cells for subcutaneous tumor transplantation in nude mice in in vivo experiments to detect tumor growth rate and pass immunization after removing the tumor tissue Histochemical staining determines the correlation between PSMD12 and KIF15 expression.Results(1)By analyzing the transcriptome of liver cancer and adjacent tissues in public databases,we found that the expression of PSMD12 in cancer tissues was significantly higher than that in adjacent tissues.The conclusions drawn from the immunohistochemical staining of liver cancer specimens and the database analysis are suppressed.In addition,we found that high expression of PSMD12 is associated with poor prognosis of patients with liver cancer.The overall survival(OS),progression-free survival(PFS),recurrence-free survival(RFS)and disease-specific survival(DSS)times of patients with high PSMD12 expression are all Low.Multivariate COX analysis with clinical factors including age,gender,and tumor stage found that PSMD12 expression is an independent prognostic risk factor for liver cancer patients(HR=1.493,95% CI: 1.018-2.191,P=0.04).(2)Knockdown and overexpression of PSMD12 protein on liver cancer cells Hepg2 and Huh7,and detect the effect of PSMD12 expression on the biological functions of liver cancer cells.It is found that PSMD12 can promote tumor cell proliferation,migration and invasiveness.(3)Through GSEA detection,we found that PSMD12 is closely related to the enrichment of the MAPK pathway in liver cancer,and analyzing the correlation between PSMD12 and the expression of key genes in the MAPK pathway,we found that PSMD12 is closely related to the expression of MAPK1 and MAP2K1 m RNA(MAPK1:R=0.66 p <0.001;MAP2K1:R=0.56 p<0.001.Using Western blotting to detect the activation of the MAPK pathway in PSMD12 knockdown cells,we found that knockdown of PSMD12 significantly down-regulates the expression of p-MEK and p-ERK,but does not affect MEK As with ERK expression,the same pathway activation trend was observed after PSMD12 was overexpressed,indicating that PSMD12 was involved in the activation of the MEK-ERK pathway.(4)Treatment of Hepg2 and Huh7 cells overexpressing PSMD12 with the ERK inhibitor U0126 showed that U0126 could partially Reduce the activation of the MEK-ERK pathway induced by the overexpression of PSMD12 and inhibit the increase in the malignant phenotype of liver cancer.It is proved that PSMD12 promotes the malignant progression of liver cancer cells by activating the MEK-ERK pathway.(5)Analysis of the TCGA database liver cancer data found that PSMD12 and KIF15 expression There is a correlation(R=0.47 P<0.001),immunohistochemical staining found that the expression of KIF15 in cancer tissues was also up-regulated in the case of high expression of PSMD12.In addition, knocking down PSMD12 in Hepg2 and Huh7 can significantly down-regulate the expression of KIF15,and in the past A consistent trend was observed in cells expressing PSMD12.KIF15 knocking down had no significant effect on PSMD12 expression,proving that KIF15 is a downstream target gene of PSMD12.(6)Analysis of liver cancer data from the TCGA database found that KIF15 expression was significantly higher in cancer tissues The expression in adjacent tissues is positively correlated with the poor prognosis of patients.KIF15 knockdown in Hepg2 and Huh7 can inhibit the proliferation and migration of tumor cells,which proves that KIF15 is a tumorpromoting gene in liver cancer.In addition,knocking down KIF15 can Down-regulate the expression of p-MEK and p-ERK in liver cancer cells,but does not affect the expression of MEK and ERK,which proves that KIF15 can promote the activation of ERK pathway in liver cancer.(7)Finally,in liver cancer Hepg2 and Huh7 cells that overexpress PSMD12 By knocking down KIF15,we observed that knocking down KIF15 can inhibit the activation of the ERK pathway induced by overexpression of PSMD12 and reduce the malignant phenotype of tumor cells,indicating that PSMD12 promotes the activation of the ERK pathway through KIF15 to promote the malignant expression of tumor cells.Type progression.Conclusion Our study proved that PSMD12 is a newly discovered oncogene in liver cancer,and its expression is positively correlated with the poor prognosis of patients.In in vitro experiments,it was found that PSMD12 can activate the MEK-ERK pathway and promote the proliferation,migration and invasion of liver cancer cells.In particular,we found that KIF15 is highly expressed in liver cancer and can activate the MEK-ERK pathway in liver cancer cells to promote the malignant phenotype of cancer cells.In addition,we found that the expression of PSMD12 is closely related to KIF15 through database analysis and immunohistochemical staining,and through western blotting,we found that KIF15 is a downstream target gene of PSMD12.In addition,knocking down KIF15 in liver cancer cells can inhibit the MEK-ERK pathway activated by overexpression of PSMD12,and restore the enhancement of tumor cellrelated malignant phenotypes.Our research shows that PSMD12 promotes the activation of the ERK pathway through KIF15,thereby promoting the progression of the malignant phenotype of tumor cells.