Correlation Study Of Screening And Validation Biomarker-DTL In Adrenocortical Carcinoma On Bioinformatics Analysis

Background and purposeAdrenocortical carcinoma(Adrenocortical carcinoma,ACC)is the primary malignant tumour of the adrenal cortex.Adrenocortical tumor(Adrenocortical tumor,ACT)is a common endocrine tumor,and adrenocortical adenocarcinoma is the main cause of adrenocortical tumor death.ACC accounts for 0.02% of malignant tumors,and the incidence rate is about 0.7-2/1 million.With the spread of the use of imaging diagnosis technology,more and more adrenal disease have found the adrenal diseases for non-adrenal disease’s examination,which called adrenal accidental tumors,although most of those were benign or no-functional,still there was a risk of cancer and endocrine disorders.According to foreign reports,Adrenocortical carcinoma accounts for as much as12% of adrenocortical tumors in some medical centers.At present,there are no clear criteria for differentiating adrenocortical malignant or benign tumors.In some cases,it was difficult to distinguish benign from malignant adrenal tumors based on their clinical features or histological criteria.Accurate marker is important for identify benign from malignant for adrenocortical tumors then for prognosis,follow-up,and treatment strategies.Weiss,including the nine-point histopathological criteria,is the most commonly used gold standard for assessing malignancy.However,ACT with a Weiss score of 3 May have problems in differentiating between adrenalcortical carcinoma and adrenalcortical adenoma.Adrenocortical adenocarcinomas were highly malignant and aggressive,most of which have surrounding tissue invasion or distant metastasis at the time of diagnosis,with a poor prognosis.Complete surgical resection in early cases is the only possible treatment to cure the cancer.According to the literature,the 5-year survival rate of ACC patients was 32% to50%,but the risk of recurrence after surgery was as high as 50% to 85%.Although mitotan is the main basis of drug therapy,it has been controversial as an adjunctive therapy in retrospective studies,and it is difficult as a therapeutic drug because of its narrow therapeutic window and large toxic and side effects.Other types of treatment,such as chemotherapy or radiation,have a limited impact on patient survival,with a 5-year survival rate of less than 30 percent.The low 5-year survival rate of adrenocortical adenocarcinoma is closely related to the following factors: 1.Lack of early detection markers.2.Histological indicators of low prognostic value.3.Currently,the therapeutic effect on adrenal cortical adenocarcinoma is limited.4.Lack of molecular markers for effective targeted therapy.It is important to screen and identify the key biomarkers and effective therapeutic targets of adrenocortical tumor.Therefore,it is necessary to identify new molecular markers to identify benign and malignant adrenalcortical tumors and to explore new targeted therapeutic drugs.Bioinformatics is an emerging interdisciplinary subject combining molecular biology and information technology,which is of great significance for screening tumor molecular markers and revealing the molecular mechanism of tumor pathogenesis.As an emerging technology,gene chip can obtain biological information efficiently and in large quantities from the expression profile data of disease gene chip.As long as we correctly carry out reasonable bioinformatics analysis,scientific algorithm and rigorous statistical analysis,we can easily and quickly find the accurate and consistent results of statistical analysis.Gynostemma pentaohyllum was a traditional Chinese herbal medicine in our country.The active ingredient was saponins,flavonoids and polysaccharide and amino acid etc.,and the gynostemma saponins(Gypsosides,GYP)was most active.The study found that gynostemma saponins had many pharmacological activities,such as inhibiting tumor,enhance immunity,reduce blood sugar,blood fat,resisting thrombus,cardiovascular and liver protection,etc.In recent years,gynostemma pentaphyllum and tumor research has been increasing in many fields including liver cancer,colon cancer,breast cancer,lung cancer,tongue cancer,etc.In this paper,bioinformatics tools were used to analyze the data of adrenocortical carcinoma and adrenocortical adenoma expression microarrays in the gene expression omnibus(GEO)database.We found candidate differentially expressed genes(DTL)and then for further experiments.Immunohistochemical staining of adrenocortical adenocarcinoma and adrenocortical adenoma tissues verified the differences in the expression of candidate genes(DTL).Further cell experiments were conducted to verify whether the targeted interference of DTL gene could enhance the effect of gynostosaponins on ACC cells and help to found the possible mechanism.Research methods and resultsThe first partDifferential expression genes(DEGs)in human adrenocortical adenocarcinoma and adrenocortical adenoma were screened by bioinformatics analysis using microarray technology from gene expression synthesis(GEO)database.The gene ontology(GO)and KEGG pathway enrichment analysis of DEGs were carried out by using DAVID database,the protein-protein interaction(PPI)network was established by using Cytoscape software,and the module analysis was carried out by using MCODE software.The overall survival(OS)analysis of the core genes was then performed by the ualcan online tool.Results a total of 2 gses were downloaded from GEO,including 28 and 55 cases,respectively.A total of 115 DEGs were obtained,among which positively regulated DEGs were significantly enriched in Mitotic nuclear division,where concentrated chromosomal centromeres bind to protein kinases.DEGs associated with negative regulation significantly enriched membrane repolarization,extracellular exosomes and heparin binding during the action potential of atrial myocytes.KEGG pathway analysis showed that significant pathways included cell cycle,oocyte meiosis,and retinol metabolism.PPI network consists of 109 nodes and 772 edges.An important module has been found in PPI network,whose enrichment functions and pathways include mitotic fission,nuclear plasma,protein kinase binding,and cell cycle.Three core genes(DTL、TPX2 and RAD51AP1)which were related to the prognosis of ACC were screened.The second partImmunohistochemistry was used to observe the expression of DTL/CDT2 protein in 18 cases of adrenal cortical adenocarcinoma,31 cases of adrenal cortical adenoma and 8 cases of paracancer normal tissues.The results showed that DTL/CDT2 protein was mainly located in the tumor cell nucleus of adrenal cortical tissues,and was not expressed in normal adrenal cortical tissues.DTL/CDT2 protein in adrenal cortical adenocarcinoma tissues was significantly higher than that in adrenal cortical adenoma and para-carcinoma normal adrenal cortical tissues(P<0.05).Positive DTL expression in adrenal cortical adenocarcinoma tissues accounted for 88.89%(16/18),and positive DTL expression in adrenal cortical adenoma tissues was 38.7%(12/31).DTL protein expression was significantly correlated with tumor size and invasion and metastasis but not with gender,age and clinical stage.DTL expression was significantly increased in patients with tumor diameter greater than 5cm or with invasion and metastasis.Studies have found that adrenocortical adenocarcinoma mostly involved the capsule,adjacent organ tissues such as kidney and peritoneal tissues,and even forms tumor thrombus through blood vessels to renal vein and inferior vena cava,thus achieving distant metastasis and colonization.Graphpad Prism7.0 was used to plot the survival curve,and log-rank test was used to test the significance of the difference between the two groups.The results showed that the survival time of the high-expression group was significantly shorter than that of the low-expression group,indicating that patients with adrenal cortical adenocarcinoma with low DTL protein expression had better surgical prognosis.The results showed that the survival time of the high-expression DTL group was(26±7.2)months,while that of the low-expression DTL group was(76.3±8.2)months.The 3-year survival rate of the high-expression DTL group was less than 30%,while that of the low-expression DTL group was more than 80%.The third part1.Synthesis of DTL RNA interference sequences The si RNA sequence of DTL was targeted to interfere with the screening.RNA interference was mainly used to inhibit the protein expression of the target gene at the transcriptional level by exogenous introduction of double-stranded RNA complementary to the m RNA of the target gene.In this study,four interference sequences for DTL were designed and synthesized according to si RNA design principle.Si RNA was transfected into sw-13 cells to detect the level of DTL m RNA and protein expression,and the sequence with the best interference effect was selected to construct si RNA knockdown DTL cells.2.Interference with transfection of DTL expressing cells and screening of interfering sequences According to the si RNA sequence that effectively inhibited the expression of DTL,the DTL interference cells DTL si-1RNA,DTL si-2 RNA,DTL si-3RNA and DTLsi RNA were constructed by using si RNA.At the same time,mock si RNA without homologous sites was constructed as si RNA control.Rt-pcr results showed that the inhibition efficiency of DTL si RNA sequence was the highest.Mock-sirna,DTL si-1 and DTL si-3 had no significant inhibitory effect on DTL m RNA.Western blot analysis showed that DTL si-1,DTL si-2,DTL si-3 and DTL si RNA all inhibited DTL protein expression.At the same time,no statistical difference was found between the mock group and the control group for DTL expression.After comprehensive consideration,DTL si RNA was selected as DTL interference sequence for follow-up study.3.The effects of DTL si RNA or/and GYP on the proliferation of sw-13 cells in DTL knockdown type and mock control group were detected by growth curve method.The results showed that DTL si RNA inhibited the proliferation rate of DTL knockdown sw-13 cells.The inhibition of GYP on sw-13 cell proliferation was time-dependent.GYP showed a time-dependent inhibitory effect on the proliferation of DTL knockdown sw-13 cells,and was more strongly inhibited than GYP alone.DTL played an important role in the proliferation of sw-13 cells.4.Effect of DTL interference on GYP-induced sw-13 cell cycle arrest After DTL knockout,sw-13 cell S stage block was induced.The action of GYP on sw-13 cells can cause its G2/M phase arrest.The effect of GYP on DTL silencing sw-13 cells resulted in cell arrest in the S and G2/M phases.It is suggested that DTL interference can enhance the cell cycle arrest of sw-13 induced by GYP.To further investigate the effect of DTL interference on gyp-induced sw-13 cell cycle block-related protein changes,we used rt-pcr and WB to detect the changes in m RNA and protein levels of P53 and P21 in sw-13 cells of DTL si RNA or/and GYP on DTL knockdown or mock control.The results suggested that the expression of P53 and P21 in DTL knockdown or mock control cells could be up-regulated by silencing DTL or/and GYP,and the expression of P53 and P21 in DTL knockdown sw-13 cells could be enhanced by targeted interference with DTL.5.DTL interference increases GYP-induced apoptosis of sw-13 cells To further explore the sensitization mechanism of DTL interference on GYP,we used FCM Annexin V/PI double staining to detect the effect of apoptosis.The results showed that DTL si RNA or/and GYP significantly induced the apoptosis of sw-13 cells in the DTL knockdown or mock control group,and DTL interference significantly enhanced the gyp-induced apoptosis,suggesting that the sensitivities of DTL interference to GYP were related to the promotion of gyp-induced apoptosis.Then,we used RT-PCR and WB to detect changes in m RNA and protein levels of Caspase 3 and Caspase 9 in sw-13 cells of the DTL knockdown or mock control group or DTL si RNA or/and GYP.The results suggested that silencing DTL or/and GYP significantly up-regulated the expression of Caspase 3 and Caspase 9 in sw-13 cells of the DTL knockdown or mock control group.Targeted interference with DTL enhanced the expression of Caspase 3and Caspase 9 in DTL knockdown sw-13 cells by GYP.ConclusionGYP could induce cell cycle arrest and apoptosis.DTL si RNA could inhibit SW-13 cell proliferation,induce cell cycle arrest and induce cell apoptosis.GYP could up-regulate the expression of cell cycle and apoptosis-related genes and proteins;and DTL si RNA can up-regulate the expression of cell cycle and apoptosis-related genes and proteins;then targeted interference DTL can enhance GYP on SW-13 cells up-regulating expression of the cell cycle and cell apoptosis-related genes and proteins.Targeted interference DTL could enhance the anti-proliferation,induce cell cycle arrest and induce apoptosis of GYP on human ACC cells.The results of this study also suggested that DTL might be a promising target for the diagnosis and treatment of adrenocortical carcinoma,and DTL might be used as an anti-tumor target for gypenoside for further study.