| Prostate cancer is one of the common clinical malignancies.It refers to the current malignant tumors of the prostate epithelium.Its pathological types include adenosquamous carcinoma,adenocarcinoma,squamous cell carcinoma,urothelial carcinoma and ductal adenocarcinoma,and more than 95%For adenocarcinoma.According to the American Cancer Society survey,as of the end of 2015,there were approximately 220,000 new cases of prostate cancer in the United States,and nearly 30,000 deaths.The analysis of prostate morbidity and mortality in my country in 2015 showed that there were about 72 million new cases of prostate cancer in the country in 2015,of which the incidence rate,the incidence rate of winning the bid and the incidence rate of the world standard were 10.23/100,000,6.59/100,000 and 6.47/10.10,000,ranking 6th in the incidence spectrum of clinical male malignant tumors;about 30,700 deaths occurred at the same time.The patient mortality rate,the standard-winning mortality rate and the world-standard mortality rate were 4.36/100,000,2.61/100,000,and 2.65/100,000 respectively.,Ranking 10th in the death spectrum of male malignant tumors,but the morbidity and mortality of patients still show a continuous upward trend.Different miRNAs in the body can exhibit the functions of tumor suppressor genes or oncogenes,and at the same time have certain temporal expression characteristics,high degree of conservation and tissue specificity.The clinical detection of miRNAs is relatively simple and can be isolated in small doses of body fluids such as breast milk,serum,saliva,urine,and plasma.Based on the above characteristics,people have generally agreed that miRNAs are potential tumor markers.Related studies have shown that miR-155 is highly expressed in prostate cancer cells and is involved in the occurrence and progression of prostate cancer.Testicular proteoglycan 1(SPOCK1)is a calcium-binding protein isolated from the seminal plasma of the testis in 1992.It plays a role in regulating changes in cell morphology,cell proliferation,adhesion,migration,and transfer.It was first discovered that SPOCK1 participated in the regulation of the central nervous system.In recent years,it has been gradually verified that SPOCK1 is highly expressed in malignant tumors such as colon cancer,prostate cancer,liver cancer and lung cancer.It is considered to be an oncogene and can accelerate tumor invasion and metastasis.Inhibit tumor cell apoptosis.The expression level of SPOCK1 in different tissues and organs of the body is not the same.Its expression level in lung tissue and liver tissue is relatively low,while the expression level in kidney tissue,prostate tissue and testis is relatively high.Recent research reports have shown that SPOCK1 is abnormally expressed in various tumors and is involved in glioblastoma cell invasion,hepatocellular carcinoma epithelial mesenchymal progression,and regulation of lung cancer metastasis(EMT).In vitro and in vivo analysis showed that SPOCK1 promotes tumor growth and metastasis in human prostate cancer through a variety of ways,such as PI3K/Akt,Wnt/catenin,Bcl-2 family and matrix metalloproteinase(MMP).However,the underlying mechanism of SPOCK1 overexpression remains unclear.Based on the data obtained from TargetScan and miRBase,miR-155-5p seems to directly target SPOCK1.Due to the carcinogenic effect of SPOCK1 in the prostate,it is speculated that miR-155-5p may inhibit the invasion and migration of prostate cancer cells by targeting SPOCK1,but At present,there are few studies on the role of miR-155-5p in prostate cancer.Therefore,this study analyzed the impact of miR-155-5p on the invasion and migration of prostate cancer by targeting SPOCK1,in order to provide more diagnosis and treatment for prostate cancer patients.Part I The expression of miR-155-5p and SPOCK1 in the tissues of prostate cancer patientsObjective:To explore the expression of miR-155-5p and SPOCK1 in the tissues of prostate cancer patients,and to determine the abnormal expression of miR-155-5p and SPOCK1 in prostate cancer.Methods:A retrospective analysis of 41 prostate cancer patients admitted to Kunshan Hospital of Traditional Chinese Medicine in January 2015 and December 2018 was performed.The surgically resected prostate cancer tissue and normal prostate tissue adjacent to the cancer were collected.Data mining based on datasets:This study selected two datasets Oncomine(https://www.oncomine.org/):(23 normal prostate tissue samples,13 prostate tissue samples,and 30 prostate intraepithelial neoplasias)Prostate cancer samples)and(41 normal prostate tissue samples and 62 prostate cancer samples),the above data set compares the expression of SPOCK1 in prostate cancer and normal tissues.Record the patient’s clinical data,record the expression of miR-155-5p and SPOCK1 in prostate cancer tissues and adjacent normal tissues,record the correlation between miR-155-5p and SPOCK1 expression in prostate cancer tissue,and record miR in prostate cancer tissue-155-5p and SPOCK1 expression and the clinical characteristics of the patient,follow up the patient by telephone or outpatient review before 3,with the occurrence of new metastatic lesions,recurrence or death as the end of follow-up,and record the level of miR-155-5p and SPOCK1 to the patient The survival prognosis affects the situation.Results:①The age of the included patients was 5276 years old,with an average of(73.54±16.02)years old.Among them,25 cases were ≥70 years old and 16 cases were<70 years old.In clinical T staging,there were 14 cases of T1c,17 cases of T2,and 10 cases of T3;PSA In terms of level,18 cases were 49ng/ml,23 cases were ≥10ng/ml;in terms of Gleason score,1 case was 2-4 points,18 cases were 5-7 points,and 22 cases were 8-10 points;37 cases were M0 and 4 cases were M1;31 cases were N0 and 10 cases were N1.②The relative expression of miR-155-5p in prostate cancer tissue is lower than that in normal tissues adjacent to cancer,the relative expression of SPOCK1 is higher than that in normal tissues adjacent to cancer,and the expression of SPOCK1 in prostate cancer tissues is higher than that of miR-155-5p.There are statistical differences Academic significance(P<0.05).③The expression of miR-155-5p and SPOCK1 in prostate cancer tissue is negatively correlated(R2=0.189,P=0.005).④The differences in the expression of miR-155-5p and SPOCK1 in the cancer tissues of prostate cancer patients with different Gleason scores,clinical T staging,lymph node metastasis,and serum PSA levels were statistically significant(P<0.05);patient age,M staging There was no significant difference in the expression of miR-155-5p and SPOCK1 in terms of N staging(P>0.05).⑤Taking the relative expression level of miR-155-5p 2-△△CT=2.18 and the relative expression level of SPOCK1 2-△△CT=6.51 as cut-off values,Kaplan-Meier survival analysis showed that patients with high miR-155-5p expression and The overall survival rates of patients with low expression were 81.33%and 68.88%,respectively.The overall survival rates of patients with high SPOCK1 expression and those with low expression were 67.06%and 80.00%,respectively,and the difference was statistically significant(P<0.05).Summary:①miR-155-5p is lowly expressed in prostate cancer tissues,SPOCK1 is highly expressed in prostate cancer tissues,and the expression levels of the two are correlated;②The expression levels of miR-155-5p and SPOCK1 and the patient’s Gleason score,T Staging,and serum PSA level are related;③The low expression of miR-155-5p and the high expression of SPOCK1 indicate the poor prognosis of patients.Part Ⅱ The effect of miR-155-5p on the proliferation,migration and invasion of prostate cancer cellsObjective:To explore the effects of miR-155-5p on the proliferation,migration and invasion of prostate cancer cells,and to clarify the biological effects of miR-155-5p on prostate cancer cells.Methods:Cell lines:normal human prostate cell line RWPE-2,human prostate cancer cell line 22Rv1,PC-3,LNCap,DU145(all purchased from the Cell Resource Center,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences).Cells were transfected after cell culture.The miR-155-5p mimic and the corresponding negative control group were purchased from Shanghai Jima Pharmaceutical Company.Cell viability was tested by CCK-8,and cell migration experiments and cell invasion experiments were performed respectively.Results:①The expression of miR-155-5p in prostate cancer cell lines PC3,22Rv1,DU145 and LNCap was lower than that of normal prostate cells RWPE-1(P<0.05),and the expression in PC3 was the lowest.PC3 was used in subsequent experiments cell.②The expression of miR-155-5p in miR-155-5p mimic group was significantly higher than that in NC group and control group after transfection(P<0.05).③CCK-8 experiment showed that the proliferation rate of prostate cancer cells after miR-155-5p transfection was significantly lower than that of the NC group,and the difference was statistically significant(P<0.05).④ After transfection of miR-155-5p mimic,the cell migration rate of miR-155-5p mimic group was significantly lower than that of NC group and control group.The cell closure rate of miR-155-5p mimic group was 72.00%,which was higher than that of NC group.56.00%and 59.00%of the control group,the difference was statistically significant(P<0.05);the number of cell invasions in the miR-155-5p mimic group was(46±5.19)cells,which was lower than the(142±10.83)cells in the NC group and In the control group(168±15.37),the difference was statistically significant(P<0.05).Summary:Overexpression of miR-155-5p can inhibit the proliferation and migration of prostate cancer cells,so miR-155-5p may be used as a new target for the diagnosis and treatment of prostate cancer.Part Ⅲ The mechanism of miR-155-5p on the invasion and migration of prostate cancerObjective:To explore the mechanism of miR-155-5p on prostate cancer invasion and migration,and to clarify the targeting relationship between miR-155-5p and SPOCK1 and related influence mechanisms.Methods:The experimental cells were the same as the second part,and luciferase reporter gene detection,western blotting test and real-time fluorescent quantitative PCR were performed respectively.Results:①Based on TargetScan and miRBase analysis,it was found that miR-155-5p directly targets SPOCK1.WT-SPOCK1 and MUT-SPOCK1 luciferase reporter gene vectors contain 7 bp mutations to construct predicted miR-155-5p binding sites.The detection of luciferase reporter gene showed that luciferase activity was reduced in the SPOCK1 3’-UTR WT group transfected with miR-155-5p mimic,thus indicating that SPOCK1 may be the direct target gene of miR-155-5p.②The expression of SPOCK1 mRNA and protein in miR-155-5p mimic group was lower than that of NC group and control group,the difference was statistically significant(P<0.05).③The expressions of vimentin,N-cadherin,β-catenin,MMP-3 and MMP-9 protein in the miR-155-5p mimic group were lower than those in the NC group and the control group,and the expression of E-cadherin was lower than that in the NC group and the control group.The control group increased,and the differences were statistically significant(P<0.05).Summary:①The target gene of miR-155-5p in prostate cancer is SPOCK1;②miR-155-5p inhibits the metastasis and invasion of prostate cancer cells by targeting SPOCK1 to regulate the Wnt/β-catenin signaling pathway and reduce the expression of MMPs... |
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