| Research background:Thymic epithelial tumor(TET)is a kind of rare tumor which originates from thymus epithelial cells.The main characteristic of TET is abnormal epithelial cell proliferation,which occurs in 0.2%to 1.5%of all malignancies.The World Health Organization(WHO)divides TET into thymomas(TM)and thymic carcinomas(TC).According to the morphology of epithelial cells and the number of non-neoplastic lymphocytes,thymocytes are further classified into five different subtypes(A,AB,B1,B2,and B3).The prognosis of thymocytes deteriorates gradually from A to B3.Histologically,thymus carcinoma can be divided into seven subtypes:squamous,mucoepidermoid,basal-cell,sarcomatoid,small cell,lymphoepithelial,and clear cell.At present,complete surgical resection is the main treatment for TET.However,because of the lack of specific symptoms in the early stage of TET,most patients are in the late stage at the time of consultation.In addition,for patients with thymic carcinoma,recurrence and metastasis are still possible after surgery,radiotherapy,and chemotherapy.Therefore,it is of great clinical value to explore the mechanisms underlying the progression of TET.Studies have shown that 90%of the human genome has transcriptional function,yet only 2%of the transcripts are translated into proteins.Non-coding RNAs(ncRNAs)occupy the majority of the transcripts in the nucleus,and can be divided into long non-coding RNAs(lncRNAs;>200nt)and microRNAs(miRNA;<200 nt)according to the transcript length.Accumulated evidence shows that ncRNAs play an indispensable role in tumor genesis and development,and may become an effective target for tumor therapy.Studies have shown that miRNA is closely related to tumor metastasis and is an important factor in the initiation of tumor cell invasion and metastasis cascade.MiR-525-5p plays an important role in tumor formation by targeting different target genes in a variety of tumors,and up-regulation of its expression can inhibit tumor cell activity,metastasis,invasion,and induce cell apoptosis.Loxll-asl is an lncRNA.Studies have shown that LOXL1-AS1 is abnormally expressed in a variety of tumors and can enter the tumorigenesis process by regulating the miRNA/mRNA axis.HSPA9(Mortalin),located on chromosome 5q31.2,mainly exists in mitochondria and plays an important role in regulating cell growth and survival.Studies have shown that down-regulation of HSPA9 expression can cause growth arrest,apoptosis and death of cells in a variety of tumors,and has tumor suppressive effects.Research objectives:To explore whether lncRNA LOXL1-AS 1 can participate in the progression of TET by regulating miR-525-5p/HSPA9.Part Ⅰ:miR-525-5p targets HSPA9 to inhibit the progression of thymoma and thymic carcinomaMethods:Bioinformatics technology was applied to predict the clinical value of the expression levels of miR-525-5p and HSPA9 in predicting the survival rates of patients with TET;real time quantitative PCR technology(qPCR)was applied to analyze the expression levels of miR-525-5p in 70 TET tissues,including 42 TM tissues and 28 TC tissues,and 30 normal thymus tissues.Western blotting(WB)technology was used to measure protein expression levels of HSPA9 in TET tissues.Lentiviral infection was used to down-regulate miR-525-5p and HSPA9,plasmid transfection was used Achieve up-regulation of miR-525-5p;dual luciferase gene reporter assay was carried out to evaluate the relationship between miR-525-5p and HSPA9;CCK-8,flow cytometry,Transwell chamber and in vivo tumor-bearing experiments were performed to assess the effect of miR-525-5p/HSPA9 axis on cell growth,apoptosis,invasiveness and in vivo tumor formation abilities of Ty-82 and Thy0517,the two TET cell lines.All of the experiments were carried out for three times,and the data were present with x±SD.Student’s t test and one-way ANOVA were used for data comparisons between 2 groups and≥3 groups,respectively.Kaplan-Meier survival curves with log-rank tests were applied for analysis of the clinical value of HSPA9 and miR-525-5p in predicting the overall survival times in patient with TET.Pearson correlation tests were used to evaluate the expression correlation between the levels of miR-525-5p and HSPA9 in TET tissue samples.SPSS21.0(SPSS,Chicago,IL,USA)statistical software was used for statistical analysis of all the results.When the P value is less than 0.05,the difference was statistically significant.Results:1.Through the TCGA database,we found that thymoma patients with low miR-525-5p expression have a lower 5-year survival rate;From the qPCR results which were used to detect the expression levels of miR-525-5p in clinical thymoma and thymic cancer samples,we found that miR-525-5p level was apparently decreased in TET tissues as compared with the normal tissues,and the low expression levels of miR-525-5p indicated a lower survival;2.CCK-8 experiment results showed that,cell viability of the mimic-miR-525-5p group was significantly reduced as compared with the control group,and increased in the inhibitor-miR-525-5p group;The results of flow cytometry showed that,cell apoptotic rate in the mimic-miR-525-5p group was significantly raised when compared with the control group,and was reduced in the inhibitor-miR-525-5p group;The results of the Transwell chamber experiment showed that upregulation of miR-525-5p significantly weakened cell invasiveness,while silencing of miR-525-5p enhanced cell invasiveness;3.The results from the luciferase report experiment and WB experiment showed that HSPA9 was a target of miR-525-5p,and miR-525-5p reduced HSPA9 expression;4.The TCGA database showed that HSPA9 expression was significantly upregulated in thymoma,and high levels of HSPA9 are closely related to poor prognosis;clinical studies also show that HSPA9 levels in TET tissues are up-regulated,and the 5-year survival rate of high-level HSPA9 is lower;consistently,the expression levels of miR-525-5p and HSPA9 at both mRNA and protein levels in human embryonic thymocyte cell line HBT8810 and thymoma cell lines Thy0517 and Ty-82 were detected using qPCR and/or WB.The results demonstrated that the mRNA and protein levels of HSPA9 in Thy0517 and Ty-82 cells were increased as compared to HBT8810 cells,while miR-525-5p expression levels were decreased;5.miR-525-5p expression levels in TET tissues were negatively correlated with HSPA9 expression levels;6.Further experiments showed that downregulation of the expression of HSPA9 obviously rescued the trend of downregulation of miR-525-5p-mediated increases in cell proliferation and invasion rates and decrease in cell apoptosis rate;7.The results of animal experiments showed that tumor weight and volume in the inhibitor-miR-525-5p+sh-HSPA9 group were significantly reduced when compared to that of the inhibitor-miR-525-5p+sh-NC group.In addition,the inhibitor-miR-525-5p+sh-HSPA9 group showed a significantly lower HSPA9 expression level as compared to the inhibitor-miR-525-5p+ sh-NC group.Conclusion:The decreased expression of miR-525-5p in TET and the increased expression of HSPA9 were related to the poor prognosis of TET patients;silencing of miR-525-5p promoted TET cell proliferation,invasion and tumor formation by targeting HSPA9,and impaired cell apoptosis rates.In conclusion,this part of the study shows that miR-525-5p can inhibit TET progression by targeting HSPA9.Part 2:LncRNA LOXL1-AS1 targets miR-525-5p/HSPA9 to promote the progression of thymoma and thymic carcinomaMethods:Bioinformatics technology evaluates the expression level of LOXL1-AS1 in TET;qPCR technology detects the difference in expression of LOXL1-AS1 in 70 TET tissues(42 TM tissues and 28 TC tissues)and 30 normal thymus tissues;WB evaluated HSPA9 protein expression;lentiviral shRNA infection was used for LOXL1-AS1 down-regulation;dual luciferase gene reporter assay was used to assess the relationship between miR-525-5p and LOXL1-AS1 in TET cells;CCK-8,flow cytometry,and Transwell chamber assays were applied to investigate the effects of LOXL1-AS1/miR-525-5p axis on the growth,apoptotic rate and invasiveness of TET cells Ty-82 and Thy0517,respectively.All of the experiments were carried out for three times,and the data were presented with x±SD.The analysis of differences between all groups was performed using statistical software SPSS21.0(SPSS,Chicago,IL,USA).Student’s t test and one-way ANOVA were used for difference comparisons between two groups and≥3 groups,respectively.Kaplan-Meier survival curves with log-rank tests were applied to analyze the correlation between LOXL1-AS1 and the overall survival time of TET patients.Pearson correlation test was applied to analyze the correlation between the expression levels of LOXL1-AS1,miR-525-5p,and HSPA9 in TET tissue samples.SPSS21.0(SPSS,Chicago,IL,USA)statistical software was used for statistical analysis of all the results.When the P value was less than 0.05,the difference was statistically significant.Results:1.Through TCGA database and the results from qPCR,we found that the expression level of LOXL1-AS1 in TET was significantly increased as compared to the normal tissues;2.Kaplan-Meier survival curves showed that the high expression level of LOXL1-AS1 predicted with shorter survival time in patients with TET;3.The expression level of LOXL1-AS1 in TET tissues was negatively correlated with miR-525-5p,but positively correlated with HSPA9 level;4.The luciferase report and WB experiment results showed that LOXL1-AS1 targeted miR-525-5p and then promoted the translation of HSPA9;5.CCK-8 results showed that,compared with sh-NC+inhibitor-NC group,sh-LOXL1-AS1+inhibitor-NC group had significantly lower cell viability,while sh-NC+inhibitor-miR-525-5p group cell viability Increase;compared with sh-LOXL1-AS1+inhibitor-NC group,sh-LOXL1-AS1+inhibitor-miR-525-5p group has increased cell activity;6.Flow cytometry results showed that,the level of cell apoptosis in the sh-LOXL1-AS1+inhibitor-NC group was elevated when compared to the sh-NC+inhibitor-NC group,while sh-NC+inhibitor-miR-525 was reduced;cell apoptotic level in the sh-LOXL1-AS1+inhibitor-miR-525-5p group was reduced when compared to the sh-LOXL1-AS1+inhibitor-NC group;7.The results of the Trans well chamber assay showed that,the sh-LOXL1-AS1+inhibitor-NC group had a reduced cell invasion ability as compared to the sh-NC+inhibitor-NC group,while cell invasiveness in the sh-NC+inhibitor-miR-525-5p group was enhanced;the sh-LOXL1-AS1+inhibitor-miR-525-5p group has an enhanced cell invasion ability as compared with the sh-LOXL1-AS1+inhibitor-NC group;8.In vivo tumor-bearing experiment showed that the tumor volume of the sh-LOXL1-AS1+inhibitor-NC group was lower than that of the sh-NC+intertior-NC group,while increased in the sh-NC+inhibitor-miR-525-5p group.Tumor volume was higher in the sh-LOXL1-AS1+inhibitor-miR-525-5p group than that of the sh-LOXL1-AS1+inhibitor-NC group.Conclusion:LOXL1-AS1 was highly expressed in TET and linked to poor prognosis;downregulation of LOXL1-AS1 suppressed cell proliferation and invasion by targeting miR-525-5p,and inhibited cell apoptosis.In conclusion,this part of the study shows that LOXL1-AS1 can promote TET progression by targeting miR-525-5p/HSPA9. |
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