| Background and PurposeHigh-grade serous ovarian cancer(HGSOC)is one of the most common ovarian epithelial malignancies,accounting for about 90% of ovarian serous carcinoma,which seriously threatens the life and health of women.Most patients are belong to terminal cancer at the time of diagnosis due to the lack of effective early screening strategy.Moreover,HGSOC surgery and chemotherapy are usually not effective.Therefore,HGSOC's mortality rate is still the highest in gynecological cancer,and the 5-year survival rate is less than 30%.At present,it is generally believed that the histological origin of ovarian epithelial carcinoma is diverse,HGSOC may originate from tubal epithelial cells,and the molecular genetic characteristics of TP53 and BRCA1/2 gene mutations are relatively common.However,our understanding of the origin,epidemiology and development process of HGSOC is very limited.In view of the high mortality rate of HGSOC,identifying its pathogenesis and finding new therapeutic targets has become an important part of gynecological oncology research.tRNA-derived fragments(t RFs)are a group of small RNA molecules derived from precursor t RNA and mature t RNA,which are ranged from 14-50 nucleotides in size and cannot encode proteins.There are six main types of t RFs: ti RNA-5,ti RNA-3,t RF-5,t RF-3,t RF-1 and it RF.With the deepening of the understanding of t RFs,previous studies found that t RFs have unique structural characteristics and specific source patterns,so scholars are no longer simply considered that t RFs are products of random degradation.Although t RFs has been widely studied in other diseases,and has been found to play an important regulatory role in the occurrence and development of many diseases,such as human non-small cell lung cancer,colorectal cancer,lung cancer and breast cancer,there are few reports about t RF effects on HGSOC.Given the critical regulatory role of t RFs in other cancers,we realized that t RFs may be a key regulator of the pathogenesis in HGSOC.In addition,due to the complex pathogenesis of HGSOC,the known molecular regulatory mechanism of HGSOC cannot improve and treat ovarian cancer very well,hence it is urgent to find new effective regulatory molecules.Therefore,studying the role of t RFs in ovarian cancer has important clinical implications.This study aims to identify t RFs related to HGSOC through high-throughput sequencing technology,and to study the function and mechanism of key t RFs in SK-OV-3 ovarian cancer cell lines,which will provide theoretical basis and new targets for subsequent treatment of HGSOC.MethodsThe first part,small RNA sequencing method was used to screen HGSOC related t RFs:(1)Serum samples from 3 patients with HGSOC and 3 healthy volunteers were collected,total RNA was extracted from above 6 serum samples and then c DNA libraries were constructed,and small RNA sequencing was performed.(2)Bioinformatics were used to analyze the sequencing data,and GO functional analysis and KEGG pathway analysis were performed on the differentially expressed t RFs.(3)To verify the reliability of the sequencing results,three differentially expressed t RFs were selected for quantitative reverse transcription PCR(q RT-PCR)experiments after expanded serum samples.Candidate genes were screened out and the relationship between gene expression and clinical characteristics was analyzed.The second part,the function of t RF-03357 in ovarian cancer cells was investigated:(1)The q RT-PCR was used to verify the expression levels of t RF-03357 and t RF-03358 in human ovarian epithelial cell(HOSEPIC),ovarian cancer cells SK-OV-3 and HO8910.The results showed that compared with HOSEPIC cells,the expression levels of t RF-03357 and t RF-03358 were significantly increased in SK-OV-3 and HO8910 cells,and the change rate of t RF-03357 was higher than that of t RF-03358.Therefore,t RF-03357 is a candidate target for subsequent experiments.(2)t RF-03357 inhibitor/NC were transfected into SK-OV-3 cells,while t RF-03357mimics/NC were transfected into HOSEPIC cells.The cells were used for subsequent experiments,respectively.(3)The qRT-PCR was used to detect the expression level of t RF-03357 in SK-OV-3 and HOSEPIC cells.(4)The CCK-8 assay was used to detect the proliferation of SK-OV-3 and HOSEPIC cells after transfected.(5)The Transwell assay was used to detect the migration and invasion of SK-OV-3 and HOSEPIC cells after transfected.(6)The TUNEL assay was used to detect the apoptosis of SK-OV-3 and HOSEPIC cells after transfected.The third part,the preliminary study with the mechanism of action of t RF-03357 targeting HMBOX1:(1)Five target genes of t RF-03357 were predicted online,and the expression of five target genes in SK-OV-3 cells transfected with t RF-03357 inhibitor/NC was detected by q RT-PCR.The most significant candidate gene HMBOX1 was selected.(2)Western blotting(WB)was used to detect the expression levels of HMBOX1 protein in SK-OV-3 cells after transfecting with t RF-03357 inhibitor/NC or mimics/NC,respectively.(3)The immunohistochemistry was used to detect the expression of HMBOX1 in ovarian cancer group and normal ovary group.(4)The dual-luciferase reporter assay was used to verify the interact relationship between HMBOX1 and t RF-03357.The HMBOX1 wild-type reporter vector and HMBOX1 mutant vector(t RF-03357 binding site mutation)were co-transfected with t RF-03357 mimics / NC to 293 T cells,and the fluorescence signals were detected.(5)Three HMBOX1 si RNA sequences,including si RNA-1,si RNA-2 and si RNA-3were designed and transfected into SK-OV-3 cells.The expression level of HMBOX1 was detected by q RT-PCR,and the si RNA-1 with best interference effect was screened out for subsequent experiments.(6)The CCK-8 and Transwell assay was used to detect the proliferation,migration and invasion of SK-OV-3 cells after transfecting with HMBOX1 si RNA-1.(7)Rescue experiment: t RF-03357 inhibitor,t RF-03357 inhibitor +HMBOX1 si RNA-1inhibitor and inhibitor NC were simultaneously transfected into SK-OV-3 cells,and the proliferation,migration and invasion of SK-OV-3 cells after transfection were detected by CCK-8 method and Transwell assays.ResultsPart ? t RF expression profiles of HGSOC:(1)An average of 8.9 million clean reads were obtained from small RNA sequencing,accounting for 85.85%,and the GC content of the six sequencing libraries were between49-52%.The database alignment found an average of 977,309 reads of t RFs,and identified 27 differentially expressed t RFs,including 22 t RFs up-regulated and 5down-regulated in HGSOC.(2)Target genes of differentially expressed t RFs were predicted and their functions were analyzed.GO functional analysis showed differentially expressed t RFs were involved in protein phosphorylation,and KEGG pathway analysis indicates that differentially expressed t RFs were mainly involved in the regulation of MAPK signaling pathway,cancer pathway and Wnt signaling pathway.(3)q RT-PCR results showed that the expression levels of t RF-03357 and t RF-03358 were significantly up-regulated in HGSOC patients compared with healthy controls,but there was no significant difference in the expression of t RF-07650.(4)Analysis of gene expression and clinical characteristics of ovarian cancer patients showed that the expression level of t RF-03357 was significantly correlated with CA125.Part ?: In vitro functions of t RF-03357 in ovarian cancer cells(1)The q RT-PCR was used to further verify the expression levels of t RF-03357 and t RF-03358 in HOSEPIC,ovarian cancer cells SK-OV-3 and HO8910.The results showed that compared with HOSEPIC cells,the expression levels of t RF-03357 and t RF-03358 were significantly increased in SK-OV-3 and HO8910 cells,and the fold change of t RF-03357 was higher than that of t RF-03358.Therefore,t RF-03357 was used as a candidate target for subsequent experiments.(2)The results of q RT-PCR showed that the expression of t RF-03357 in SK-OV-3 cells transfected with t RF-03357 inhibitor was lower than that in NC group and the expression of t RF-03357 in HOSEPIC cells transfected with t RF-03357 mimics was higher than that in NC group,indicating that the transfection efficiency was reliable.(3)CCK-8 assay showed that the proliferation of SK-OV-3 cells transfected with t RF-03357 inhibitor was lower than that of NC group and the proliferation of HOSEPIC cells transfected with t RF-03357 mimics was higher than that of NC group,indicating that t RF-03357 promotes cell proliferation.(4)Transwell assay showed that the migration and invasion of SK-OV-3 cells transfected with t RF-03357 inhibitor was lower than that of NC group,and the migration and invasion of HOSEPIC cells transfected with t RF-03357 mimics was higher than that of NC group,indicating that t RF-03357 promote cell migration and invasion.(5)TUNEL assay showed no significant difference in cell apoptosis between the groups,indicating that t RF-03357 did not affect cell apoptosis.Part ?: The potential mechanism of t RF-03357 action in ovarian cancer(1)The results of q RT-PCR showed that compared with NC group,the expression level of HMBOX1 was significantly increased in cells transfected with t RF-03357 inhibitor,while the expression levels of PKN2,KLF3,PTPN13 and ESR2 were not significantly changed.Further study by q RT-PCR showed that the expression level of HMBOX1 gene was significantly down-regulated in cells transfected with t RF-03357 mimics,indicating that t RF-03357 down-regulated the expression of HMBOX1 gene.(2)WB showed that the expression level of HMBOX1 protein in SK-OV-3 cells transfected with t RF-03357 inhibitor was significantly higher than that in NC group and the expression level of HMBOX1 protein in SK-OV-3 cells transfected with t RF-03357 mimics was significantly lower than that in NC group,indicating that t RF-03357down-regulated expression of HMBOX1 protein.(3)Immunohistochemistry assays showed that the expression of HMBOX1 was significantly decreased in tumor tissues compared with normal tissues.(4)Dual luciferase reporter gene assay showed that the t RF-03357 significantly inhibited HMBOX1 activation,whereas this effect was abolished by mutation of the binding site.(5)q RT-PCR in SK-OV-3 cells transfected with si RNA-1,si RNA-2 and si RNA-3showed that the expression level of HMBOX1 was significantly decreased in the HMBOX1 si RNA-1 group,indicating that si RNA-1 had the best interference effect and could be used for subsequent experiments.(6)CCK-8 and Transwell assay showed that,compared with the NC group,the proliferation,migration and invasion of SK-OV-3 cells transfected with HMBOX1 si RNA-1 were significantly increased,indicating that HMBOX1 inhibited the proliferation,migration and invasion of the SK-OV-3 cells.(7)Rescue experiment: The results showed that inhibition of t RF-03357 expression significantly reduced the proliferation,migration and invasion of SK-OV-3 cells,while transfection of HMBOX1 si RNA significantly reversed these effects.This indicates that tRF-03357 regulates the proliferation,migration and invasion of SK-OV-3 cells by HMBOX1.ConclusionsThis study revealed the t RF expression profiles of HGSOC,and identified a molecule of t RF-03357 that closely related to the development of ovarian cancer.Moreover,t RF-03357 can promote the cell proliferation,migration and invasion of HGSOC by regulated HMBOX1.t RF-03357 is a new molecule we discovered and the first reported its function in ovarian cancer.t RF-03357 plays an important role in the development of HGSOC and may act as a novel potential target for the diagnose and treatment of ovarian cancer patients. |
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