The Expression Pattern And Functional Mechanism Exploration Of Antisense Long Non-Coding RNA SPINT1-AS1 In Colorectal Cancer

Background:Colorectal cancer(CRC)is one of the most important causes of human death caused by malignant tumors,and its incidence in China is increasing year by year.Because of the low prevalence of colonoscopy,lack of effective tumor markers and low public awareness,early diagnosis of CRC is difficult,and most CRC patients were found in the middle or late stages with poor prognosis.Natural antisense transcripts(NATs),as a new kind of biological macromolecule that has been proved to affect the development of many kinds of cancers,have great significance in exploring their potential as tumor biomarkers and studying their mechanism of action for the development of new early diagnostic indicators and therapeutic targets of cancers.Objective:To study the expression level and clinical relevance of serine peptidase inhibitor,Kunitz type 1 antisense RNA1(SPINT1-AS1),a natural antisense transcript,in CRC.To evaluate the prognostic and therapeutic monitoring value of SPINT1-AS1 in CRC.Methods:1 Microarray analysis:Screening NATs differentially expressed in 6 pairs CRC and adjacent normal tissues by microarray analysis,and identifying SPINT1-AS1 as the biomarker according to relevant literature,and verifying its differential expression in large clinical samples.2 Sample collection:According to inclusion criteria,150 pairs of CRC tissues and adjacent normal tissues were selected to extract total RNA.3 Detection of SPINT1-AS1:Detecting SPINT1-AS1 expression levels in CRC and adjacent normal tissues by strand-specific RT-qPCR and analysising their expression differences.4 Interaction between SPINT1-AS1 and its complementary sense RNA:The interaction between SPINT1-AS1 and its complementary sense RNA SPINT1 mRNA was demonstrated by dual luciferase reporter assay.5 Prognostic value of SPINT1-AS1 in CRC:According to the median expression of SPINT1-AS1,150 patients with CRC were divided into SPINT1-AS1 high expression group and SPINT1-AS1 low expression group.Survival analysis was used to evaluate the impact of clinical parameters on disease-free survival(DFS)of CRC patients.6 Detection of serum exosomal SPINT1-AS1:Collecting blood samples from patients with CRC before and after operation,isolating serum exosomes,detecting differences before and after operation,and evaluating the predictive effect of SPINT1-AS1 on surgical treatment of CRC.Results:1 Screening and identification of NATs differentially expressed in CRC.The expression of antisense IncRNA SPINT1-AS1 in CRC tissues was significantly increased(P=0.015)as suggested by microarray analysis of 6 pairs of CRC tissues and adjacent normal tissues.The expression of SPINT1-AS1 was further detected in 150 pairs of CRC and adjacent normal tissues.The results of strand-specific RT-qPCR showed that the expression of SPINT1-AS1 in CRC tissues was significantly higher than that in adjacent normal tissues(P<0.001),and the expression of SPINTI-AS1 in 116 CRC tissues increased more than 2-fold.Receiver operating characteristic(ROC)curve showed that the expression of SPINT1-AS1 could be used to differentiate CRC tissues from adjacent normal tissues.Area under the ROC curve(AUC)was 0.865(95%CI,0.821-0.902).2 SPINT1-AS1 was negatively correlated with the expression of its sense transcript SPINT1 mRNA.We also detected the expression of SPINT1 mRNA in the same 150 pairs of CRC and adjacent normal tissues,and found that the expression of SPINT1 mRNA in CRC tissues was significantly lower than that in adjacent normal tissues(P<0.001),and there was an obviously negative correlation between SPINT1-AS1 expression and its sense transcript(r=-0.701,P=0.001).The result of dual luciferase reporter assay showed that SPINT1-AS1 overexpression decreased the luciferase activity of the overlapping region in SPENT mRNA 5’UTR(P=0.020).3 Increased expression of SPINT1-AS1 in patients with CRC is associated with regional lymph node metastasis(P<0·001)and distant metastasis(P<0.001).There was no significant correlation between SPINT1-AS1 expression level and age(P=0.747),gender(P=0.218),location of tumors(P=0.959),differentiation of tumors(P=0.647),T stage(P=0.430)and size of tumors(P=0.410).4 Survival analysis showed that there was no significant correlation between the expression of SPINT1-AS1 and the overall survival(OS)time(HR=0.5861;95%CI,0.282-1.271;P=0.152),but the high expression of SPINT1-AS1 was related to the shortened DFS(HR=0.2547;95%CI,0.143-0.452;P<0.001).Univariate Cox proportional hazards regression model analysis showed that T stage(HR=2.259;95%CI,1.013-5.036;P=0.046),regional lymph node metastasis(HR=3.657;95%CI,1.820-7.348;P<0.001),distant metastasis(HR=5.606;95%CI,3.010-10.439;P<0.001)and high expression level of SPINT1-AS1(HR=4.355;95%CI,2.219-8.549;P<0.001)were correlated with DFS.Multivariate Cox analysis showed that SPINT1-AS1 expression(HR=2.519;95%CI,1.180-5.378;P=0.017)and distant metastasis(HR=2.770;95%C1,1.398-5.489;P=0.003)could be used as independent prognostic indicators for CRC patients’ DFS.5 The expression of SPINT1-AS1 and SPINT1 mRNA in exosomes was detected by strand-specific RT-qPCR.The results showed that the expression level of serum exosomal SPINT1-AS1 after operation was significantly lower than that before operation(P=0.001)in CRC patients,while the expression level of SPINT1 mRNA had no significant change(P=0.062).Conclusions:1 SPINT1-AS1 is associated with adverse clinicopathological characteristics and poor prognosis of CRC.2 SPINT1-AS1 can be used as an independent prognostic and therapeutic monitoring biomarker for CRC.Objective:Natural antisense transcripts(NATs),as the hotspot of non-coding RNA research,have been proved to play an important role in the occurrence and development of cancers.The study of its function and mechanism in cancer is of great significance in screening new biomarkers and finding new targets for cancer treatment.Our previous studies have shown that high expression of SPINT1-AS1 in colorectal cancer(CRC)tissues is associated with poor prognosis.In this study,we intend to explore the carcinogenic effect and mechanism of SPINT1-AS1 in CRC through in vitro and in vivo experiments,and to evaluate the potential of exosomal SPINT1-AS1 as a biomarker for diagnosis and prognosis of CRC by large clinical samples.Methods:1 Lentivirus vectors were constructed to overexpress or knock down SPINT1-AS1 and overexpress SPINT1 mRNA,and stablely transfected CRC cells were screened after lentivirus transfection;2 CCK-8 assay was used to detect the effect of SPINT1-AS1 and SPINT1 mRNA on the proliferation of CRC cells;3 Colony formation assay was used to detect the effect of SPINT1-AS1 and SPINT1 mRNA on colony formation ability of CRC cells;4 CRC cells stably overexpressing SPINT1-AS1 were used to carry out xenograft experiment in nude mice,to detect the effect of SPINT1-AS1 on CRC cells tumorigenic ability in vivo;5 Flow cytometry was used to detect the effect of SPINT1-AS1 and SPINT1 mRNA on apoptosis of CRC cells;6 TUNEL assay was used to detect the effect of SPINT1-AS1 and SPINT1 mRNA on apoptosis of CRC cells;7 Flow cytometry was used to detect the effect of SPINT1-AS1 and SPINT1 mRNA on cell cycle distribution of CRC cells;8 Western blot and confocal microscopy were used to detect the effect of SPINT1-AS1 and SPINT1 mRNA on autophagy of CRC cells.9 Exosomes secreted by CRC cells after overexpression of SPINTI-AS1 were isolated and the effect of exosome-transmitted SPINTI1-AS1 on CRC cells was explored;10 The efficacy of exosomal SPINT1-AS1 as a diagnostic and prognostic biomarker for CRC was validated by a large cohort of clinical samples.Results:1 Recombinant viruses with SPINT1-AS1 overexpression/knockdown and SPENT1 mRNA overexpression were constructed using lentiviral vectors,and stably transfected CRC cell lines were screened;2 Both in vitro and in vivo exp.eriments showed that SPINT1-AS1 promoted proliferation ability of CRC cells:CCK-8 assay and colony formation assay proved that overexpression of SPINT1-AS1 could promote the proliferation ability of CRC cells in vivo;xenograft experiment showed that the increased expression of SPINT1-AS1 enhanced the tumorigenicity of HCT116 cells in vivo.Overexpression of SPINT1-AS 1 in HCT116 cells significantly increased the tumor volume of mice;3 SPINT1-AS1 overexpression inhibited CRC cell apoptosis and promoted cyto-protective autophagy;4 SPINT1-AS1 down-regulated CRC cells had similar cellular phenotypes as CRC cells stably overexpressing SPINT1 mRNA which showed up as diminished proliferation ability,increased apoptotic cell proportion and decreased autophagy activity;5 Exosome mediates SPINT1-AS1 transfer and promotes CRC cells malignant behaviors;6 Large clinical samples analysis showed that serum exosomal SPINT1-AS1 could be used as a non-invasive biomarker for diagnosis and prognosis of CRC.Conclusion:1 In CRC,SPINT1-AS1 plays an oncogenic role by negatively regulating the expression of SPINT1 mRNA;2 Exosomete-mediated intercellular communication promotes the cancer-promoting effect of SPINT1-AS1;2 Serum exosomal SPINT1-AS1 can be used as a non-invasive biomarker for diagnosis and prognosis of CRC.