The Molecular Mechanism Of Long Noncoding RNA SATB2-AS1 By Regulating SATB2 Expression In Progression Of Colorectal Carcinoma

BACKGROUND&OBJECTIVEColorectal cancer(CRC)is the second most common human malignancy.The prevalence of CRC is 9.7%in total population worldwide.Although since 1990 colorectal cancer mortality decline gradually at the rate of about 3%a year,but it is still the third caused of death in cancer,for social development and brought great threat to human life and health.Along with economic development and change of residents' diet structure,the morbidity of CRC increases at an annual.At present,treatment strategy for colorectal cancer are surgery,radiation and chemotherapy.Though after years of hardwork,the clinical curative effect had no significant improvement and the 5-year survival rate was increased in patients.Eventually,colorectal cancer patients inevitably appear tumor recurrence and metastasis.Therefore,looking for a new early diagnosis of colorectal cancer markers,to clarify the molecular mechanism of colorectal cancer development,identification of new therapeutic targets is crucial important for inhibition of colorectal tumor evolution,and reducing recurrence and mortality.The occurrence of malignant tumor is a function,multiple genes involved in multiple factors and multiple stages to the final formation of the extremely complex biological phenomenon.Past researches had been focused on tumor related genes coding protein gene.However,with the continuous development of high-throughput sequencing technology,large amount of long noncoding RNA were detected,their important value in activities of life was also revealed gradually.Providing a new perspective and important source for the study of new tumor related gene and has become a research hotspot.LncRNAs is a RNA molecular which do not have the complete open reading frame and can't encode protein.The length of transcription of LncRNAs is 200nt.Studies had shown that LncRNAs involved chromosome silence,genomic imprinting,chromatin modification,transcription activation,transcription regulation,protein function regulation,and other important life activities control process,as well as plays an extremely important role in a variety of human tumor development.In our previous studies,SATB2-ASl,one of the discriminating genes,was down-regulated in colorectal cancer tissues especially in which associated with lymph node metastasis,which tested by IncRNAs Microarray in without lymph node metastasis,associated with lymph node metastasis of colorectal cancer tissues and normal intestinal mucosal tissues.This finding shows that the disorder or dysfunction of SATB2-AS1 gene may has close relationship with colorectal cancer.However,the molecular role and mechanism of SATB2-AS1 in development and progression of colorectal cancer is not clear.In this study,we aim to to clarify the possible role and mechanism of SATB2-AS1 gene in the proliferation,ivasion and metastasis of colorectal cancer and it will provide new strategies for the prevention and treatment of colorectal cancer metastasis,which has important scientific significance and clinical application value.METHODS1.Real-time fluorescent quantitative PCR(Real-time PCR)was used to detect the expression of SATB2-AS1 in 50 pairs colorectal cancer and its ml.First we utilized real-time fluorescent quantitative PCR(Real-time PCR)to detect the expatched normal mucosa of fresh tissues as well as colorectal cancer cell lines and normal intestinal epithelial cell line;2.Stable expression of SATB2-AS1 cell lines and control cell lines were established by lentivirus;And then the biological function of LncR NA SATB2-AS1 in cell proliferation ability was investigated by CCK-8 assay,plate co-lony formation assay.The effect on cell cycle was detected by cell cycle experiments.Cell invasion ability was tested by Transwell invasion assay and scratch wound healing assay.Meanwhile,Western blot was used to detecte the expression of biomarkers related with proliferation and migration ability in stable overexprssed and knockdown of SATB2-AS1 cells.3.SATB2-AS1 knockdown CRC cell lines was established by RNAi.In vitro functional experiment were utilized to observe the biology changement of colorectal cancer cell.4.The expression of SATB2 in stable overexpressed SATB2-AS1 CRC cells was detected,in order to confirm the regulating effect of SATB2 expression and SATB2-AS1.The expression of SATB2 were detected in colorectal cancer tissues to further confirm the the relationship of SATB2 expression and SATB2-AS1.5.The bioinformatics analysis,RIP and RNA pull down were used to discovery the epigenetic trim that binding to SATB2-AS1.Furthermore,we identified the effects of SATB2-AS1 and the epigenetic trim that regulate the SATB2.6.Knockdown of the expresion of the epigenetic trim or SATB2 and then functional experiments were carried to observe the reversal effects to SATB2-AS1.RESULTS1.The detection of SATB2-AS1 in colorectal cancer tissues and cell linesReal-time PCR was used to detect the expression of SATB2-AS1 in nine colorectal cancer cell lines and normal intestinal mucosal epithelial cells,as well as 50 paired colorectal cancer tissues and the adjacent normal colorectal mucosa tissues.The results showed that SATB2-AS1 was downregulation in colorectal cancer cell lines compared with normal epithelial cells,in cancerous tissue the expression of SATB2-AS1 was also reduced compared with the matched normal tissues,and its expression in lymph node metastasis colorectal cancer tissues was lower than without lymph node metastasis tissues.2.The impact of SATB2-AS1 overexpression on the biological functions in colorectal cancer cell linesTo validate the biological functions of SATB2-AS1 in colorectal cancer cell line,we develpoed in vivo and in vitro experiments in established the stable overexpressioned SATB2-AS1 CRC cell lines.The results of CCK-8 array showed that:compared with the control cells,the proliferation rate of overexpressioned cells(M5/SATB2-AS1,LOVO/SATB2-AS1)slowed significantly in vitro(FLOVO=43.135,P<0.0001,FM5=61.586,P<0.0001);The results of plate clone formation experiments showed that:compared with the control cells,the clony number of overexpressioned cells(M5 and LOVO)reduced significantly(t LOVO=14.00,PLOVO=0.0002;t M5=3.675,PM5=0.0213);Flow cytometry cycle test results demonstrated that overexpression of SATB2-AS1 induced the percentage of cells in G1 phase increased dramaticly compared with the control grouptLOVO=31.14,PLOVO<0.0001;t M5=3.14,PM5=0.0348);Scratch healing experiment results showed that the movement ratio of the overexpresed SATB2-AS1 decreased significantly(tLOVO=7.106,PLOVO=0.0001;t M5=5.494,PM5=0.0006);Transwell invasion experiment results show that the number of cells invasion through the matrix decreased significantly in the overexpresed CRC cells than the control groups(tLOVO=11.13,PLOVO=0.0004;t M5=7.684,P M5=0.0015);In vivo animal experiment results illustrated that:In Nude mice subcutaneously into tumor experiment the tumor volume of overexpressioned cells reduced dramaticly compared with control group(F=13.707,P=0.01)and the positive rate of Ki67 was decreased significantly than the control group(t=14.60,P=0.0001).3.The effect of SATB2-AS1 knockdown on the biological functions in colorectal cancer cell linesThe expression of SATB2-AS1 was silenced in colorectal cancer cell lines SW480,and obtained SW480/NC and SW480/si cell lines.CCK-8 results showed that inhibited of SATB2-AS1 the proliferation rate increased significantly compared with the negative control(F=7.671,PSW480=0.009);Clone formation experiment showed that knockdown of SATB2-AS1 the clony number increased significantly compared with the negative control(tSW480=3.452,P SW480=0.0260);The result of Flow cytometry confirmed that cells proportion of G1 phase reduced in SATB2-AS1 knockdown cells,and S/G2/M cell proportion increased(t sW480=4.281,PSW480=0.0128);Scratches healing experiments showed that knockdown of SATB2-AS1 the migration distance of CRC cells was dramatically increased(t SW480=2.228,PSW480=0.0405).Transwell experiment showed that the number of migration cell that migrated from the polycarbonate membrane was significantly increased in downregulation of SATB2-AS1 cells(t SW480=4.217,PSW480=0.0056).4.The correlation of SATB2-AS1 and SATB2The mRNA expression of SATB2-AS1 and SATB2 were dctected by the Real-time PCR and the consequences demonstrated that there is significant correlation of SATB2-AS1and SATB2(r 2=0.3123,P<0.0001).After overexpression of SATB2-AS1 the expression of SATB2 was also increased both in mRNA and protein level(tLOVO=15.03,P LOVO=0.0001;tM5=11.62,P M5?0.003).These results showed that the SATB2-AS1 might positively regulate the expression mRNA and protein of SATB2.5.The mechanism of SATB2-AS1 regulation the expression of SATB25.1 The relationship of SATB2-AS1 and the stablity of SATB2RNA block experiment was carried to observe the effect of overexpression of SATB2-AS1 to SATB2 mRNA stability.The results upregulation of SATB2-AS1 the half-life of SATB2 mRNA had no significant changes(F=3.192,P=0.091).5.2 SATB2-AS1 can combined with acetyltransferase p300,but did not change its expression levelBioinformatics analysis found that SATB2-AS1 and SATB2 overlap parts(i.e.,both transcription SATB2 transcription start)near the histone locus and H3K9,H3K27 acetylation has high level state,the sites of H3K4 methylation went into a state of high level;Potential transcriptional regulation factor and its surroundings.The combination of SATB2-AS1 with acetyltransferase p300 was confirmed by RNA-IP and RNA-pulldown,however upregulation of SATB2-AS1 did not change the expression level of p300.5.3 Overexpression of SATB2-AS1 regulated the acetylation level of histone and enhanced the transcription SATB2Western blot was used to detect acetylation level of histone H3K9,H3K27,and the methylation level of H3K4.The results show that the overexpression of SATB2-AS1 promotes histone acetylation of H3K9,H3K27 loci and the expression of SATB2,but had no obvious effect on H3K4me3.5.4 The expression of SATB2 is regulated by the acetylation levelAcetylation enzyme(Trichostatin A,TSA)and acetyltransferase(C646)inhibitors were used to observe the expression changes of SATB2 in colorectal cancer cell.The results showed that the TSA treatment and overexpression of SATB2-AS1 can promote SATB2 mRNA expressiont(tSATB2-AS1=3.121,PSATB2-AS1=0.0355;tTSA=2.918,PTSA=0.0433),however the enhanced of SATB2 in overexpression of SATB2-AS1 can be reversed by C646,the same results was shown in the protein level.5.5 Inhibition of p300 or SATB2 can reversed the founction of SATB2-AS1We established the p300 or SATB2 knowndown cell lines in the overexpressed SATB2-AS1 cells.The funtional experiments showed that:CCK-8 array showd that when overexpression of SATB2-AS 1 the proliferation of CRC was inhibited(FLOVO/SATB2-AS1=24.035,PLOVO/SATB2-AS1<0.0001;FM5/SATB2-AS1=9.260,PM5/SATB2-AS1=0.004),whereas knockdown of p300 or SATB2 the proliferation of CRC was enhanced(FLOVO/SATB2-AS1/Sip300=121.734,P LOVO/SATB2-AS1/Sip300<0.0001,FLOVO/SATB2-AS1/SiSATB2-4.653,PLOVO/SATB2-AS1/SiSAB2=0.0096;/FM 5/SATB2-AS1/Sip3-00=93.461,PM5/SATB2-AS1/Sip300<0.0001;FM 5/SATB2-AS1/SiSATB2=0.138,PM5/SATB2-AS1/SiSATB2=0.713).These implied that overexpression of SATB2-AS1 decreased the proliferation ability in LOVO cell lines,whereas its proliferation ability get reply(increased again)when further knockdown the expression of p300 or SATB2.The plate clone formation experiment showed that when overexpression of SATB2-AS1 the colony number of CRC was decreased(tLOVO/SATB2-AS1=5.339,P LOVO/SATB2-AS1=0.0059;tM5/SATB2-AS1=6.841,PM5/SATB2-AS1=0.0024),however knockdown of p300 or SATB2 colony number of CRC was increased.Flow cytometry of the cell cycle demonstrated that upregulation of SATB2-AS1 the percentage of G1 phase in LOVO and M5 were increased(tLOVO=31.14,PLOVO<0.0001;t M5=3.14,PM5=0.0348),whereas knockdown the p300 or SATB2 in overexpressioned SATB2-AS1 cells accelerated the cell cycle and dismissed the G1 phase arrest(tLOVO/SATB2-AS1/Sip300=7.543,PLOVO/SATB2-AS1/Sip300=0.001 7;tLOVO/SATB2-AS1/SiSATB2=12.41,PLOVO/SATB2-AS1/SiSATB2-0.0002;tM5/SATB2-AS1/Sip300=2 1.06,PM5/SATB2-AS1/Sip300<0.0001;tM5/SATB2-AS1/SiSATB2=5.823,PM5/SATB2-AS1/SiSATB2=0.0043).Wound healing assays showed that overexpression of SATB2-AS1 the migration of LOVO and M5 were decreased significantly,and p300 or SATB2 downregulation increased the ability of migration(tLOVO/SATB2-AS1/Sip300=6.946,PLOVO/SATB2-AS1/Sip300=0.0023;tLOVO/SATB2-AS1/SiSATB2=4.654,PLOVO/SATB2-AS1/SiSATB 2=0.0096;tM5/SATB2-ASI/Sip300=20.95,P M5/SATB2-AS1/Sip300<0.0001;t M5/SATB2-AS1/SiSATB2=1 5.1 7,PM5/SATB2-AS1/SiSATB2=0.0001).Transwell showed that overexpresion of SATB2-AS1 the number of cells that migrated from the polycarbonate membrane were decreased(tLOVO=8.523,PLOVO=0.0010;t M5=17.49,PM5<0.0001),while knockdown of p300 or SATB2 the migration cells were increased(t LOVO/SATB2-AS1/Sip300=12.19,PLOVO/SATB2-AS1/Sip300=0.0003;tLOVO/SATB2-AS1/SiSATB2=6.603,PLOVOO/SATB2-AS1/SiSATB2=0.0037;tM5/SATB2-AS1/Sip300=25.36,PM5/SATB2-AS1/Sip300<O.0001;tM5/SATB2-AS1/SiSATB2=14.50,PM5.SATB2-AS1/SiSATB2 <0.0001).All these results further confirmed that SATB2-AS1 regulated the expression of SATB2 by acetylated histone H3K9 and H3K27 near the transcription start site of SATB2 though recruiting p300,so as to exert its biological functions in colorectal cancer.CONCLUSION1.SATB2-AS1 was downregulated in CRC.2.Downregulation of SATB2-AS1 can promote the proliferation,miagration and invasion of CRC cells.3.There was a positive correlation between SATB2-AS1 and its positive-sense chain SATB2,and SATB2-AS1 can increase the expression of SATB2.4.SATB2-AS1 can increase the expression of SATB2 by acetylated histone H3K9 and H3K27 near the transcription start site of SATB2 though recruiting p300.5.SATB2-AS1 medicates the expression of SATB2 through the epigenetic modification and participate in the development and progression of colorectal cancer.