MicroRNA-22 Suppresses The Growth,Migration And Invation Of Colorectal Cancer Cells Through A Sp1 Negative Feedback Loop

BackgroundIn recent years,the research of Micro RNAs(MiRNAs)were gradually valued,and became one of the hot topics in the study of tumor.MiRNAs are small(approximately 18-24 nucleotides in length)non-coding RNAs that recognize and bind to partially complementary sequences of their target m RNA,resulting in either m RNA degradation or inhibition of its translation.Our previous studies have suggested that miR-22 is significantly down-regulated in CRC tissues and low expression of miR-22 correlated with liver metastases and poor prognosis.Specificity protein 1(Sp1)plays an important role in colorectal cancer(CRC),including growth and metastasis.Sp1(a member of transcription factor family)is a sequence specific DNA binding protein and play a important role in regulating gene expression.In colorectal cancer,Sp1 promotes cancer cell proliferation,invasion and migration.PTEN is a key tumor suppressor gene.Our previous studies discovered that PTEN can inhibited colorectal cancer cell growth and metastasis.Previous studies confirmed that PTEN by blocking AKT signaling pathway inhibits tumor growth and metastasis.A large number of studies have shown that miR-22 and PTEN were positively correlated.According to miR-22,Sp1 and PTEN play an important regulation role in colorectal cancer cell growth and metastasis,and the study found that miR22/Sp1 had a negative feedback loop in breast cancer,so we speculate that miR-22/Sp1 may be have a negative feedback loop in colorectal cancer,and then promote AKT phosphorylation by inhibiting PTEN expression to promote colorectal cancer cell growth and metastasis,but at present there is no report.ObjectiveDetecting miR-22 expression in colorectal cancer tissues,liver metastatic tissues and cells,studying the function of miR-22 on CRC cells growth and metastasis in vitro and vivo,studying the corelation of miR-22 and Sp1 in CRC cells,studying the effect of miR-22 on sigal pathway of PTEN/AKT?Through the above study We confrmed the mechanism of miR-22/Sp1 how to regulate the growth and metastasis of colorectal cancer,and then provided theoretical basis for miR-22 as a new target gene treatment of colorectal cancer.Methods1.MiR-22 expression in colorectal cancer tissues,liver metastatic tissues,normal tissues and cells was detectedQuantitative reverse transcription-PCR(q RT-PCR)was used to detecte the expression of miR-22 in primary colorectal cancer(CRC),their matched normal tissues and Liver metastatic tissues.Cell lines including SW620,Lo Vo,SW480,HT-29 and Caco-2 were detected also.2.The influence of miR-22 on CRC cells biological characteristics in vitro and in vivo(1)Over expression of miR-22 or miR-Nc SW620 and Lo Vo cell lines and anti-miR-22 or anti-miR-Nc SW480 and HT-29 cell lines were obtained by using Lipofectamine transfection.miR-22 virus supernatant was obtained by using lentivirus packaging system,and then infected it to SW620 to get stable over expression miR-22 cells.(2)Up-regulating miR-22 and down-regulate miR-22,using MTT assays,plate colony formation assays and Transwell migration and invasion assays to detect cell growth,colony formation,migration and invasion in vitro.(3)To assess the effect of over expression miR-22 on tumor growth and metastatic ability in vivo,we used nude mice subcutaneous forming tumor and metastases assays.3.Confirming Sp1 is the target gene of miR-22(1)We used bioinformatics algorithms of Target Scan,Pictar and micro RNA.org.to analysis potential molecular targets of miR-22.(2)The expression of Sp1 were analyzed in miR-22 or miR-nc and anti-miR-22 or miR-nc SW620 cell by Western blot.(3)We performed a luciferase reporter assay to test whether the miR-22 can bind to Sp1 3,UTR to testiy Sp1 is the target gene of miR-22.(4)To confirm the interaction between miR-22 and Sp1,we performed rescue experiment.We transfected SW620 and Lo Vo cell with miR-22 or miR-nc or miR-22+Sp1 or Sp1,the cell function assays were carried out to detect cell growth,colony formation,migration and invasion.4.Confirming miR-22/Sp1 negative feedback loop(1)We finded the promoter sequence of miR-22,and then predicting the transcription factor binding site using TRAVSFAC ?JASPAR and PROMO.(2)We transfected Sp1 or si Sp1 or Nc or si Nc to SW480 and HT-29 and detected the expression of miR-22 by q RT-PCR.(3)We performed a luciferase reporter assay to test whether the Sp1 can directly bind to miR-22 promoter in SW480 and HT-29 cells.(4)The direct association of Sp1 with miR-22 promoter was validated by Chromatin Immunoprecipitation(CHIP)analyses in CRC cells.(5)To confirm the interaction between miR-22 and Sp1,we performed rescue experiment.We transfected SW480 and HT-29 cell with Sp1 or vector or Sp1+miR-22,the MTT assays,plate colony formation assays and Transwell migration and invasion assays were carried out to detect cell growth,colony formation,migration and invasion.5.To confirm miR-22 suppress PTEN/AKT pathway by targeting Sp1 expression(1)Finding the promoter sequence of PTEN,and predicting the transcription factor binding site using TRAVSFAC?JASPAR and PROMO.(2)We transfected miR-22 or miR-nc or miR-22+Sp1 to SW480 and SW620 and detected the expression of p-AKT,T-AKT and PTEN by westernblotting.(3)We performed a luciferase reporter assay to validate the Sp1-binding sites in the PTEN promoter.6.Statistical analysisFor continuous variables,data are expressed as mean ± standard deviation(SD).Statistical significance between groups was analyzed by Student's t-test or Mann–Whitney U test,while categorical data were studied using chi-square test.The postoperative survival rate was analyzed with Kaplan–Meier method and the survival differences were compared by the log-rank test.Statistical analyses were conducted using IBM SPSS Statistics.Statistical significance was defined as P < 0.05.Results1.Down-regulation of miR-22 in human CRC was associated with disease progression and metastasis(1)MiR-22 was down-regulated in CRC tissueswe investigated the miR-22 expression level in an expanded CRC cohort consisting of 118 pairs of primary CRC and their matched normal 5.To confirm miR-22 suppress PTEN/AKT pathway by targeting tissues.76%(83 of 118)of the CRC patients had reduced miR-22 expression by at least 4-fold as compared with their matched normal tissues,and miR-22 correlated with tumor progression as it was decreased in the sequence from stage I to stage IV CRC.In addition,we found that the miR-22 expression levels were significantly down-regulated in the tissues of CRC patients with lymph node metastasis compared with those without lymph node metastasis(2)The expression of miR-22 was lower in the liver metastatic tissues than in primary tumors.(3)The expression of miR-22 was down-regulated in high metastatic cells.The expression of miR-22 was lower in high metastatic cells of SW620 and Lo Vo than in low metastatic cells of SW480,HT-29 and Caco-2.(4)The patients with low expression of miR-22 were more prone to recurrence and metastatic.Postoperative recurrence was observed in 28.2% cases(31/110)among 110 patients with stage I-III disease who underwent curative resection.Clinical investigations showed that the patients with low miR-22 levels possessed a higher risk of CRC recurrence and metastatic rate.Using Kaplan-Meier analysis,the results also demonstrated that CRC patients with low miR-22 levels displayed a higher recurrence rate than patients with high miR-22 expression.2.MiR-22 suppress colorectal cancer cells growth and metastatic in vitro an vivo(1)MiR-22 suppresses CRC cell proliferation,colony formation,migration and invasion abilities in vitro.We transfected SW620 and Lo Vo cell with miR-22 or miR-nc and transfected SW480 and HT-29 cell with anti-miR-22 or anti-miR-nc.Cell function assays(MTT,plate colony formation,Transwell migration and invasion)showed that over-expression miR-22 could down-regulate cell growth,colony formation,migration and invasion,but miR-22 expression inhibition could up-regulate cell growth,colony formation,migration and invasion.(2)Nude mice subcutaneous forming tumor trial showed that the subcutaneous tumor volume in miR-22 group was significantly decreased compared with control group.(3)Metastatic trial showed that 6 cases(total 6 cases)were detected small and less lung metastatic in SW620/miR-22 group and 6 cases were detected large and more lung metastatic(total 6 cases)in SW620/Control group.3.Sp1 is the target of miR-22(1)Using bioinformatics algorithms in the three commonly used databases including Target Scan,Pictar and micro RNA.org.predicated that Sp1 was a potential molecular target of miR-22.(2)Sp1 expression were detected with westernblotting in SW620 cells which transfected with miR-22 or miR-nc and in SW480 cells which transfected with anti-miR-22 or anti-miR-nc.The results indicated that miR-22 can suppress the expression of Sp1.(3)Wt Sp1 3'UTR and Mut Sp1 3'UTR dual-luciferase reporter vector were constructed.We inserted Wt Sp1 3'UTR and Mut Sp1 3'UTR sequences immediately downstream of the luciferase reporter gene and co-expressed these with either miR-22 or anti-miR-22 in HEK293 TT cells.miR-22 over-expression caused a significant decrease in relative luciferase activity,whereas miR-22 silencing increased the luciferase activity.In addition,mutation of the binding site of miR-22 in the 3'UTR of Sp1 abolished both the effect of miR-22 and anti-miR-22,confirming that miR-22 can bind to the Sp1 3'UTR.(4)MiR-22 suppresses CRC cell growth and invasion through target gene Sp1: We transfected SW620 and Lo Vo cell with miR-22 or miR-nc or miR-22+Sp1 and Sp1,the cell function trial showed that miR-22 can suppress cancer cell growth,colony formation,migration and invasion,but ectopic Sp1 can promote it.We performed a rescue experiment showed that reintroduction of Sp1 could partly reverse the inhibitory effects induced by miR-22 in CRC cells.4.Sp1 suppresses miR-22 expression to promote CRC cell growth and aggressiveness(1)Predicting the transcription factor binding site using TRAVSFAC?JASPAR and PROMO in the promoter sequence of miR-22,we found three binding sites of Sp1.(2)We transfected SW480 and HT-29 cells with Sp1 or Nc or si Sp1 or si Nc and detected the expression of miR-22 by q RT-PCR.The results showed that ectopic expression of Sp1 reduced miR-22 level,whereas depletion of Sp1 resulted in an increased miR-22 level.(3)In the miR-22 promoter,we generated amplicon A which overlaps site 1 and 2,amplicon B which overlaps site3 and inserted their into a PGL3-luciferase reporter as PGL3-Wt-Luc.The PGL3-Wt-Luc vectors and the pc DNA3.1-Sp1 plasmid were co-transfected into SW480 or HT-29 cells.The results showed that a reduction of the wild-type miR-22 promoter luciferase activity was observed upon up-regulation of Sp1.So confirming Sp1 could directly bind to the promoter sequence of miR-22.(4)CHIP experiment indicated that the binding of Sp1 protein in the miR-22 promoter is site A.(5)Sp1 promoted cells growth,migration and invasion of CRC cells through miR-22.we performed a rescue experiment by introducing pc DNA3.1-Sp1 plasmid or empty vector in the presence or absence of ectopic miR-22 expression in SW480 and HT29 cells.The results showed that miR-22 incompletely reversed the Sp1-induced proliferation,colony formation,migration and invasion in CRC cells.5.MiR-22 suppresses PTEN/AKT pathway by targeting Sp1 expression(1)Predicting the transcription factor binding site using TRAVSFAC?JASPAR and PROMO in the promoter sequence of PTEN,we found binding site of Sp1.(2)We transfected miR-22 or miR-nc or miR-22+Sp1 to SW480 and SW620 and detected the expression of p-AKT,T-AKT and PTEN by westernblotting.we found that miR-22 significantly increased PTEN expression and correspondingly reduced the expression of the phosphorylated AKT(p-AKT)without changing the total level of AKT(T-AKT).However,Sp1 over-expression attenuated the roles of miR-22 in the above effects.(3)The dual-luciferase reporter vector result showed that Sp1 could directly bind to PTEN promoter,so Sp1 can inhibited CRC cell growth and metastatic by inhibiting the expression of PYEN.Conclusion1.miR-22 Down-regulation in human CRC was associated with disease progression and metastasis;miR-22 expression was significantly lower in highly metastatic CRC cell lines;miR-22 expression was lower in the metastases than their primary tumors;CRC patients with low miR-22 levels had a higher recurrence rate than patients with high miR-22 expression.We reasoned that miR-22 play a suppressor in CRC.2.miR-22 could suppress the CRC cell growth,colony formation,migration and invasion in vivo and vitro.3.Sp1 was the target of miR-22.4.miR-22 suppressed the growth,migration and invasion of CRC cells through a Sp1 negative feedback loop.5.miR-22 suppressed the PTEN/AKT pathway by targeting Sp1 expression and then suppressed CRC cells growth and metastatic.