Studing Of The Mechanism Of CNEs Upstream And Downsream Of SHOX Regulating SHOX

ObjectiveTo establish U2 OS cell line with Conserved noncoding DNA elements(CNEs)knocked out is to Study the control activity of downstream CNE9 and CNE10 of short stature homeobox-containing gene(SHOX)by the functional deletion strategy;To study the relationship of upstream and downstream CNEs of SHOX gene and SHOXpromoter2,and analyze the regulatory mechanisms of CNES.Methods1.We established U2 OS cell line with CNE9 knocked out and U2 OS cell line with CNE10 knocked out by the usage of CRISPR/Cas9 technique;Sanger sequencing verified positive cell lines;Western-blot tested the protein expression of SHOX gene.2.To construct the recombinant vector by the replacement of the T7 promoter in the PsiCHECK-2 vector with SHOX promoter2(PsiCHECK-2-SHOXpromoter2);various CNEs were cloned into the recombinant vector of PsiCHECK-2-SHOXpromoter2.Recombinant vectors and the blank control group(Psi CHECK-2-SHOXpromoter2)were transfected into HEK293 T cells(human embryonic kidney 293 T cells),the relative activity of fluorescence was measured,and the measured values were statistically analyzed.Results1.Sanger sequencing verified positive cell lines,and U2 OS cell line with CNE9 knocked out and U2 OS cell line with CNE10 knocked out were acquired.2.The protein expression decreased and had a significant statistical difference among U2 OS cell line with CNE9 knocked out and U2 OS cell line with CNE10 knocked out compared with blank control group(without the processing of U2 OS cells)(CNE9,P=0.000<0.01;CNE10,P=0.003<0.01).3.PsiCHECK-2-SHOXpromoter2 vector was successfully constructed,and the recombinant vector containing CNE-5,CNE-3,CNE-2,CNE9,CNE10,CNE11,(PsiCHECK-2-SHOXpromoter2-CNE)were constructed respectively.4.Compared with Rluc/Fluc of the control group,the relative activity of the recombinant vector containing CNE-3,CNE-2 rose(P=0.000 <0.01,P=0.006<0.01),and the relative activity of the recombinant vector containing CNE 9,CNE 10,CNE 11 reduced(P=0.000<0.01,P=0.000<0.01,P=0.000<0.01).It had a significant statistical difference between Each of the recombinant vector containing CNE-3,CNE-2,CNE 9,CNE 10,CNE 11 and the control group.The recombinant vector containing CNE-5 compared with the control group was no obvious variation(P=0.323>0.05),the recombinant vector containing CNE-5 had no statistically significant difference.Conclusion1.We established U2 OS cell line with CNE9 knocked out and U2 OS cell line with CNE10 knocked out by the usage of CRISPR/Cas9 technique.In Western blot experiments,the expression quantity of SHOX protein of blank control group is higher than that of U2 OS cell line with CNE9 knocked out and U2 OS cell line with CNE10 knocked out,and that illustrated the lack of CNE9 or CNE10 reduced the SHOX protein expression.It proved that CNE9 CNE10 strengthened the transcription and translation of SHOX gene in loss of functional level,CNE9 and CNE10 had enhanced activity and that provided a cell model for further study of the regulatory mechanism of CNEs and SHOX gene.2.By dual luciferase reporter gene(dual-luciferase report gene,DLR)system it was verified that CNE-3,CNE-2 enhancing the activity of SHOXpromoter2 may control the expression of SHOX through SHOXpromter2 and CNE9,CNE10 inhibiting the activity SHOXpromoter2 may inhibit the transcription and translation of SHOX gene through SHOXpromoter2;In short,the upstream and downstream CNEs of SHOX gene may regulate the expression of SHOX gene by using a different promoter.