Study On The Regulation Mechanism Of Sp1 Negative Feedback To CDKN2B-AS1 In Human Gastric Carcinoma Cell Proliferation

Background:Transcription factor Sp1 regulate target gene expression by binding GC/GT box of promoter region with specific DNA binding protein sequence and play key role in the process of tumor proliferation, apoptosis, differentiation and formation. Thus it has been regarded as an important prognosis factor. The promoter of Sp1 contain a variety of transcription factor binding sites include Sp1 itself, and its status affect Sp1 transcriptional activity. It is remain unknown that whether abnormalities of Sp1 promoter initiate cell malignant transformation in gastric cancer or not. Furthermore, it has been indicated that long non-coding RNA(lnc RNA) was "disability" products of protein-coding genes and play important role in tumor carcinogenesis and development by gene modulation at genetic, transcriptional and post-transcriptional level. It has been reported that protein-coding m RNA transcribed from some transcription sites as well as lnc RNA at some location in genome, and lnc RNA often play feedback regulation in the expression of homologous protein-coding genes. Its very important and necessary. to screening lnc RNA that related to Sp1 and explore the mechanism between Sp1 and lnc RNA gene expression as Sp1 was critical for gastric cancer.Objetct: In the present study, correlation between mutation occurrence and expression of Sp1 protein will be established based on the mutations detection of Sp1 promoter; also lnc RNA closely related to Sp1 were screened with "expression" and "interference" combining strategy, and we explored effect of egulation mechanism of Sp1 negative feedback to CDKN2B-AS1 in human gastric carcinoma cell proliferation.Methods: In the present study, DNA of 101 pathological tissues cases of gastric cancer were extracted and clone sequencing. IHC was undertaken for scoring of Sp1 protein expression and standardized score, The correlation between expression of Sp1 and mutation were analyzed by Fisher’s exact test.Luciferase assay were carried out after lucifer reporter plasmid which contain 3 kinds mutation promoter have been constructed and transfected in 293 cells and The mutation type were not located in transcription factor binding sites with bioinformatics software analysis In order to explore the mechanism of Sp1 abnormal expression, we selected 33 candidate lnc RNAs which closed to carcinogenesis from long non-coding RNA for q RT-PCR detection after overexpression or RNA interference of Sp1 by plasmid or si RNA transfection with lipo2000 in SGC7901. Also the m RNA and protein of Sp1 were detected by q RT-PCR and western blot after DKN2B-AS1 RNAi in SGC7901 cell line.CCK8 counting exprement, flow cytometry, cell scratch and Transwell experiments were carried out to evaluate effects of CDKN2B-AS1 on gastric cancer cell biological function after CDKN2B-AS1 RNAi with SGC7901 cell line.Result: In the present study, altogether 3 kinds promoter mutation—-684C→T,-637 T→C and-617 T→G—of Sp1 were detected in 12 cases, the occurrence frequency were 3.96%(4 cases), 5.94%(6 cases) and 1.98%(2 cases) respectively. The correlation between expression of Sp1 and mutation were analyzed by Fisher’s exact test and the result showed no statistically significant difference(p>0.05). Luciferase assay showed that fluorescence of-684 C→T and-617 T→G group was significantly higher than control(p<0.05), but not to-637 T→C group(p=0.319). The mutation type were not located in transcription factor binding sites with bioinformatics software analysis CDKN2B-AS1, H19, KCNQ1OT1, MIR31 HG and XIST 5 were negative correlation with Sp1 expression level And CDKN2B-AS1 RNAi expression promote Sp1 m RNA and protein expression levels, and enhance fluorescence intensity of Lucifer report plasmid which contain Sp1 promoter,no matter mutation group or control.CCK8 counting exprement, flow cytometry, cell scratch and Transwell experiments showed that CDKN2B-AS1 promote the proliferation of SGC7901, but not to its apoptosis, cell migration and invasion.Conclusion:The present data indicates abnormal expression of Sp1 correlate with CDKN2B-AS1, and there is negative feed-loop between them. Also CDKN2B-AS1 affect activity of Sp1 promoter and promote the proliferation of SGC7901 cell line.