Effect of MiR-1264 on the proliferation and migration of Hep2 cell line / 医学研究生学报

Objective Previous study shows that the expression of mir-1264 is down-regulated in laryngeal carcinoma. This study aimed to investigate the effect of miR-1264 on the biological function of laryngeal carcinoma Hep2 cells and verify whether miR-1264 could down-regulate the expression of tumor suppressor gene la-ryngeal carcinoma related gene 1(LCRG1)in laryngeal carcinoma. Methods Real-time quantitative PCR was used to assess the miR-1264 expression in human laryngeal cancer tissues. There are three groups in the experiment:miR-1264 mimic/inhibitor group,mimic/inhibitor NC group and untransfected group. MTT and Transwell were applied to observe the effect of miR-1264 on the proliferation,migration and invasion capabilities of Hep2 cells. Luciferase experiment was used to verify the combination of miR-1264 and LCRG1 3'UTR.RT-PCR and Western blot were used to test the expression of miR-1264 and LCRG1 protein respectively. Results Compared with the adjacent laryngeal tissue,miR-1264 expression in human laryn-geal cancer tissues was significantly increased(P<0.05).Compared with NC control group and blank group,the proliferation,migration and invasion capabilities of Hep2 cells were significantly enhanced after they were transiently transfected with miR-1264 mimic(P<0.05);however,after Hep2 cells were transiently transfected with miR-1264 inhibitor,their proliferation,migration and invasion ca-pabilities were significantly inhibited(P<0.05). Luciferase experiment showed miR-1264 mimic could significantly decrease LCRG1 3'UTR luciferase activity,which was of statistical significance. After the verification of successful transient transfection,the results of further Western blot on LCRG1 protein expression showed that there was no significant difference between the experimental group and the mimic NC control group as well as the blank group(P>0.05). Conclusion miR-1264 is highly expressed in laryngeal cancer tissues. Though miR-1264 may not participate in down-regulating the expression of tumor suppressor gene LCRG1 in laryngeal carcino-ma,it can promote the proliferation,migration and invasion capabilities of Hep2 cells.

Cloning and Identification of Promoter of Human LCRG1 Gene / 生物化学与生物物理进展

Laryngeal carcinoma related gene 1(LCRG1) is a candidate tumor suppressor gene of Laryngeal carcinoma. To further investigate its transcriptional regulation, the transcriptional start sites for LCRG1 gene have been identified by 5′ RACE (rapid amplification of cDNA ends) based on the bioinformation analysis of LCRG1. Then eleven luciferase expression vectors which contained potential human LCRG1 gene promoter were constructed. Luciferase reporter assay indicated that LCRG1 promoter region was mainly located in -169～+127 region nearby the major transcriptional start site. These results suggested that the region (-169～+127) includes an essential promoter for human LCRG1 gene transcription.

Characterization of Key Regulatory Elements of LCRG1 Promoter / 生物化学与生物物理进展

LCRG1(laryngeal carcinoma related gene1,LCRG1),a new candidate tumor suppressor gene of laryngeal carcinoma.However,it is known little about the possible regulatory mechanisms of LCRG1 gene expression.Restriction endonuclease digestion was used to obtain a set of the 5',or 3'deletion mutants from the region(-169～+127) of the LCRG1 gene.It has been found that the minimal promoter of the LCRG1 gene is mapped at the region from-169～-57.Linker scanning mutational analysis in the region(-169～+127) of the LCRG1 gene was used to identify the crucial cis-elements within the promoter region,The key cis-elements are within the region from-137～-122.SP1,E2F1/DP1,EKLF and ZF9 transcription factor binding site sites were predicted in the region by bioinformatics analysis.Co-transfection with each of a panel of the expression plasmids of the known transcription factors with the relevant reporter construct indicates Sp1 is potent transcription factor for enhancement of the promoter activity,SP1 can also up-regulate the endogenous expression of LCRG1 gene.Electrophoretic mobility shift assay(EMSA) was applied to verify that the key cis-elements of LCRG1 gene exist sequence of Sp1 binding sites.The findings,which showed that the key cis-elements within the region from 137～-122 play an important role in expression of the LCRG1 gene,provide a novel evidence for further study of the function of LCRG1 gene.

Regulation of LCRG1 promoter transcription activity by Sp1 and Egr-1 / 医学研究生学报

Objective:The regulatory mechanisms of LCRG1 gene expression is undear,this study was to investigate the transcription regulation of the promoter of human LCRG1 gene by transcription factors Sp1 and Egr-1.Methods:Analysis of putative binding sites of transcription factors in the promoter of human LCRG1 gene by online program MatInspector,Co-transfection with the expression plasmids of the known transcription factors such as Sp1、wtEgr-1、mtEgr-1with the LCRG1 reporter construct analyze potent transcription factor for enhancement of the promoter activity.Results:SP1 and Egr-1 transcription factor binding site sites were predicted in the region by bioinformatics analysis,mtEgr-1 can enhance the promoter activity.Conclusion:mtEgr-1 may be involved in the regulatory mechanisms of LCRG1 gene expression

Cloning of 5'flank upstream regulation region of LCRG1 and the measurement of promoter activity / 中国癌症杂志

Background and purpose:The molecular regulation mechanism of the LCRG1 gnen is unclear;The study was designed to clarify the regulatory elements of LCRG1 gene in its 5'flanking region.Methods:Bioinformatics approaches were adopted and a putative promoter region was found in 5'flank upstream fragment of LCRG1 gene,5'flank upstream regulation fragment 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 gene was amplified by PCR using genomic DNA as template,construct was obtained by cloning DNA fragments into pGL3 reporter vector.The construct was then introduced into COS7 cells by Lipofectemin method for transient expression of reporter gene,and luciferase activities was measured by luciferase assay.Results:The sequence of 5'flank upstream regulation fragment 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 gene was successfully cloned and proved to be correct by DNA sequencing,the activity of pGL3-1776 was about 0.16-fold higher than that of pGL3-control cotransfection with PRL-TK and 35-fold higher than that of pGL3-basic cotransfection with PRL-TK.Conclusions:5'flank upstream regulation region 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 was cloned successfully,the fragment presented promoter activity.