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Investigating the role of introns in the regulation of regenerating gene 1 expression.

Chai Y, Sun Y, Guo L, Li D, Ding Y - Oncol Lett (2014)

Bottom Line: The results indicated that the luciferase activity was significantly higher in cells transfected with recombinant vectors containing introns 2, 3, 4 or 5 than that in the cells transfected with an empty vector (P<0.05).Following incubation with gastrin, no significant difference was identified (P>0.05).In conclusion, gastrin may regulate the expression of the Reg1 gene via the interaction of the introns by binding to the transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Histology and Embryology, College of Basic Medicine, Zhengzhou University, Zhengzhou, Henan 450001, P.R. China.

ABSTRACT

Gastrin is a hormone that physiologically regulates gastric acid secretion and contributes to the maintenance of gastric epithelial architecture by regulating the expression of genes such as regenerating gene 1 (Reg1). Reg1 is involved in gastric carcinogenesis as an antiapoptotic factor. The current study explores the molecular mechanism of gastrin-regulated Reg1 expression in human gastric cancer cells. In total, five intron fragments of the Reg1 gene were cloned by polymerase chain reaction and inserted into luciferase reporter vector pGL3 to construct intron-luciferase reporter vectors. After confirmation by Xho I/Hind III digestion and DNA sequencing, the five constructs were transfected into the SGC7901 gastric cancer cell line. The luciferase activity of the cells transfected with each of the five constructs was detected following incubation without or with gastrin. The five intron fragments of Reg1 were also randomly labeled with digoxin as a probe, and nuclear proteins of gastric cancer cells were extracted following treatment with or without gastrin. Southwestern blotting was subsequently performed to detect transcription factors that bind to the introns. The results indicated that the luciferase activity was significantly higher in cells transfected with recombinant vectors containing introns 2, 3, 4 or 5 than that in the cells transfected with an empty vector (P<0.05). However, no statistically significant difference in luciferase activity was identified between cells transfected with pGL3-intron 1 and those transfected with pGL3-Basic (P>0.05). Following incubation with gastrin, no significant difference was identified (P>0.05). The five introns of Reg1 can bind a number of transcription factors and gastrin may affect this interaction. Introns 2-5 of Reg1 potentially have transcriptional control over gene expression in gastric cancer cells. In conclusion, gastrin may regulate the expression of the Reg1 gene via the interaction of the introns by binding to the transcription factors.

No MeSH data available.


Related in: MedlinePlus

Analysis of transcription factors by Southwestern blotting with the five introns of the Reg1 gene as probes: (A) intron 1, (B) intron 2, (C) intron 3, (D) intron 4 and (E) intron 5. Lane i, SGC7901 cells without gastrin incubation, as a control; lane ii, SGC7901 cells incubated with 1×10−8 mol/l gastrin; lane iii, SGC7901 cells incubated with 1×10−7 mol/l gastrin.
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f2-ol-09-02-0875: Analysis of transcription factors by Southwestern blotting with the five introns of the Reg1 gene as probes: (A) intron 1, (B) intron 2, (C) intron 3, (D) intron 4 and (E) intron 5. Lane i, SGC7901 cells without gastrin incubation, as a control; lane ii, SGC7901 cells incubated with 1×10−8 mol/l gastrin; lane iii, SGC7901 cells incubated with 1×10−7 mol/l gastrin.

Mentions: The five intron fragments were cloned by PCR and labeled with digoxin. The sensitivity of the five intron probes was 1 pg/μl, which was effective for detection. Results of the analysis of transcription factors through Southwestern blotting are shown in Figs. 2 and 3. For the probe of intron 1, 15 major bands of Tfs with different molecular weights were detected. Following gastrin treatment, the density of certain bands was evidently changed. Compared with the control group (without gastrin treatment), the density of bands 1, 2, 3, 4 and 7 was decreased significantly, and that of band 6 was increased significantly (P<0.05). For the cells incubated with 10−8 mol/l gastrin, the density of bands 1, 3, 4, 6, 9 and 10 was increased significantly when compared with that of the cells incubated with 10−7 mol/l gastrin (P<0.05).


Investigating the role of introns in the regulation of regenerating gene 1 expression.

Chai Y, Sun Y, Guo L, Li D, Ding Y - Oncol Lett (2014)

Analysis of transcription factors by Southwestern blotting with the five introns of the Reg1 gene as probes: (A) intron 1, (B) intron 2, (C) intron 3, (D) intron 4 and (E) intron 5. Lane i, SGC7901 cells without gastrin incubation, as a control; lane ii, SGC7901 cells incubated with 1×10−8 mol/l gastrin; lane iii, SGC7901 cells incubated with 1×10−7 mol/l gastrin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4301469&req=5

f2-ol-09-02-0875: Analysis of transcription factors by Southwestern blotting with the five introns of the Reg1 gene as probes: (A) intron 1, (B) intron 2, (C) intron 3, (D) intron 4 and (E) intron 5. Lane i, SGC7901 cells without gastrin incubation, as a control; lane ii, SGC7901 cells incubated with 1×10−8 mol/l gastrin; lane iii, SGC7901 cells incubated with 1×10−7 mol/l gastrin.
Mentions: The five intron fragments were cloned by PCR and labeled with digoxin. The sensitivity of the five intron probes was 1 pg/μl, which was effective for detection. Results of the analysis of transcription factors through Southwestern blotting are shown in Figs. 2 and 3. For the probe of intron 1, 15 major bands of Tfs with different molecular weights were detected. Following gastrin treatment, the density of certain bands was evidently changed. Compared with the control group (without gastrin treatment), the density of bands 1, 2, 3, 4 and 7 was decreased significantly, and that of band 6 was increased significantly (P<0.05). For the cells incubated with 10−8 mol/l gastrin, the density of bands 1, 3, 4, 6, 9 and 10 was increased significantly when compared with that of the cells incubated with 10−7 mol/l gastrin (P<0.05).

Bottom Line: The results indicated that the luciferase activity was significantly higher in cells transfected with recombinant vectors containing introns 2, 3, 4 or 5 than that in the cells transfected with an empty vector (P<0.05).Following incubation with gastrin, no significant difference was identified (P>0.05).In conclusion, gastrin may regulate the expression of the Reg1 gene via the interaction of the introns by binding to the transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Histology and Embryology, College of Basic Medicine, Zhengzhou University, Zhengzhou, Henan 450001, P.R. China.

ABSTRACT

Gastrin is a hormone that physiologically regulates gastric acid secretion and contributes to the maintenance of gastric epithelial architecture by regulating the expression of genes such as regenerating gene 1 (Reg1). Reg1 is involved in gastric carcinogenesis as an antiapoptotic factor. The current study explores the molecular mechanism of gastrin-regulated Reg1 expression in human gastric cancer cells. In total, five intron fragments of the Reg1 gene were cloned by polymerase chain reaction and inserted into luciferase reporter vector pGL3 to construct intron-luciferase reporter vectors. After confirmation by Xho I/Hind III digestion and DNA sequencing, the five constructs were transfected into the SGC7901 gastric cancer cell line. The luciferase activity of the cells transfected with each of the five constructs was detected following incubation without or with gastrin. The five intron fragments of Reg1 were also randomly labeled with digoxin as a probe, and nuclear proteins of gastric cancer cells were extracted following treatment with or without gastrin. Southwestern blotting was subsequently performed to detect transcription factors that bind to the introns. The results indicated that the luciferase activity was significantly higher in cells transfected with recombinant vectors containing introns 2, 3, 4 or 5 than that in the cells transfected with an empty vector (P<0.05). However, no statistically significant difference in luciferase activity was identified between cells transfected with pGL3-intron 1 and those transfected with pGL3-Basic (P>0.05). Following incubation with gastrin, no significant difference was identified (P>0.05). The five introns of Reg1 can bind a number of transcription factors and gastrin may affect this interaction. Introns 2-5 of Reg1 potentially have transcriptional control over gene expression in gastric cancer cells. In conclusion, gastrin may regulate the expression of the Reg1 gene via the interaction of the introns by binding to the transcription factors.

No MeSH data available.


Related in: MedlinePlus