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Apolipoprotein M increases the expression of Vitamin D receptor mRNA in colorectal cancer cells detected with duplex fluorescence reverse transcription-quantitative polymerase chain reaction

Yu, Miao mei; Yao, Shuang; Luo, Kai Ming; Mu, Qin feng; Yu, Yang; Luo, Guang-Hua and Xu, Ning LU (2017) In Molecular Medicine Reports 16(2). p.1167-1172
Abstract

Apolipoprotein M (ApoM) and the Vitamin D receptor (VDR) are apolipoproteins predominantly presenting in high-density lipoprotein (HDL) and a karyophilic protein belonging to the steroid-thyroid receptor superfamily, respectively. Previous studies have demonstrated that ApoM and VDR are associated with cholesterol metabolism, immune and colorectal cancer regulation. In order to investigate whether ApoM affected the expression of VDR in colorectal cancer cells, a single-tube duplex fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) system was developed to simultaneously detect the mRNA levels of VDR and GAPDH in HT-29 cells overexpressing ApoM. The results demonstrated that the amplification products were... (More)

Apolipoprotein M (ApoM) and the Vitamin D receptor (VDR) are apolipoproteins predominantly presenting in high-density lipoprotein (HDL) and a karyophilic protein belonging to the steroid-thyroid receptor superfamily, respectively. Previous studies have demonstrated that ApoM and VDR are associated with cholesterol metabolism, immune and colorectal cancer regulation. In order to investigate whether ApoM affected the expression of VDR in colorectal cancer cells, a single-tube duplex fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) system was developed to simultaneously detect the mRNA levels of VDR and GAPDH in HT-29 cells overexpressing ApoM. The results demonstrated that the amplification products were confirmed as the specific fragment of VDR/GAPDH using the DNA sequencing instrument. The sensitivity, linear range, correlation coefficient, amplification efficiency, intra-assay and inter-assay coefficients of variation were 40 copies/μl, 4.00×101-4.00×105 copies/μl, 0.999, 92.42%, 0.09-0.34% and 0.32-0.65% for VDR, and 40 copies/μl, 400×101-4.00×105 copies/μl, 0.999, 98.07%, 0.19-0.43% and 0.40-0.75% for GAPDH, respectively. The results indicated that the expression of VDR mRNA was significantly higher in HT-29 cells overexpressing ApoM, compared with the negative control group (P<0.05). In conclusion, the current study successfully developed the single-tube duplex RT-qPCR to simultaneously detect VDR and GAPDH expression in colorectal cancer cells. The methodology results demonstrated that the duplex RT-qPCR system with high sensitivity and specificity could ensure the objectivity and credibility of the detection. The present study confirmed that ApoM significantly increased the expression of VDR in HT-29 cells. In addition, it was hypothesized that ApoM may be involved in antineoplastic activity via the upregulation of VDR expression, which may provide novel directions for the investigation of ApoM in cancer.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Apolipoprotein M, Colorectal cancer, Glyceraldehyde-3-phosphate dehydrogenase, Polymerase chain reaction, QPCR, Vitamin D receptor
in
Molecular Medicine Reports
volume
16
issue
2
pages
6 pages
publisher
Spandidos Publications Ltd
external identifiers
  • scopus:85022338984
  • wos:000405079300024
ISSN
1791-2997
DOI
10.3892/mmr.2017.6716
language
English
LU publication?
yes
id
99d4562a-8724-4c84-a80a-dd3c6deec1ff
date added to LUP
2017-07-26 13:42:35
date last changed
2017-09-18 11:39:54
@article{99d4562a-8724-4c84-a80a-dd3c6deec1ff,
  abstract     = {<p>Apolipoprotein M (ApoM) and the Vitamin D receptor (VDR) are apolipoproteins predominantly presenting in high-density lipoprotein (HDL) and a karyophilic protein belonging to the steroid-thyroid receptor superfamily, respectively. Previous studies have demonstrated that ApoM and VDR are associated with cholesterol metabolism, immune and colorectal cancer regulation. In order to investigate whether ApoM affected the expression of VDR in colorectal cancer cells, a single-tube duplex fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) system was developed to simultaneously detect the mRNA levels of VDR and GAPDH in HT-29 cells overexpressing ApoM. The results demonstrated that the amplification products were confirmed as the specific fragment of VDR/GAPDH using the DNA sequencing instrument. The sensitivity, linear range, correlation coefficient, amplification efficiency, intra-assay and inter-assay coefficients of variation were 40 copies/μl, 4.00×10<sup>1</sup>-4.00×10<sup>5</sup> copies/μl, 0.999, 92.42%, 0.09-0.34% and 0.32-0.65% for VDR, and 40 copies/μl, 400×10<sup>1</sup>-4.00×10<sup>5</sup> copies/μl, 0.999, 98.07%, 0.19-0.43% and 0.40-0.75% for GAPDH, respectively. The results indicated that the expression of VDR mRNA was significantly higher in HT-29 cells overexpressing ApoM, compared with the negative control group (P&lt;0.05). In conclusion, the current study successfully developed the single-tube duplex RT-qPCR to simultaneously detect VDR and GAPDH expression in colorectal cancer cells. The methodology results demonstrated that the duplex RT-qPCR system with high sensitivity and specificity could ensure the objectivity and credibility of the detection. The present study confirmed that ApoM significantly increased the expression of VDR in HT-29 cells. In addition, it was hypothesized that ApoM may be involved in antineoplastic activity via the upregulation of VDR expression, which may provide novel directions for the investigation of ApoM in cancer.</p>},
  author       = {Yu, Miao mei and Yao, Shuang and Luo, Kai Ming and Mu, Qin feng and Yu, Yang and Luo, Guang-Hua and Xu, Ning},
  issn         = {1791-2997},
  keyword      = {Apolipoprotein M,Colorectal cancer,Glyceraldehyde-3-phosphate dehydrogenase,Polymerase chain reaction,QPCR,Vitamin D receptor},
  language     = {eng},
  month        = {08},
  number       = {2},
  pages        = {1167--1172},
  publisher    = {Spandidos Publications Ltd},
  series       = {Molecular Medicine Reports},
  title        = {Apolipoprotein M increases the expression of Vitamin D receptor mRNA in colorectal cancer cells detected with duplex fluorescence reverse transcription-quantitative polymerase chain reaction},
  url          = {http://dx.doi.org/10.3892/mmr.2017.6716},
  volume       = {16},
  year         = {2017},
}