Establishment of Real-time PCR for Detection of <i>CHRNA7</i> Gene

LIU Yiqiao; SUN Zhihua; QIAN Jiang; ZHANGSUN Dongting; LUO Sulan

doi:10.15886/j.cnki.rdswxb.2019.02.013

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Volume 10 Issue 2

Jun. 2019

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Article Navigation > Journal of Tropical Biology > 2019 > 10(2): 178-183

LIU Yiqiao, SUN Zhihua, QIAN Jiang, ZHANGSUN Dongting, LUO Sulan. Establishment of Real-time PCR for Detection of CHRNA7 Gene[J]. Journal of Tropical Biology, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013

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LIU Yiqiao, SUN Zhihua, QIAN Jiang, ZHANGSUN Dongting, LUO Sulan. Establishment of Real-time PCR for Detection of CHRNA7 Gene[J]. Journal of Tropical Biology, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013

Establishment of Real-time PCR for Detection of CHRNA7 Gene

doi: 10.15886/j.cnki.rdswxb.2019.02.013

College of Marine, Hainan University/Key Laboratory of Tropical Biological Resources of Ministry of Education/Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China

Received Date: 2019-03-25
Rev Recd Date: 2019-04-05

Abstract

In order to detect the expression of α7 nicotine acetylcholine receptor（nAChR） subunit gene（CHRNA7）, the primers were designed according to the sequence of human CHRNA7 gene, and then CHRNA7 gene was amplified by PCR. After being cloned into pMD-18 T vector, the recombinant plasmid was sequenced and prepared. The recombinant plasmids were gradient diluted and used as templates to establish standard curves for real-time PCR. The sensitivity and repeatability of the real-time PCR were carried out. A real-time PCR method for detecting CHRNA7 was successfully established. With this method CHRNA7 gene was detected 10 copies·μL^-1 at the lowest. The variation coefficients of five replications of the three templates at 10~4, 10⁵ and 10⁶ copies·μL^-1 were 1.22%, 1.90% and 2.63%, respectively, which indicated the real-time PCR detection had a good repeatability. Furthermore, the detection of α7 nAChR subunit gene expression in human cervical cancer cell line SiHa and human normal cervical cell line Ect1/E6 E7 showed that the expression of α7 nAChR subunit in SiHa cells was significantly lower than that in Ect1/E6 E7 cells（P=0.015）.
- α7 nAChR,
- cervical cancer,
- real-time PCR

References

[1]	KALAMIDA D, POULAS K, AVRAMOPOULOU V, et al.Muscle and neuronal nicotinic acetylcholine receptors:Structure, function and pathogenicity[J].FEBS J., 2007, 274 (15):3799-3845.
[2]	EGLETON R D, BROWN K C, DASGUPTA P.Nicotinic acetylcholine receptors in cancer:multiple roles in proliferation and inhibition of apoptosis[J].Trends Pharmacol Sci., 2008, 29 (3):151-158.
[3]	DANG N, MENG X, SONG H.Nicotinic acetylcholine receptors and cancer[J].Biomed Rep., 2016, 4 (5):515-518.
[4]	SUZUKI T, HIDE I, MATSUBARA A, et al.Microglial alpha7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role[J].J.Neurosci Res., 2006, 83 (8):1461-1470.
[5]	MEI D, ZHAO L, CHEN B, et al.α-Conotoxin ImI-modified polymeric micelles as potential nanocarriers for targeted docetaxel delivery to alpha7-nAChR overexpressed non-small cell lung cancer[J].Drug Deliv, 2018, 25 (1):493-503.
[6]	CHEN R J, HO Y S, GUO H R, et al.Rapid Activation of Stat3 and ERK1/2 by Nicotine Modulates Cell Proliferation in Human Bladder Cancer Cells[J].Toxicological Sciences, 2007, 104 (2):283-293.
[7]	WEI P L, CHANG Y J, HO Y S, et al.Tobacco-specific carcinogen enhances colon cancer cell migration through alpha7-nicotinic acetylcholine receptor[J].Ann Surg, 2009, 249 (6):978-985.
[8]	WANG W, CHIN-SHENG H, KUO L J, et al.NNK enhances cell migration through alpha7-nicotinic acetylcholine receptor accompanied by increased of fibronectin expression in gastric cancer[J].Ann Surg Oncol, 2012, 19 (S3):580-S588.
[9]	JIN T, HAO J, FAN D.Nicotine induces aberrant hypermethylation of tumor suppressor genes in pancreatic epithelial ductal cells[J].Biochem Biophys Res Commun, 2018, 499 (4):934-940.
[10]	MARTINEZ A K, JENSEN K, HALL C, et al.Nicotine promotes cholangiocarcinoma growth in xenograft mice[J].Am J.Pathol, 2017, 187 (5):1093-1105.
[11]	CALLEJA-MACIAS I E, KALANTARI M, BERNARD H U.Cholinergic signaling through nicotinic acetylcholine receptors stimulates the proliferation of cervical cancer cells:an explanation for the molecular role of tobacco smoking in cervical carcinogenesis?[J].Int.J.Cancer, 2009, 124 (5):1090-1096.
[12]	王晓楠, 程民, 胡世莲.鳞状细胞癌抗原1 mRNA实时荧光定量RT-PCR检测方法建立与应用[J].中华肿瘤防治杂志, 2018, 25 (7):471-475.
[13]	张敬梅, 顾亦韧, 李江淩, 等.乌金猪、青峪猪和成华猪免疫器官多种免疫调节基因表达的比较[J].畜牧与兽医, 2018, 50 (10):43-48.
[14]	PAPKE R L, PORTER PAPKE J K.Comparative pharmacology of rat and human alpha7 nAChR conducted with net charge analysis[J].Br.J.Pharmacol, 2002, 137 (1):49-61.
[15]	UTESHEV V V.alpha7 nicotinic ACh receptors as a ligand-gated source of Ca2+ ions:the search for a Ca2+ optimum[J].Adv Exp.Med.Biol., 2012, 740:603-638.
[16]	GRANDO S A.Connections of nicotine to cancer[J].Nat.Rev.Cancer, 2014, 14 (6):419-429.
[17]	ZHAO Y.The Oncogenic Functions of Nicotinic Acetylcholine Receptors[J].J.Oncol, 2016 (2).doi: 10.1155/2016/9650481.
[18]	CUI H, LAN X, LU S, et al.Preparation of monoclonal antibody against human KIAA0100 protein and Northern blot analysis of human KIAA0100 gene[J].J.Pharm Anal, 2017, 7 (3):190-195.
[19]	GUO Y, XIE X, GUO C, et al.Effect of electro-acupuncture on gene expression in heart of rats with stress-induced pre-hypertension based on gene chip technology[J].J.Tradit Chin Med, 2015, 35 (3):285-294.
[20]	LI W F, DAI H, OU Q, et al.Overexpression of microRNA-30a-5p inhibits liver cancer cell proliferation and induces apoptosis by targeting MTDH/PTEN/AKT pathway[J].Tumour Biol., 2016, 37 (5):5885-5895.
[21]	NAVARRO E, SERRANO-HERAS G, CASTANO M J, et al.Real-time PCR detection chemistry[J].Clin Chim Acta, 2015, 439:231-250.
[22]	RODRIGUEZ A, RODRIGUEZ M, CORDOBA J J, et al.Design of primers and probes for quantitative real-time PCR methods[J].Methods Mol Biol., 2015, 1275:31-56.
[23]	MALANDRAKI I, BERIS D, ISAIOGLOU I, et al.Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR[J].Plos One, 2017, 12 (7):1-14.
[24]	SINGH A, PANDEY G K.Primer design using Primer Express (R) for SYBR Green-based quantitative PCR[J].Methods Mol Biol., 2015, 1275:153-164.

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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Establishment of Real-time PCR for Detection of CHRNA7 Gene

College of Marine, Hainan University/Key Laboratory of Tropical Biological Resources of Ministry of Education/Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China

Received Date: 2019-03-25

Rev Recd Date: 2019-04-05

Key words:

Abstract: In order to detect the expression of α7 nicotine acetylcholine receptor（nAChR） subunit gene（CHRNA7）, the primers were designed according to the sequence of human CHRNA7 gene, and then CHRNA7 gene was amplified by PCR. After being cloned into pMD-18 T vector, the recombinant plasmid was sequenced and prepared. The recombinant plasmids were gradient diluted and used as templates to establish standard curves for real-time PCR. The sensitivity and repeatability of the real-time PCR were carried out. A real-time PCR method for detecting CHRNA7 was successfully established. With this method CHRNA7 gene was detected 10 copies·μL^-1 at the lowest. The variation coefficients of five replications of the three templates at 10~4, 10⁵ and 10⁶ copies·μL^-1 were 1.22%, 1.90% and 2.63%, respectively, which indicated the real-time PCR detection had a good repeatability. Furthermore, the detection of α7 nAChR subunit gene expression in human cervical cancer cell line SiHa and human normal cervical cell line Ect1/E6 E7 showed that the expression of α7 nAChR subunit in SiHa cells was significantly lower than that in Ect1/E6 E7 cells（P=0.015）.

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Reference (24)

[1]	KALAMIDA D, POULAS K, AVRAMOPOULOU V, et al.Muscle and neuronal nicotinic acetylcholine receptors:Structure, function and pathogenicity[J].FEBS J., 2007, 274 (15):3799-3845.
[2]	EGLETON R D, BROWN K C, DASGUPTA P.Nicotinic acetylcholine receptors in cancer:multiple roles in proliferation and inhibition of apoptosis[J].Trends Pharmacol Sci., 2008, 29 (3):151-158.
[3]	DANG N, MENG X, SONG H.Nicotinic acetylcholine receptors and cancer[J].Biomed Rep., 2016, 4 (5):515-518.
[4]	SUZUKI T, HIDE I, MATSUBARA A, et al.Microglial alpha7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role[J].J.Neurosci Res., 2006, 83 (8):1461-1470.
[5]	MEI D, ZHAO L, CHEN B, et al.α-Conotoxin ImI-modified polymeric micelles as potential nanocarriers for targeted docetaxel delivery to alpha7-nAChR overexpressed non-small cell lung cancer[J].Drug Deliv, 2018, 25 (1):493-503.
[6]	CHEN R J, HO Y S, GUO H R, et al.Rapid Activation of Stat3 and ERK1/2 by Nicotine Modulates Cell Proliferation in Human Bladder Cancer Cells[J].Toxicological Sciences, 2007, 104 (2):283-293.
[7]	WEI P L, CHANG Y J, HO Y S, et al.Tobacco-specific carcinogen enhances colon cancer cell migration through alpha7-nicotinic acetylcholine receptor[J].Ann Surg, 2009, 249 (6):978-985.
[8]	WANG W, CHIN-SHENG H, KUO L J, et al.NNK enhances cell migration through alpha7-nicotinic acetylcholine receptor accompanied by increased of fibronectin expression in gastric cancer[J].Ann Surg Oncol, 2012, 19 (S3):580-S588.
[9]	JIN T, HAO J, FAN D.Nicotine induces aberrant hypermethylation of tumor suppressor genes in pancreatic epithelial ductal cells[J].Biochem Biophys Res Commun, 2018, 499 (4):934-940.
[10]	MARTINEZ A K, JENSEN K, HALL C, et al.Nicotine promotes cholangiocarcinoma growth in xenograft mice[J].Am J.Pathol, 2017, 187 (5):1093-1105.
[11]	CALLEJA-MACIAS I E, KALANTARI M, BERNARD H U.Cholinergic signaling through nicotinic acetylcholine receptors stimulates the proliferation of cervical cancer cells:an explanation for the molecular role of tobacco smoking in cervical carcinogenesis?[J].Int.J.Cancer, 2009, 124 (5):1090-1096.
[12]	王晓楠, 程民, 胡世莲.鳞状细胞癌抗原1 mRNA实时荧光定量RT-PCR检测方法建立与应用[J].中华肿瘤防治杂志, 2018, 25 (7):471-475.
[13]	张敬梅, 顾亦韧, 李江淩, 等.乌金猪、青峪猪和成华猪免疫器官多种免疫调节基因表达的比较[J].畜牧与兽医, 2018, 50 (10):43-48.
[14]	PAPKE R L, PORTER PAPKE J K.Comparative pharmacology of rat and human alpha7 nAChR conducted with net charge analysis[J].Br.J.Pharmacol, 2002, 137 (1):49-61.
[15]	UTESHEV V V.alpha7 nicotinic ACh receptors as a ligand-gated source of Ca2+ ions:the search for a Ca2+ optimum[J].Adv Exp.Med.Biol., 2012, 740:603-638.
[16]	GRANDO S A.Connections of nicotine to cancer[J].Nat.Rev.Cancer, 2014, 14 (6):419-429.
[17]	ZHAO Y.The Oncogenic Functions of Nicotinic Acetylcholine Receptors[J].J.Oncol, 2016 (2).doi:10.1155/2016/9650481.
[18]	CUI H, LAN X, LU S, et al.Preparation of monoclonal antibody against human KIAA0100 protein and Northern blot analysis of human KIAA0100 gene[J].J.Pharm Anal, 2017, 7 (3):190-195.
[19]	GUO Y, XIE X, GUO C, et al.Effect of electro-acupuncture on gene expression in heart of rats with stress-induced pre-hypertension based on gene chip technology[J].J.Tradit Chin Med, 2015, 35 (3):285-294.
[20]	LI W F, DAI H, OU Q, et al.Overexpression of microRNA-30a-5p inhibits liver cancer cell proliferation and induces apoptosis by targeting MTDH/PTEN/AKT pathway[J].Tumour Biol., 2016, 37 (5):5885-5895.
[21]	NAVARRO E, SERRANO-HERAS G, CASTANO M J, et al.Real-time PCR detection chemistry[J].Clin Chim Acta, 2015, 439:231-250.
[22]	RODRIGUEZ A, RODRIGUEZ M, CORDOBA J J, et al.Design of primers and probes for quantitative real-time PCR methods[J].Methods Mol Biol., 2015, 1275:31-56.
[23]	MALANDRAKI I, BERIS D, ISAIOGLOU I, et al.Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR[J].Plos One, 2017, 12 (7):1-14.
[24]	SINGH A, PANDEY G K.Primer design using Primer Express (R) for SYBR Green-based quantitative PCR[J].Methods Mol Biol., 2015, 1275:153-164.

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Establishment of Real-time PCR for Detection of CHRNA7 Gene

doi: 10.15886/j.cnki.rdswxb.2019.02.013

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通讯作者: 陈斌, bchen63@163.com

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Establishment of Real-time PCR for Detection of CHRNA7 Gene

doi: 10.15886/j.cnki.rdswxb.2019.02.013

College of Marine, Hainan University/Key Laboratory of Tropical Biological Resources of Ministry of Education/Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China

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Establishment of Real-time PCR for Detection of CHRNA7 Gene

doi: 10.15886/j.cnki.rdswxb.2019.02.013

Abstract

References

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通讯作者: 陈斌, bchen63@163.com

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Establishment of Real-time PCR for Detection of CHRNA7 Gene

doi: 10.15886/j.cnki.rdswxb.2019.02.013

College of Marine, Hainan University/Key Laboratory of Tropical Biological Resources of Ministry of Education/Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China

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