An efficient method to genotype the polymorphisms of cholinergic nicotinic receptor subunit genes and their associations with COPD onset risk

<p><b>Background:</b> Single-nucleotide polymorphisms (SNPs) in the cholinergic nicotinic receptor subunit genes on chromosome 15q25.1, including <i>CHRNA3</i>, <i>CHRNB4</i> and <i>CHRNA5</i>, are well-established biomarkers of chronic obstructive pulmonary disease (COPD) and lung cancer. Thus, there is great demand for a rapid, easy and inexpensive method to detect these variations for purpose of risk prediction in large populations. <b>Aim of the Study:</b> The aim of this study was to establish an accurate and efficient method for genotyping <i>CHRN</i> SNPs and testing their association with age at onset of COPD in Chinese population as well as the clinical stage in COPD patients. <b>Materials and Methods:</b> We designed a method to specifically genotype 5 SNPs of <i>CHRN</i> genes based on a modified high-resolution melt (HRM) method and then validated the genotyping results by direct sequencing of 120 samples. We further used the HRM method to genotype these 5 SNPs in 1,013 COPD patients. <b>Results:</b> Requiring little time, few material costs and only a simplified protocol, the modified HRM method could accurately distinguish the genotypes of <i>CHRN</i> SNPs, demonstrating kappa coefficients >0.96 based on the results from direct sequencing. Furthermore, the data showed that the GG genotype of SNP rs56218866 was associated with a significantly earlier age of COPD onset than A (AA+AG) genotypes (61.0 ± 8.93 vs. 67.8 ± 9.88; <i>P</i> = 0.031), which was not found for the other SNPs. No significant association was observed between the COPD stages and any of the above SNPs. <b>Conclusion:</b> A simple, rapid and efficient HRM method was introduced for <i>CHRN</i> SNP genotyping and a suggestion that the SNP rs56218866A>G is associated with early-onset COPD in a Chinese population was found.</p

The effects of the Akt pathway on the nicotine-induced proliferation of RASMCs.

<p>(A) RASMCs were treated with 10 μM nicotine for 24 hours in the presence or absence of the PI3K inhibitor LY294002 or the GSK3β inhibitor SB216763. The cells were harvested by trypsinization and counted. In the LY294002 +10 μM nicotine-treated group, the cell numbers were significantly decreased, whereas the cell numbers in the SB216763 +10 μM nicotine-treated group increased significantly compared with the 10 μM nicotine-treated group. (B) RASMCs were treated with nicotine for 24 hours in the presence of LY294002. The EDU incorporation assays revealed that the percentage of S-phase cells dramatically decreased, whereas the percentage of S-phase cells in the SB216763 +10 μM nicotine-treated group increased compared with the nicotine-treated group. (C, D) RASMCs were treated with nicotine in the presence or absence of LY294002 or SB216763. The levels of Cyclin D1 expression at the 12 h time point and of RB phosphorylation at the 24 h time point were detected by Western blot analysis. The levels of Cyclin D1 expression and RB phosphorylation in the LY294002 +10 μM nicotine-treated group were significantly decreased, whereas they were significantly increased in the SB216763 +10 μM nicotine-treated group compared with the nicotine-treated group. *P<0.05, compared with the control group, N = 3; ^#P<0.05, compared with the nicotine-treated group(10 μM), N = 3.</p

The effects of nicotinic antagonists on the activation of Akt in RASMCs.

<p>(A) RASMCs were treated with 10 μM nicotine for 30 min in the presence or absence of the nonspecific nAchR subunit inhibitor MCA and the α7-antagonist MG624. The levels of Akt phosphorylation in MCA +10 μM nicotine-treated and MG624 +10 μM nicotine-treated groups were decreased significantly compared with the nicotine-treated group. (B) RASMCs were treated with 10 μM nicotine for 30 min in the presence of MCA or MG624. The levels of GSK3β phosphorylation were also decreased; the role of MG624 was particularly significant. *P<0.05, compared with the control group, N = 3; ^#P<0.05, compared with the nicotine-treated group (10 μM), N = 3.</p

RT-PCR Primers.

<p>Unique primers for each nAchR subunit were used for the relevant PCR reactions. All of the results were sequenced and compared with the known subunit sequences to confirm that the correct subunit was being amplified.</p

Nicotine promotes the proliferation of airway smooth muscle cells (RASMCs).

<p>(A) The cell numbers in the nicotine-treated groups were significantly increased, and the nicotine-mediated cell proliferation was time-dependent. (B) The percentage of the nicotine-treated cells in S-phase was significantly increased compared with the control group. (C) Nicotine promoted DNA replication; the DNA replication was increased by the nicotine treatment at every time point examined. The role of nicotine was particularly significant at the 24 h time point. *P<0.05, compared with the control group, N = 3; ^#P<0.001, compared with the control group, N = 3.</p

The effect of nAchRs on the nicotine-induced proliferation of RASMCs.

<p>(A, B) The RASMCs were treated with 10 μM nicotine for 24 h in the presence or absence of the nAchR subunit inhibitors MCA and MG624. The cells were harvested by trypsinization and then counted. The cell numbers in the nicotine- treated groups were significantly increased, whereas in the MCA + 10 μM nicotine-treated and MG624 +10 μM nicotine-treated groups, the cell numbers decreased significantly compared with the 10 μM nicotine-treated group. (C) Nicotine increased the levels of EDU incorporation at the 24 h time point. When the RASMCs were treated with nicotine in the presence of MCA or MG624, the percentage of S-phase cells was sharply reduced. (D) Nicotine increased the levels of Cyclin D1 at the 12 h time point, as detected by Western blot. When the RASMCs were treated with nicotine in the presence of MCA or MG624, the levels of Cyclin D1 decreased significantly compared with the nicotine-treated group. (E) Nicotine increased the level of RB phosphorylation at the 24 h time point as detected by Western blot. When the RASMCs were treated with nicotine in the presence of MCA or MG624, the levels of RB phosphorylation were significantly decreased compared with the nicotine-treated group. The role of MG624 was particularly significant. *P<0.05, compared with the control group, N = 3; ^#P<0.05, compared with the nicotine-treated group (10 μM), N = 3.</p

The nicotine-induced phosphorylation of Akt and GSK3β in RASMCs.

<p>(A, B) RASMCs were exposed to 10 μM nicotine for different treatment periods (0, 5, 15, 30, and 60 min). Nicotine treatment increased the Akt and GSK3β phosphorylation in a time-dependent manner in the RASMCs, as assessed by Western blot analysis. (C, D) RASMCs were exposed to the indicated concentrations of nicotine (0.1 μM–100 μM) for 30 min. The phosphorylation of Akt and GSK3β was increased by the nicotine treatment at every concentration examined. (E) RASMCs were treated with 10 μM nicotine for 30 min in the presence or absence of the PI3K inhibitor LY294002, or the GSK3β inhibitor SB216763, and the Akt phosphorylation was assessed by Western blot. Treatment with LY294002 +10 μM nicotine dramatically decreased the levels of phosphorylated Akt compared with treatment with 10 μM nicotine alone. In contrast, the GSK3β inhibitor SB216763 elicited no effect. When LY294002 was added to the culture medium without nicotine stimulation, the level of phosphorylated Akt was reduced compared with that of the control group. *P<0.05, compared with the control group, N = 3; ^#P<0.05, compared with the nicotine group (10 μM), N = 3.</p

The effects of the Akt pathway on the nicotine-induced proliferation of RASMCs.

<p>(A) RASMCs were treated with 10 μM nicotine for 24 hours in the presence or absence of the PI3K inhibitor LY294002 or the GSK3β inhibitor SB216763. The cells were harvested by trypsinization and counted. In the LY294002 +10 μM nicotine-treated group, the cell numbers were significantly decreased, whereas the cell numbers in the SB216763 +10 μM nicotine-treated group increased significantly compared with the 10 μM nicotine-treated group. (B) RASMCs were treated with nicotine for 24 hours in the presence of LY294002. The EDU incorporation assays revealed that the percentage of S-phase cells dramatically decreased, whereas the percentage of S-phase cells in the SB216763 +10 μM nicotine-treated group increased compared with the nicotine-treated group. (C, D) RASMCs were treated with nicotine in the presence or absence of LY294002 or SB216763. The levels of Cyclin D1 expression at the 12 h time point and of RB phosphorylation at the 24 h time point were detected by Western blot analysis. The levels of Cyclin D1 expression and RB phosphorylation in the LY294002 +10 μM nicotine-treated group were significantly decreased, whereas they were significantly increased in the SB216763 +10 μM nicotine-treated group compared with the nicotine-treated group. *P<0.05, compared with the control group, N = 3; ^#P<0.05, compared with the nicotine-treated group(10 μM), N = 3.</p

A schematic depicting the proliferative signaling induced by nicotine in RASMCs.

<p>Here, we show that nicotine functions similarly to a growth factor. The binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway. Akt triggers a network that positively regulates cell cycle progression through G1/S by phosphorylating and inactivating GSK3β, resulting in increased Cyclin D1. Cyclin D1 translocates into the nucleus and forms a holoenzyme with CDK4/6 to phosphorylate RB protein, resulting in the release of E2F. The E2F remains bound to the proliferative gene promoters, thereby facilitating S-phase entry. Thus, the signaling pathways induced by nicotine in the proliferation of RASMCs resemble those involved in growth factor stimulation. (This figure is referred to <a href="http://pathwaymaps.com/maps/474_map.png" target="_blank">http://pathwaymaps.com/maps/474_map.png</a>)</p