We cultivated human lung carcinoma A549 cells in FK12 medium containing 10% fetal calf serum and plated them into 6-well dishes (5x10^5/well), grown for 20 h, and transfected them by the lipofectin method (New England Biolabs, Ipswich, Mass). We cotransfected test plasmid (HSPA1A-pGL3 4.0 mg) and internal Renilla luciferase control plasmid (phRL-TK, 1.0 mg) into cells, which were grown for 48 h. The cells were incubated at 42°C for 1 h and harvested 5 h after heat treatment. We prepared cell lysates, and measured luciferase activity with a luminometer by using a dual luciferase assay system. We measured each promoter activity directly by the ratio of the firefly luciferase level to the Renilla luciferase level. For each reporter construct, we performed five independent DNA extractions and phenotype measurements.

We drew blood samples, anticoagulated with 2% ethylenediaminetetraacetic acid, for DNA extraction and biochemical examination. We extracted genomic DNA from white blood cells using the proteinase K/phenol/chloroform purification method, followed by ethanol precipitation, and stored it in 10 mmol/L Tris-HCl, 1 mmol/L Na-EDTA, pH 8.0. We calibrated body weight and height, as well as blood pressure levels according to the last measurement at the time of survey. We calculated body mass index (BMI) as weight in kilograms divided by the square of height in meters. We measured blood pressure at the right arm with a conventional mercury sphygmomanometer after the subjects rested for at least 5 min. We determined plasma triglyceride, total cholesterol, high-density lipoprotein cholesterol, apolipoprotein A, and apolipoprotein B concentrations enzymatically using available kits and an autoanalyzer in Xinjiang Medical University. We estimated plasma low-density lipoprotein cholesterol using Friedewald's formula. We detected fasting plasma glucose by the Glucorder analyzer (Roche, Basel, Switzerland).

We determined the genotypes of the three studied variants by polymerase chain reaction (PCR) and restriction fragment length polymorphism techniques. With respect to the HSPA1L 2437 T/C variant, the sequences of the forward and reverse primers were GTC CCT GGG GCT GGA GAC GG and GTG ATG ATA GGG TTA CAC ATC TGC T, respectively. We carried out PCR amplification in a PTC-200 MJ Research Peltier Thermal Cycler (Bio-Rad Laboratories, Mass) for 35 cycles with denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 45 s. All amplifications were performed with 50-100 ng of DNA in a total volume of 25 μL containing 10 pmol of each primer, 200 mmol/L dNTPs, and 2.0 mmol/L.

We conducted database management and simple statistical calculations using SAS software version 9.1.3 (Institute Inc., Cary, NC). We compared the means of continuous variables using an unpaired t-test. We used the c2Age, years: 95.11 ± 4.81, 96.26 ± 5.89*, 94.21 ± 5.03, p=0.054 (T2437C TT (n=130), TC (n=54), CC (n=7) P trend). Age, years: 98.29 ± 6.40, 93.80 ± 4.41, 95.94 ± 5.72*, p=0.020. P<0.01 for A1267 G compared with AA; for G190C compared with CC; for T2437C compared with TC.

Because the G190C variant was located in the promoter region of HSPA1A, we assessed the promoter activity by measuring the ratio of firefly luciferase activity to Renilla luciferase activity in A549 cells (Fig 2). No significant differences were observed between 190 G and 190C alleles in both control (p=0.562) and heat-inducible (p=0.463) A549 cells.

The haplotype A-C-C was significantly lower (p=0.027) than haplotypes A-G-T, A-G-C and A-C-T in long-lived people (Table III). Haplotypes A-G-C and A-C-T had a significant 3.46- and 4.51-fold increased tendency for longevity, respectively (p=0.025 and p=0.016, respectively), when using the most common haplotype A-G-T as a statistical reference.

Table IV compares the age distributions by Hsp70 genotypes among long-lived people. Compared with 1267AA homozygotes, carriers with 1267GG genotype had a tendency to live longer (p<0.05). The same result was noted for 190GC heterozygotes in comparison with 190CC genotype carriers. As for T2437C variant, the different allele carriers (2437TT and CC) both had an increased tendency for longevity, especially for the DISCUSSION.

It is generally believed that all organisms at the cellular level respond to stress by synthesizing heat shock proteins at the expense of other proteins, and the ability of human cells to respond to heat stress decreases with aging. The possibility could not be excluded that longevity might be partly heat-stress mediated. Therefore, proteins that participate in heat-stress cascade and its regulation are potential candidates whose encoded genes may contain sequence variation contributing to the inherited tendency for longevity. We performed a candidate gene-longevity association study as well as the promoter activity assessment to explore the possible involvement of Hsp70 gene variants in susceptibility to longevity in a Xinjiang Hetian Uygur population. The Hetian city was listed as one of the four longevity districts in the world by the Institute of International Natural Medicine. This special population can offer us an interesting opportunity to establish an ideal model to decipher genetic underpinnings of natural longevity.

The amount of information gained about longevity status by putting two variants together using the MDR function was analyzed. The interaction information analysis indicated that variants G190C and T2437C had a strong synergistic effect, whereas the A1267 G variant was connected with the other by a green line, which suggests a loss of information that can be interpreted as redundancy or correlation. The pattern of high-risk (dark gray) and low-risk (light gray) genotype combinations was nonlinear across the nine two-locus genotype cells, which represented interaction as suggested by the interaction analysis.