Activation of a calcium‐sensing receptor (Ca‐SR) leads to increased intracellular calcium concentration and altered cellular activities. The expression of Ca‐SR has been identified in both nonexcitable and excitable cells, including neurons and smooth muscle cells. Whether Ca‐SR was expressed and functioning in cardiac myocytes remained unclear. In the present study, the transcripts of Ca‐SR were identified in rat heart tissues using RT‐PCR that was further confirmed by sequence analysis. Ca‐SR proteins were detected in rat ventricular and atrial tissues as well as in isolated cardiac myocytes. Anti‐(Ca‐SR) Ig did not detect any specific bands after preadsorption with standard Ca‐SR antigens. An immunohistochemistry study revealed the presence of Ca‐SR in rat cardiac as well as other tissues. An increase in extracellular calcium or gadolinium induced a concentration‐dependent sustained increase in [Ca 2+ ] i in isolated ventricular myocytes from adult rats. Spermine (1–10 m m ) also increased [Ca 2+ ] i . Pre‐treatment of cardiac myocytes with thapsigargin or U73122 abolished the extracellular calcium, gadolinium or spermine‐induced increase in [Ca 2+ ] i . The blockade of Na + /Ca 2+ exchanger or voltage‐dependent calcium channels did not alter the extracellular calcium‐induced increase in [Ca 2+ ] i . Finally, extracellular calcium, gadolinium and spermine all increased intracellular inositol 1,4,5‐triphosphate (IP 3 ) levels. Our results demonstrated that Ca‐SR was expressed in cardiac tissue and cardiomyocytes and its function was regulated by extracellular calcium and spermine.
Objective: To explore the effects of both folic acid, p16 protein expression and their interaction on progression of cervical cancerization. Methods: Participants were pathologically diagnosed new cases, including 80 women with normal cervical (NC), 55 patients with low-grade cervical intraepithelial neoplasia (CINⅠ), 55 patients with high-grade cervical intraepithelial neoplasia (CINⅡ/Ⅲ) and 64 patients with cervical squamous cell carcinoma (SCC). Serum folate levels were detected by microbiological assay method while p16 protein expression levels were measured by Western-blot. In vitro, cervical cancer cell lines C33A (HPV negative) and Caski (HPV16 positive) were treated with different concentrations of folate. Proliferation and apoptosis of cells and the levels of p16 protein expression were measured in groups with different folic acid concentrations. Results: Results showed that the levels of serum folate were (5.96±3.93) ng/ml, (5.08±3.43) ng/ml, (3.92±2.59) ng/ml and (3.18±2.71) ng/ml, and the levels of p16 protein were 0.80±0.32, 1.33±0.52, 1.91±0.77, and 2.09±0.72 in the group of NC, CINⅠ, CINⅡ/Ⅲ and SCC, respectively. However, the levels of serum folate decreased (trend χ2=32.71, P<0.001) and p16 protein expression increased (trend χ2=56.06, P<0.001) gradually along with the severity of cervix lesions. An additive interaction was seen between serum folate deficiency and high expression of p16 protein in the CINⅠ, CINⅡ/Ⅲ and SCC group. Results in vitro showed that, with the increase of folate concentration, the inhibition rate of cell proliferation (C33A: r=0.928, P=0.003; Caski: r=0.962, P=0.001) and the rate on cell apoptosis (C33A: r=0.984, P<0.001; Caski: r=0.986, P<0.001) all increased but the levels of p16 protein expression (C33A: r=-0.817, P=0.025; Caski: r=-0.871, P=0.011) reduced. The proliferation inhibition rate (C33A: r=-0.935, P=0.002; Caski: r=-0.963, P=0.001) and apoptosis rate of cells (C33A: r=-0.844, P=0.017; Caski: r=-0.898, P=0.006) were negatively correlated with the levels of p16 protein expression. Conclusions: Our findings indicated that both serum folate deficiency and high expression of p16 protein could increase the risk of cervical cancer and cervix precancerous lesion, and there was an additive interaction between them. Our findings suggested that folic acid supplementation could reverse the abnormal expression of p16 protein, and effectively promote apoptosis and inhibit proliferation in cervical carcinoma cells.
To explore the effect of folic acid and DNA methyltransferase 1 (DNMT1) on cervical cancer and cervix precancerous lesion.100 patients with cervix squamous cell carcinoma (SCC), 101 patients with cervical intraepithelial neoplasm (CIN) and 109 patients with cervix inflammation (CI) diagnosed by histology were included in this study. Radioimmunoassay (RIA), polymerase chain reaction (PCR) and Western blot were used to detect the levels of serum folate, HPV16 infection and the expression of DNMT1 protein, respectively.The average levels of serum folate were (2.60 ± 1.61) ng/ml, (3.14 ± 2.08) ng/ml and (3.32 ± 1.74) ng/ml, and the expression of DNMT1 protein were 2.40 ± 0.99, 1.88 ± 0.33 and 0.89 ± 0.29 in the group of SCC, CIN and CI, respectively. The relationship of folate levels and DNMT1 protein expression showed inverse correlation (r = -0.186, P = 0.001). The results in our study indicated that there was an additive interaction between low-level of serum folate and high-expression of DNMT1 protein related to the risk of CIN and SCC, with OR value as 2.50 (95%CI: 1.21 - 9.22) and 6.03 (95%CI: 2.79 - 21.72) respectively. The relative excess risk of interaction (RERI), attributable proportion of interaction (API) and synergy index (S) were 0.92, 0.36 and 2.59 in the CIN group while 2.47, 0.41 and 1.96 in the SCC group.The low level of serum folate and high expression of DNMT1 protein seemed to be associated with high risk of cervical cancer and its precancerous lesion. It suggested that there might be a synergistic action between serum folate and DNMT1 in the progression of cervix carcinogenesis.
Objective To explore the effects of aberrant expression of DNA methyltransferase 1 (DNMT1) in cervical cancerization tissue and cervical cancer cells.Methods Cervical tissues were collected from 80 cases with a diagnosis of invasive cervix squamous cell carcinoma (SCC),53 cases with high-grade cervical intraepithelial neoplasia (CIN Ⅱ/Ⅲ),52 cases with low-grade cervical intraepithelial neoplasia (CIN Ⅰ)and 53 cases with normal cervix (NC).Meanwhile,Caski (HPV16-positive) and C33A (HPV-negative) cells selected from cervical cancer cell lines were cultured routinely in vitro.The expression of DNMT1 protein and mRNA were examined by Western blot analysis and real-time PCR (qRT-PCR) in the tissues and cells,respectively.Results The levels of DNMT1 protein were 1.33,1.84 and 2.28,and the Ct-ratios (DNMT1/β-actin) of DNMT1 mRNA were 1.27,1.27 and 1.26 in CIN Ⅰ,CIN Ⅱ/Ⅲand SCC group,respectively.Comparing with NC group,the expression of DNMT1 protein or mRNA was elevated in deficient cervical groups,with statistical significance (F =110.57,P ＜ 0.001,F =2.68,P =0.048).The expression levels of DNMT1 protein were increased steadily according to severity of the cervix lesions (x2tend =50.80,P ＜ 0.001),however,the expression of DNMT1 mRNA was not observed the same tendency (x2tend =3.63,P ＞ 0.05).The results from experiment in vitro showed that the levels of DNMT1 protein or mRNA were both higher in Caski cell than in C33A cell,especially for DNMT1 mRNA with significantly difference (t =7.134,P =0.002).Conclusion Aberrant expression of DNMT1 protein or mRNA could link with the risk of cervical cancerization by both transcriptional and posttranscriptional mechanisms.There would be a synergistic effect between overexpression of DNMT1 and HPV16 infection in the progression of cervix carcinogenesis.
Key words:
DNA methyltransferase 1; Aberrant expression; Cervical cancerization; Cervical cancer cells
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