The effect of silencing the endoplasmic reticulum stress-related protein calnexin on the proliferation, invasion, and migration of tongue squamous cell carcinoma cells / 口腔疾病防治

Objective@#To investigate the effect of silencing the endoplasmic reticulum stress-related protein calnexin on the proliferation, invasion, and migration of tongue squamous cell carcinoma cells. @* Methods @#Calnexin siRNA was transfected into SCC-9 and SCC-25 tongue squamous cell carcinoma cells, and the expression of calnexin was detected by qRT-PCR. The silencing effect of calnexin siRNA was further verified by Western blotting. CCK-8 assay was applied to detect the effect of silencing calnexin on the proliferation of tongue squamous cell carcinoma cells; Transwell assay was used to detect the effect of silencing calnexin on the invasion and migration of tongue squamous cell carcinoma cells.@* Results @#qRT-PCR showed that calnexin siRNA could effectively downregulate the expression of calnexin. Western blot analysis further confirmed the silencing effect of calnexin siRNA on calnexin. The CCK-8 assay showed that silencing calnexin expression on the 4th and 5th days could inhibit the proliferation of tongue squamous cell carcinoma cells, and the difference was statistically significant (P < 0.01). The Transwell assay showed that knockdown of calnexin could inhibit the invasion and migration of tongue squamous cell carcinoma cells (P < 0.001).@*Conclusion@#Knockdown of calnexin can inhibit the proliferation, invasion, and migration of tongue squamous cell carcinoma cells.

The potential role of calnexin in the activation of cardiac fibroblasts / 生物医学工程学杂志

Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca homeostasis, and Ca signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts , and the cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Age-related decline in expression of calnexin

Aging is accompanied by the changes in the cells that decrease their capacity to respond to various forms of stress. Cells are known to respond to stresses through expression of stress- response proteins, heat-shock proteins composed of molecular chaperones. Recent studies suggest that chaperone level and stress-induced chaperone expression could decrease with aging. The aim of the present study is to identify chaperones that show a significant change in protein expression with aging. We used an in vitro aging model system of human diploid fibroblasts (HDF). Proteome analysis of HDF showed that endoplasmic reticulum (ER) chaperone, calnexin, significantly decreased with aging. Oxidative stress-induced expression of calnexin also attenuated in old HDF compared to young cells. These findings suggest calnexin decreases with aging and might contribute to a cytoprotection in a variety of human age-related diseases.