الملخص
Objective: To observe the influence of TRPV6 gene silence on SW480 colon cancer cell biological behavior, change in the in-tracellular concentration of calcium, as well as influence of 1,25 (OH)2D3CaCl2and CuCl2on SD rat colonic neoplasm models. Method: SW480 colon cancer cells were infected using lentivirus particles. TRPV6 protein and mRNA expression was detected using immunohistochemical tests, Western blot, and PCR. Moreover, the proliferation, metastasis, and apoptosis of SW480 colon cancer cells were detected through MTT assay and metastasis and apoptosis experiments, and the concentration of Ca2+in SW480 colon cancer cells was measured using high-speed ionic imaging. The SD rat colon cancer model was established based on DMH, and were assigned into experimental group (DMH group, 15) and intervention group (DMH+1,25 (OH)2D3group, DMH+CuCl2group) and control group, 10 in each group. The SD rat colon cancer model is established based on DMH, given 1,25(OH)2D3(37.5 nmol/kg) and CuCl2(375 μmol/kg) separately as intervention. The occurrence of colonic neoplasms and glandular cancers in each group of rats was observed, and Western blot was employed for detection of the TRPV6 protein expression. Results: After the transfection of SW480 colon cancer cells by TRPV6-RNAi, the expression of TRPV6 mRNA and protein decreased, intracellular concentration of Ca2+decreased, proliferation and metastasis rate of SW480 colon cancer cells decreased, and apoptosis rate of these cells increased. The differences between the groups with intervention and the blank control group and negative control group showed statistical significance (P<0.05). The colon cancer occurrence rate in the control group was 0, while that of the DMH+1,25 (OH)2D3 group, DMH group, and DMH+CuCl2were 100%, 84.62%, and 33.33%, respectively. The TRPV6 protein expression was detected in all groups, while DMH+1,25(OH)2D3group was observed to exhibit the highest level of expression, followed by the DMH group, DMH+CuCl2group, and control group. The differences were of statistical significance (P<0.05). Conclusions: The proliferation and metastasis of SW480 colon cancer cells can be prohibited by lowering the concentration of Ca2+in the cells. Thus, the apoptosis of the cells can be induced. 1,25 (OH)2D3can help improve the expression of TRPV6 protein in experimental rat colon tissues and promote the formation of colon neoplasms. CuCl2can help lower the expression of TRPV6 protein in experimental rat colon tissues and prevent the formation of colon neoplasms.
الملخص
Objective:To investigate the role of TRPV5 and TRPV6 in intracellular calcium regulation and biological behaviors of SW480 colon cancer cells. Methods:qRT-PCR, Western blot, and immunocytochemistry were applied to determine the mRNA and protein ex-pression levels of TRPV5 and TRPV6 in SW480 colon cancer cell line upon treatment with TRPV5 and TRPV6 agonist, 1-25(OH)2D3, and inhibitor, CuCl2. The change of intracellular Ca2+level was examined with a confocal laser scanning microscope. Scratch test, MTT, and TUNEL assays were used to analyze the cell migration, proliferation, and apoptosis, respectively. Results:As an agonist of TRPV5 and TRPV6, 1-25(OH)2D3 significantly up-regulated the mRNA and protein expression levels of TRPV5 and TRPV6 in SW480 cell lines. On the other hand, CuCl2, being an inhibitor of TRPV5 and TRPV6, effectively down-regulated the TRPV5 and TRPV6 mRNA and protein expres-sion levels (P<0.05). The intracellular calcium concentration in SW480 cell line significantly increased upon treatment with 1-25 (OH)2D3, and significantly decreased with CuCl2 treatment (P<0.05). 1-25(OH)2D3 promoted cell proliferation and migration, and inhibit-ed apoptosis of SW480 cell in a time-and dose-dependent manner (P<0.05). However, CuCl2 significantly repressed cell proliferation and migration and induced apoptosis (P<0.05). Conclusion: TRPV5 and TRPV6 can affect the biological behaviors of colon cancer SW480 cells by regulating intracellular Ca2+level.
الملخص
Objective To create transient receptor potential vanilloid 6 (Trpv6) gene knockout mouse model, so as to pave a way for further research of its biological function and its role in bone metabolism in vivo. Methods Mouse genomic DNA sequence of Trpv6 gene was obtained from Ensembl database. Trpv6 gene knockout vector (pBR322-MK-Trpv6) was constructed. Trpv6 knockout vector was transferred into the embryonic stem (ES) cells by electroporation and screening of both G418 and Ganciclovoir resistant clones were performed routinely. The homologous recombined ES cell clones were identified by PCR. The correct homologously recombined ES cells were microinjected into C57BL/6J mouse blastocysts to obtain chimera mouse. Male mice with a chimera rate of 50% were mated with C57BL/6J female mice; the offsprings with gray fur were obtained, which were identified as heterozygote mice by PCR. Heterozygote mice were intercrossed to generate homozygote mice. Results Targeting vector PBR322-MK-Trpv6 were successfully constructed. A total of 24 correct homologously recombined clones were gained after electroporation. The efficiency of homologous recombination was 25%. Four male mice with a chimera rate of more than 50% were acquired after homologously recombined clones through microinjection. After the chimera mice were mated with C57BL/6J mice,57 grey-fur mice originated from ES cell were gained, including 17 (29. 8%) with heterozygous genotype. Heterozygote mice were intercrossed to generate homozygote mice. Western blotting analysisshowed no Trpv6 protein expression in homozygote mice. Conclusion We have successfully established Trpv6 gene knockout mouse model, and there is no embryonic lethality in homologous mutant mice.
الملخص
The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel transient receptor potential vanilloid 6 (TRPV6). The putatively-selected TRPV6 haplotype contains three candidate sites for functional differences, namely derived non-synonymous substitutions C157R, M378V and M681T. Functional electrophysiological characteristics between wild-type and mutant (C157R, M378V and M681T) TRPV6 proteins were investigated by cloning the mutant TRPV6 forms, transfecting cell lines, and carrying out electrophysiology experiments via patch clamp analysis. No statistically significant differences in biophysical channel function were found although one property, namely Ca2+-dependent inactivation, may show functionally-relevant differences between the wild-type and mutant TRPV6 proteins. This study shows that Ca2+-dependent inactivation is one of the good differentiation characteristics in TRPV6, and will be useful in an advancing our knowledge about TRPV6.