Hepatocyte growth factor （HGF） is a glycoprotein that induces prostate cancer cell proliferation, migration and invasion. The activation of transient receptor potential canonical 6 （TRPC6） channels is considered i...Hepatocyte growth factor （HGF） is a glycoprotein that induces prostate cancer cell proliferation, migration and invasion. The activation of transient receptor potential canonical 6 （TRPC6） channels is considered important in promoting prostate cancer cell proliferation. In this study, we assessed the role of endogenous TRPC6 channels in the HGF-induced cell proliferation of prostate cancer. Reverse transcription-PCR and Western blotting were used to investigate TRPC6 expression. Electrophysiological techniques （whole-cell patch clamp configuration） and Ca^2＋ imaging analysis were used to investigate the channel activity in cells. The effects of TRPC6 channels on cell cycle progression, cell apoptosis and cell growth were also examined. TRPC6 and c-MET were expressed in DU145 and PC3 cells. In addition, functional TRPC6 channels were present in DU145 and PC3 cells, and TRPC6 knockdown suppressed TRPC-Iike currents evoked by oleoyl-2-acetyl-sn-glycerol （OAG）. Inhibition of TRPC6 channels in DU145 and PC3 cells abolished OAG- and HGF-induced Ca^2＋ entry. Furthermore, inhibition of TRPC6 channels arrested DU145 and PC3 cells at the G2/M phase and suppressed HGF-induced cell proliferation. Collectively, our results indicate that TRPC6 has an important role in HGF-induced DU145 and PC3 cell proliferation.展开更多
We investigated the expression of transient receptor potential canonical 6 （TRPC6） protein in benign and malignant human prostate tissues and in prostate cancer cell lines and the association with the stage, grade a...We investigated the expression of transient receptor potential canonical 6 （TRPC6） protein in benign and malignant human prostate tissues and in prostate cancer cell lines and the association with the stage, grade and androgen responsiveness of the tumors. Immunohistochemical techniques, Western blot and reverse transcription polymerase chain reaction （RT-PCR） were used to investigate TRPC6 expression. TRPC6 protein was detected in 9 of 20 （45.0%） of benign prostatic hyperplasia （BPH） cases, and there was a significant difference compared with prostate cancer （129 of 149 [86.6%]） （P 〈 0.01）. TRPC6 expression was associated with the histological grade and extraprostatic extension （P 〈 0.01）. Tumors of higher stage tended to have a higher frequency of TRPC6 protein staining, but the difference was not significant among T2, T3 and T4. TRPC6 expression difference between androgen-independent （AI） tumors and androgen-dependent （AD） tumors was not statistically significant. TRPC6 was also observed in prostate cancer cell lines. In summary, TRPC6 is detected in benign and malignant human prostate tissues and prostate cancer cell lines and is associated with the histological grade, Gleason score and extraprostatic extension of prostate cancer.展开更多
Objective:To study the expression of TRPC6 among prostate cancer cells,establish high expression cell lines of TRPC6,and to provide potential cell mode for prostate cancer oncogenesis and development.Methods:Occurrenc...Objective:To study the expression of TRPC6 among prostate cancer cells,establish high expression cell lines of TRPC6,and to provide potential cell mode for prostate cancer oncogenesis and development.Methods:Occurrence and development of prostate cancer cells,PC3,PC—3m DU145,22 rvl,LNCaP and normal prostate epithelial cells in the PrEC TRPC6 expression level were detected by QPCR method.Calcium phosphate transfection method was used to package retrovirus pLEGFP-Nl-TRPC6 and pLEGFP-Nl-vector and infect the prostate cancer cells,a stable high expression of TRPC6 prostate cancer cells.Sable cell lines of TRPC6,matrix metalloproteinase(MMP)2,MMP9 expression was detected by QPCR and Western blot.Change of cell invasion ability was detected by Transwell.Results:The expression level of prostate cancer cells TRPC6 were higher than control group PrEC cells.Among TPRC6 the expression of cell line PC 3 transfer potential wre the lowest,and high transfer cell tone PC-3M express was the highest.Real-time fluorescent quantitative PCR and western blot results showed that after filter,the seventh generation of cell TRPC6 protein and mRNA expression levels were higher than the control group obviously.Transwell experimental results showed that the overexpression of TRPC6could promote the invasion ability of PC.3 prostate cancer cells.Conclusions:TRPC6 expressed in prostate cancer cells is in disorder,and its action may be associated with the invasion and metastasis of prostate cancer cells;successful establishment of stable high expression of TRPC6prostate cancer cells primarily confirm the invasion-trigger ability of TRPC6 on prostate cancer,and lay down the foundation for exploring the TRPC6's role in the occurrence and development of prostate cancer展开更多
Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes ...Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.展开更多
This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced ...This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein （GFP）-contained plasmids （pGC） to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN＋TRPC6 shRNA transfected group and PAN＋scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.展开更多
文摘Hepatocyte growth factor （HGF） is a glycoprotein that induces prostate cancer cell proliferation, migration and invasion. The activation of transient receptor potential canonical 6 （TRPC6） channels is considered important in promoting prostate cancer cell proliferation. In this study, we assessed the role of endogenous TRPC6 channels in the HGF-induced cell proliferation of prostate cancer. Reverse transcription-PCR and Western blotting were used to investigate TRPC6 expression. Electrophysiological techniques （whole-cell patch clamp configuration） and Ca^2＋ imaging analysis were used to investigate the channel activity in cells. The effects of TRPC6 channels on cell cycle progression, cell apoptosis and cell growth were also examined. TRPC6 and c-MET were expressed in DU145 and PC3 cells. In addition, functional TRPC6 channels were present in DU145 and PC3 cells, and TRPC6 knockdown suppressed TRPC-Iike currents evoked by oleoyl-2-acetyl-sn-glycerol （OAG）. Inhibition of TRPC6 channels in DU145 and PC3 cells abolished OAG- and HGF-induced Ca^2＋ entry. Furthermore, inhibition of TRPC6 channels arrested DU145 and PC3 cells at the G2/M phase and suppressed HGF-induced cell proliferation. Collectively, our results indicate that TRPC6 has an important role in HGF-induced DU145 and PC3 cell proliferation.
文摘We investigated the expression of transient receptor potential canonical 6 （TRPC6） protein in benign and malignant human prostate tissues and in prostate cancer cell lines and the association with the stage, grade and androgen responsiveness of the tumors. Immunohistochemical techniques, Western blot and reverse transcription polymerase chain reaction （RT-PCR） were used to investigate TRPC6 expression. TRPC6 protein was detected in 9 of 20 （45.0%） of benign prostatic hyperplasia （BPH） cases, and there was a significant difference compared with prostate cancer （129 of 149 [86.6%]） （P 〈 0.01）. TRPC6 expression was associated with the histological grade and extraprostatic extension （P 〈 0.01）. Tumors of higher stage tended to have a higher frequency of TRPC6 protein staining, but the difference was not significant among T2, T3 and T4. TRPC6 expression difference between androgen-independent （AI） tumors and androgen-dependent （AD） tumors was not statistically significant. TRPC6 was also observed in prostate cancer cell lines. In summary, TRPC6 is detected in benign and malignant human prostate tissues and prostate cancer cell lines and is associated with the histological grade, Gleason score and extraprostatic extension of prostate cancer.
基金supported by Sichuan Province Department of Health(Grant Project:130188)
文摘Objective:To study the expression of TRPC6 among prostate cancer cells,establish high expression cell lines of TRPC6,and to provide potential cell mode for prostate cancer oncogenesis and development.Methods:Occurrence and development of prostate cancer cells,PC3,PC—3m DU145,22 rvl,LNCaP and normal prostate epithelial cells in the PrEC TRPC6 expression level were detected by QPCR method.Calcium phosphate transfection method was used to package retrovirus pLEGFP-Nl-TRPC6 and pLEGFP-Nl-vector and infect the prostate cancer cells,a stable high expression of TRPC6 prostate cancer cells.Sable cell lines of TRPC6,matrix metalloproteinase(MMP)2,MMP9 expression was detected by QPCR and Western blot.Change of cell invasion ability was detected by Transwell.Results:The expression level of prostate cancer cells TRPC6 were higher than control group PrEC cells.Among TPRC6 the expression of cell line PC 3 transfer potential wre the lowest,and high transfer cell tone PC-3M express was the highest.Real-time fluorescent quantitative PCR and western blot results showed that after filter,the seventh generation of cell TRPC6 protein and mRNA expression levels were higher than the control group obviously.Transwell experimental results showed that the overexpression of TRPC6could promote the invasion ability of PC.3 prostate cancer cells.Conclusions:TRPC6 expressed in prostate cancer cells is in disorder,and its action may be associated with the invasion and metastasis of prostate cancer cells;successful establishment of stable high expression of TRPC6prostate cancer cells primarily confirm the invasion-trigger ability of TRPC6 on prostate cancer,and lay down the foundation for exploring the TRPC6's role in the occurrence and development of prostate cancer
基金supported by grants from the NationalNatural Science Foundation of China(No.30500245 and No.30871174)the Science Research Foundation of HealthDepartment of Hubei Province(No.NX200510)
文摘Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.
基金supported by grants from the National Natural Science Foundation of China(No.31050110433,No.81170600 and No.81170662)
文摘This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein （GFP）-contained plasmids （pGC） to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN＋TRPC6 shRNA transfected group and PAN＋scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
