Humans ; Large-Conductance Calcium-Activated Potassium Channels ; TRPC Cation Channels ; Vasodilation

OBJECTIVE: To investigate the clinical implications of abnormal expression of miR-607 in hepatocellular carcinoma (HCC) and its influence on HCC cell proliferation and migration. METHODS: The expression of miR-607 in 45 pairs of HCC and adjacent tissues were detected with real-time PCR, and the correlation between miR-607 expression and clinicopathological features of the patients was analyzed. The effects of transfection with miR-607 mimics on proliferation, apoptosis, migration and invasion of two HCC cell lines (Huh-7 and HCCLM3) were evaluated using CCK-8 assay, flow cytometry, wound-healing assay and Transwell assay. A dual-luciferase reporter system was used to detect the direct binding between miR-607 and 3'-UTR of TRPC5 mRNA. Western blotting was used to measure the expressions of TRPC5, CCND1, MMP2 and phosphorylated Akt in the HCC cells. RESULTS: The expression of miR-607 was significantly decreased in HCC tissues (P=0.029) and HCC cell lines (P < 0.05). In HCC patients, a low expression of miR-607 was associated with a larger tumor size (>5 cm, P=0.031), vascular invasion (P=0.027) and advanced TNM stages (Ⅲ + Ⅳ, P=0.015). In the two HCC cell line, overexpression of miR-607 significantly inhibited cell proliferation, migration, and invasion and enhanced cell apoptosis (P < 0.05). The results of dualluciferase reporter assay confirmed that TRPC5 was a direct target of miR- 607 in HCC cells. Overexpression of miR-607 significantly inhibited the expressions of TRPC5, CCND1, and MMP2 and suppressed Akt phosphorylation in HCC cells (P < 0.05). CONCLUSION A low expression of miR-607 in HCC is associated with poor clinicopathological phenotypes of HCC. Overexpression of miR-607 inhibits HCC growth and metastasis possibly by down- regulating TRPC5, which causes Akt signaling inactivation.
Humans ; Carcinoma, Hepatocellular/genetics* ; Liver Neoplasms/genetics* ; Cell Line ; Cell Proliferation ; 3' Untranslated Regions ; MicroRNAs/genetics* ; TRPC Cation Channels/genetics*

The TRPC family consists of multiple important cationic channels in mammals that participate in a variety of physiological and pathological processes. Our previous studies have shown that transforming growth factor-β1 (TGF-β1) increases the expression of TRPC6 in podocytes, but the roles of other members of the TRPC family in podocytes require further investigation. In this study, we investigated the effect of TGF-β1 on the expression of the TRPC family and the role of the TRPC family in the changes of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in podocytes induced by TGF-β1. The model of podocyte injury was established by treatment with TGF-β1 in immortalized glomerular podocytes (MPC5) in vitro. qRT-PCR and Western blot were used to detect the effect of TGF-β1 on the mRNA and protein expression of each TRPC family member. After the expression of each TRPC family member was knocked down by a siRNA-based approach and blocked by SKF96365, respectively, free cytosolic Ca²⁺ was measured using the fluorescent Ca²⁺ indicator Fluo-3/AM, and the dynamic change of [Ca²⁺]_i in podocytes was detected by a dynamic high-speed calcium imaging system. The results showed that TGF-β1 increased the protein expression of TRPC1/3/6 in podocytes, but had no effects on the protein expression of TRPC4. The protein expression levels of TRPC5/7 were only affected by 4 ng/mL and 8 ng/mL TGF-β1, respectively. TGF-β1 increased TRPC1/3/6 mRNA levels in podocytes, however had no effects on TRPC4/5/7 mRNA. TGF-β1 significantly increased [Ca²⁺]_i in podocytes. Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the [Ca²⁺]_i induced by TGF-β1, but TRPC3/6 knockdown significantly decreased the [Ca²⁺]_i. There was no significant difference in the [Ca²⁺]_i between the TRPC6 siRNA-treated group and SKF96365-treated group, but the [Ca²⁺]_i of the TRPC3 siRNA-treated group was significantly higher than that of SKF96365-treated group. These results demonstrate that TGF-β1 increases the expression of the TRPC1/3/6 in podocytes. TGF-β1 increases [Ca²⁺]_i in podocytes, which is dependent on the TRPC3/6 expression. Our results also suggest that the effect of TRPC6 on [Ca²⁺]_i in podocytes may be greater than that of TRPC3.
Animals ; TRPC6 Cation Channel/metabolism* ; Calcium/metabolism* ; TRPC Cation Channels/metabolism* ; Podocytes/metabolism* ; Transforming Growth Factor beta1/metabolism* ; RNA, Small Interfering/metabolism* ; RNA, Messenger/metabolism* ; Mammals/metabolism*

Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this study were to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potential mechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension of longitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion with Krebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8 in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. The results showed that the contractions induced by cold stimuli (from 37 degrees C to 12 degrees C stepwise) were inversely proportional to the temperature with a maximum contraction at 17 degrees C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed the expression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced colonic contractions were specially inhibited by TRPA1 blocker, ruthenium red (30 μmol/L), in the proximal and distal colon (P<0.05). The cold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by the pretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 μmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellular calcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 μmol/L) and IP(3) receptor blocker (2-aminoethoxydiphenyl borate, 2-APB, 30 μmol/L) all decreased the contractions evoked by the cooling at 17 degrees C in the proximal and distal colon (P<0.001, P<0.05, P<0.001). Atropine (1 μmol/L) had no effects on these contractions. L-type Ca(2+) channels blocker nifedipine (1 μmol/L) and neurotoxin tetrodotoxin (TTX, 2 μmol/L) decreased the contractile response in the distal colon (P<0.01, P<0.05), but not in the proximal colon. In conclusion, TRPA1 contributes to cold-induced contractions of the rat colon smooth muscle, and the mechanism of TRPA1 activation involves PLC/IP(3)/Ca(2+) pathway. L-type Ca(2+) channel and neurogenic mechanism other than muscarinic receptor might be partially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal colon compared with that of proximal colon.
Animals ; Calcium Channels, L-Type ; metabolism ; Cold Temperature ; Colon ; metabolism ; physiology ; In Vitro Techniques ; Male ; Muscle Contraction ; physiology ; Muscle, Smooth ; metabolism ; physiology ; Physical Stimulation ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; TRPA1 Cation Channel ; TRPC Cation Channels ; genetics ; metabolism

OBJECTIVETo study the roles of stromal interaction molecule 2 (STIM2) and transient receptor potential canonical 3 (TRPC3) in extracellular Ca(2+)-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC).

METHODS(1) The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. (2) The expressions of STIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmids. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. (3) The second to fifth passage of HUVEC were divided into: STIM2-002 short hairpin RNA (STIM2-002 shRNA ) + spermine + Ca2+ group and TRPC3-004 short hairpin RNA (TRPC3-004 shRNA ) + spermine + Ca2+ group; control group (spermine + Ca2+ group) and vehicle+ spermine + Ca2+ group. The four groups of cells were incubated with CaR agonist spermine, the intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, and the production of NO was determined by DAF-FM (NO fluorescent probe) of each group in HUVEC.

RESULTS(1) Immunofluorescence technique results showed that STIM2 and TRPC3 proteinswere present in the cytoplasm of HUVEC. (2) The results of transfection constructed STIM2 and TRPC3 RNA interference plasmids demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRPC3 mRNA levels by 88.2% and 74.0%, respectively (P < 0.05), simultaneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively (P < 0.05). (3) Compared with spermine + Ca2+ group, the [Ca2+]i and the net NO fluorescence intensity of spermine + Ca(2+) + ShSTIM2-002 group, spermine + Ca(2+) + ShTRPC3-004 group and spermine + Ca2+ Vehicle group were not changed (P > 0.05).

CONCLUSIONSTIM2 and TRPC3 do not participate in CaR-mediated Ca2+ influx and NO production individually.

Calcium ; metabolism ; Cell Adhesion Molecules ; physiology ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; physiology ; Humans ; Nitric Oxide ; metabolism ; Stromal Interaction Molecule 2 ; TRPC Cation Channels ; physiology

OBJECTIVETo detect the expression of transient receptor potential canonical 1 (TRPC1) in a mouse model of ozone-induced lung inflammation and explore its role in lung inflammation.

METHODSIn a mouse model of lung inflammation established by ozone exposure, the expression of TRPC1 in the inflammatory lung tissues was detected by RT-PCR, Wstern blotting and immunohistochemistry.

RESULTSCompared to the control mice, the mice exposed to ozone showed significantly increased expression level of TRPC1 mRNA and protein in the inflammatory lung tissues (P<0.05). Immunohistochemistry showed increased TRPC1 protein expressions in the alveolar epithelial cells, bronchial epithelial cells, and inflammatory cells in the inflammatory lung tissues (P<0.05). The mRNA and protein expression levels of TRPC1 were positively correlated with the counts of white blood cells, macrophages, neutrophils and lymphocytes in the bronchoalveolar lavage fluid of the exposed mice (P<0.01).

CONCLUSIONTRPC1 may play a role in ozone-induced lung inflammation in mice.

Animals ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Gene Expression ; Inflammation ; pathology ; Lung ; metabolism ; pathology ; Mice ; Ozone ; adverse effects ; Pneumonia ; metabolism ; pathology ; RNA, Messenger ; TRPC Cation Channels ; metabolism

BACKGROUND AND OBJECTIVETransient receptor potential canonical (TRPC) proteins, a group of Ca2' permeable nonselective cation channels, are thought to constitute store-operated calcium channels (SOCC) and mediate store-operated calcium entry (SOCE) in various cell types. Members of TRPC have been found to be involved in abnormal proliferation, differentiation, and growth of cancer cells. The aim of this study is to detect the mRNA and protein expression of TRPC in non-small cell lung cancer (NSCLC).

METHODSReal-time quantitative PCRwas performed to screen the expression of TRPC mRNA in NSCLC tissue. Protein expression of TRPC was detected by Western blot.

RESULTSAmong the seven family members of TRPC so far identified (TRPC1-7), we detected the expression ofTRPC1, TRPC3, TRPC4, TRPC6 mRNA in 24 cases of NSCLC tissue; TRPC2, TRPC5 and TRPC7 mRNA were not detectable. The relative abundance of the expressed TRPC was TRPC1 approximately equal TRPC6 > TRPC3 > TRPC4. Western blot confirmed the protein expression of TRPC1, TRPC3, TRPC4 and TRPC6 in NSCLC tissue.

CONCLUSIONOut of the seven members of TRPC, we found TRPC1, TRPC3, TRPC4, TRPC6 mRNA and protein were selectively expressed in human NSCLC tissue. This study could provide a basis for future exploration of the individual role of these TRPC proteins in mediating SOCE and in the progression of lung cancer.

Adult ; Aged ; Blotting, Western ; Calcium ; metabolism ; Carcinoma, Non-Small-Cell Lung ; metabolism ; Female ; Humans ; Lung Neoplasms ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; analysis ; TRPC Cation Channels ; genetics ; physiology

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.
Animals ; Ankyrin Repeat ; Humans ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Protein Subunits ; TRPC Cation Channels ; chemistry ; genetics ; physiology

Our previous study found that some trigeminal ganglion (TG) nerve endings in the inner walls of rat anterior chambers were mechanosensitive, and transient receptor potential ankyrin 1 (TRPA1) was an essential mechanosensitive channel in the membrane. To address the effect of cannabinoids on the mechanosensitive TG nerve endings in the inner walls of anterior chambers of rat eye, we investigated the effect of the (R)-(+)-WIN55, 212-2 mesylate salt (WIN), a synthetic cannabinoid on their cell bodies in vitro. Rat TG neurons innervating the inner walls of the anterior chambers were labeled by 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfona (FAST DiI). Whole cell patch clamp was performed to record the currents induced by drugs and mechanical stimulation. Mechanical stimulation was applied to the neurons by buffer ejection. WIN evoked inward currents via TRPA1 activation in FAST DiI-labeled TG neurons. WIN enhanced mechanosensitive currents via TRPA1 activation in FAST DiI-labeled TG neurons. Our results indicate that cannabinoids can enhance the mechanosensitivity of TG endings in the inner walls of anterior chambers of rat eye via TRPA1 activation.
Action Potentials ; drug effects ; Animals ; Anterior Chamber ; drug effects ; innervation ; Cannabinoids ; administration & dosage ; Eye ; drug effects ; innervation ; Neurons ; drug effects ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; TRPA1 Cation Channel ; TRPC Cation Channels ; biosynthesis ; genetics ; Trigeminal Ganglion ; drug effects ; physiology

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 expression 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.
Animals ; Cell Membrane Permeability ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Cells, Cultured ; Gene Knockdown Techniques ; Mice ; Mice, Knockout ; Podocytes ; drug effects ; physiology ; Puromycin Aminonucleoside ; pharmacology ; TRPC Cation Channels ; genetics ; metabolism