Expression of GLUT4 in endometrium of rats with polycystic ovary syndrome

AIM:To explore the expression of glucose transporter 4 (GLUT4) in the endometrium of rats with polycystic ovarian syndrom (PCOS) and evaluate the relationship between GLUT4 expression and insulin resistance (IR). METHODS:54 female SD rats of 85 days were randomized to control group (n=20), PCOS model group (n=17) and metformin treatment group (n=17). The rats in the latter two groups were induced by Poretsky’s method for PCOS model, followed by placebo or metformin, respectively. After 14 days of treatment, the rats were sacrificed and the expression of GLUT4 in endometrium was detected by Elivision^TM Plus two steps immunohistochemical staining. RESULTS:The expression of GLUT4 and insulin receptor(INS-R) proteins of endometrial glandulan epitheliu in PCOS rats were significantly lower (P<0.01,P<0.05) than those in control group, however, the expression of insulin(INS) protein in PCOS rats was higher than that in control group (P<0.01). The expression of GLUT4 in the treatment group increased (P<0.01), but was still lower than that in control group (P<0.01). However, compared with PCOS group, the expression of INS protein was decreased (P<0.05), but was still higher than that in control group (P<0.05). There was no GLUT4 expression in interstitial cells in endometrium, and the changes of the expressions of INS and INS-R proteins in those cells were similar with those in glandulan epitheliu. CONCLUSION:The decrease in GLUT4 expression of endometrium in PCOS rats is related with endometrial insulin resistance.     

Establishment and verification of insulin resistance model in mouse skeletal muscle cells

AIM:To establish a stable and repeatable insulin resistance model of skeletal muscle cells in vitro, so as to promote the exploration of the pathological mechanism of insulin resistance and the development and screening of related drugs. METHODS:C2C12 mouse myoblasts were used to induce differentiation in normal differentiation medium and differentiation medium containing glucose at 40 and 60 mmol/L, respectively. The effects of glucose at different concentrations on cell convergence, fusion and formation of multinucleated myotubes were observed under phase contrast microscope every day. After 1, 3, 5 and 7 d of differentiation, 2-NBDG assay was used to detect the effects of different interventions on C2C12 basal glucose uptake and insulin-stimulated glucose uptake. The effects of different interventions on the protein expression of glucose transporter 4 (GLUT4) after 5 d and 7 d of differentiation were determined by Western blot. The effects of different interventions on the distribution of GLUT4 protein after 5 d of differentiation were detected by immunofluorescence staining. RESULTS:After treated with glucose at 60 mmol/L, the morphological observation showed that high glucose treatment significantly inhibited the growth and differentiation of C2C12 cells after 3 d. High glucose treatment significantly inhibited basal glucose uptake and insulin-stimulated glucose uptake of the C2C12 cells after 5 d and 7 d (P<0.01). No difference between insulin-stimulated GLUT4 expression and basal GLUT4 expression after 5 d and 7 d of high glucose treatment was observed (P>0.05), but there was significant difference between control group and 60 mmol/L group (P<0.05) determined by Western blot. Immunofluorescence staining observation showed that the distribution of GLUT4 protein in the C2C12 cell membrane was significantly decreased after 5 d of high glucose treatment (P<0.01). Glucose treatment (40 mmol/L) also played a role to some extent, but the effect was not as obvious and stable as 60 mmol/L glucose. CONCLUSION:A stable insulin resistance model of mouse skeletal muscle cells in vitro was successfully established by high glucose stimulation. The treatment of glucose at 60 mmol/L for 5 d was the best. Morphological observation and detection of basic and insulin-stimulated glucose uptake and GLUT4 protein expression and distribution evaluates the insulin resistance level of skeletal muscle cells in vitro.     

中国园艺学会第七届青年学术讨论会

中国园艺学会第九届第8次常务理事扩大会决定,“中国园艺学会第七届青年学术讨论会”由山东农业大学园艺科学与工程学院和山东省园艺学会承办,将于2006年7月或8月在山东泰安举行。会议交流主题:(1)园艺作物种质资源、遗传育种与生物技术;(2)园艺作物有机、无公害及标准化安全生     

Icariin attenuates high glucose-induced insulin resistance in C2C12 myotubes

AIM: To observe the potential effects of icariin on high glucose-induced insulin resistance in C2C12 myotubes and to investigate its underlying mechanisms. METHODS: The insulin resistance model was induced by high glucose (25 mmol/L) in the C2C12 myotubes. The effects of icariin on Akt phosphorylation at T308, glucose transporter 4 (GLUT4) membrane translocation, and glucose uptake were investigated in high glucose-treated C2C12 myotubes. The protein levels of phosphorylated proteins were determined by Western blot. The glucose uptake was measured by colorimetric method. The small interfering RNA (siRNA) was used to knockdown the expression of p38 MAPK. RESULTS: Icariin significantly increased insulin-stimulated Akt T308 phosphorylation in C2C12 myotubes treated with high glucose. Treatment with icariin at 25, 50 and 75 μmol/L for 24 h increased Akt T308 phosphorylation in a dose-dependent manner (P<0.05 or P<0.01). Treatment with icariin at 50 μmol/L for 12, 24 and 36 h increased Akt T308 phosphorylation in a time-dependent manner (P<0.05 or P<0.01). In addition, treatment with icariin at 50 μmol/L for 24 h significantly enhanced the expression of GLUT4 on plasma membrane (P<0.01) and 2-deoxyglucose (2-DG) uptake (P<0.01). Treatment with icariin recovered high glucose-reduced p38 MAPK phosphorylation (P<0.01). Pharmacological or genetic inhibition of p38 MAPK abolished the protective impacts of icariin on insulin-stimulated Akt T308 phosphorylation (P<0.01), GLUT4 plasma membrane translocation (P<0.01), and 2-DG uptake under high glucose condition (P<0.05). CONCLUSION: Icariin attenuates high glucose-induced insulin resistance in C2C12 myotubes by activating p38 MAPK.     

Effects of tetramethylpyrazine on calcinerin and proliferating cell nuclear antigen gene expression in the proliferation of vascular smooth muscle cells

AIM：To evaluate the effects of tetramethylpyrazine (TMP) on calcineurin (CaN) and proliferating cell nuclear antigen (PCNA) gene expression in the proliferation of vascular smooth muscle cells (VSMCs) treated by angiotensin Ⅱ (AngⅡ).METHODS：A cell proliferating model of VSMCs induced by AngⅡ was established.PCNA gene exprersion was observed by immunocytochemical staining and image analysis technique;Calcineurin (CaN) activity was detected by enzyme reaction phosphorus measurement.RESULTS：AngⅡ significantly stimulated the proliferation of VSMCs,cell proliferation activity,CaN activity and the expression levels of PCAN were higher than those in control (P<0.01).While treated with TMP,the CaN activity and PCNA expression were obviously lower than those in AngⅡ group (P<0.01).CONCLUSION：The VSMCs proliferation induced by AngII can be inhibited by tetramethylpyrazine significantly,and the inhibiting mechanism of TMP may be related to inhibiting CaN activity and restraining the expression of PCNA in a dose and time-dependent manner.     

Role of reactive oxygen species and ERK1/2 signaling pathway in proliferation and apoptosis of hypoxic rat pulmonary arterial smooth muscle cells

AIM:To investigate the change of reactive oxygen species (ROS) production in hypoxic pulmonary arterial smooth muscle cells (PASMCs) of rats, the effect of ROS on the expression of extracellular signal-regulated kinase (ERK)1/2 protein, and the role of ROS and ERK1/2 in the imbalance between proliferation and apoptosis of PASMCs.METHODS: Primary cultures of PASMCs were established and cells between passages 2 to 3 were used for experiments. PASMCs were treated with tiron, a membrane permeable ROS scavenger, and PD98059, an ERK1/2 inhibitor, under normoxia or hypoxia condition. The ROS production was measured by DCFH-DA and NBT reduction. The expression of phosphorylated-ERK1/2 (p-ERK1/2) protein was detected by immunofluorescence. Cell proliferation was examined by MTT colorimetric assay and the expression of PCNA. Cell apoptosis was detected by TUNEL.RESULTS: (1)Compared with control group, the ROS levels in hypoxia group were significantly increased (P<0.01). (2) In hypoxia group, the proliferative capacity was higher and the apoptosis index was lower than those in control group (P<0.01). Tiron significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). (3) The expression of p-ERK1/2 in hypoxia group were higher than that in control group (P<0.01), which were significantly suppressed by tiron (P<0.01)．(4) PD98059 significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). The proliferative capacity and apoptosis index was similar in hypoxia+tiron+PD98059 group to those in hypoxia+tiron group (P>0.05).CONCLUSION:The hypoxia-mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2. ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.     

PAF promotes proliferation of smooth muscle cells of rat airway

AIM: To study the effect of platelet-activating factor(PAF) on proliferation of cultured rat airway smooth muscle cells(ASMCs).METHODS: The cells were divided into control group and PAF group. The cells in PAF group were subdivided into four small groups by concentrations of PAF 10-6, 10-7, 10-8, 10-9 mol·L-1, MTT assay was used not only to investigate the effects of PAF on proliferation of ASMC but also to confirm the optimal concentration. Flow cytometry and immuneohistochemistry for proliferating cell nuclear antigen (PCNA) were also used to analyse its function on proliferation of ASMC.RESULTS: PAF (10-6-10-9mol·L-1) stimulated the cell proliferation and 10-7mol·L-1 PAF reached the maximal effect. The cell percentage of the ASMCs of 107 mol·L-1 PAF subgroup at G0/1 phase (68.67%) was much lower than that of control group (85.57%, P<0.01), in this subgroup, the percentage of expression of PCNA at 48 h (71.05%±1.22%) was significantly increased compared with the control group (53.27%±2.56%, P<0.05).CONCLUSION: PAF can stimulate the proliferation of cultured ASMC in a time-dependent, but not dose-dependent manner.     

Effects of Kv1.5 on proliferation and apoptosis of rat PASMCs under hypoxia+hypercapnia condition and relationship with MAPK pathway

AIM：To investigate the effects of voltage-dependent K＋ channel 1.5 (Kv1.5) on the proliferation and apoptosis of rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia+hypercapnia condition and the relationship with mitogen-activated protein kinase(MAPK) signal pathway. METHODS：The PASMCs isolated from the male SD rat were cultured under hypoxia+hypercapnia condition, and randomly divided into normal group (N group), hypoxia+hypercapnia group (HH group), hypoxia+hypercapnia+DMSO incubation group (HD group), hypoxia+hypercapnia+U0126 (an extracellular signal-regulated kinase 1/2 inhibitor) incubation group (HU group), hypoxia+hypercapnia+SB203580 (a p38 mitogen-activated protein kinase inhibitor) incubation group (HS group), and hypoxia+hypercapnia+anisomycin (an agonist of MAPK) incubation group (HA group). Cell Counting Kit-8 was used to detect the cell viability. The protein expression of Kv1.5, PCNA and Bax was detected by Western blotting. RESULTS：Compared with N group, the cell viability and PCNA protein expression in HH group and HD group were significantly raised (P<001), but Kv1.5 and Bax proteins were significantly decreased (P<0.01). No difference between HH group and HD group was observed (P>005). Compared with HD group, the cell viability and PCNA protein expression in HU group, HS group and HA group were decreased (P<0.05 or P<0.01), but Kv1.5 protein and Bax protein were raised (P<0.01), with the most significant changes in HA group. ^{CONCLUSION：}The regulation of Kv1.5 to the proliferation and apoptosis of PASMCs under hypoxia+hypercapnia condition might have a relationship with the activation of MAPK signal pathway.     

Effects of hypoxia-inducible factor-1 signal pathway on proliferation in hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the effect of hypoxia on the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC); and to evaluate the role of hypoxia-inducible factor-1α(HIF-1α), iNOS, P-ERK1/2 protein expression in hypoxic pulmonary hypertension (HPH) pathogenesis.METHODS: Cultured rat PASMC were divided into normoxic group; hypoxic group; hypoxia+ADM(adrenomedulin) group, hypoxia+L-NAME(iNOS inhibitor) group; hypoxia+PD98059 group. Proliferation was investigated by MTT and PCNA. Apoptosis was examined by flow-cytometry. Westen blotting was used to measure protein expression of HIF-1α, P-ERK1/2 and iNOS. RESULTS: (1) A value of 24 h-hypoxia was significantly higher than that in the normoxic group (P<0.01). In the hypoxia+PD98059 group, ADM was significantly lower than that in hypoxia group, whereas A value of the hypoxia+L-NAME was significantly higher than that in hypoxic group and normoxic group (P<0.01). (2) PCNA was positive in PASMC after 24 h hypoxia (P<0.01). PD98059, ADM inhibited the expression of PCNA significantly (P<0.01), whereas L-NAME increased the expression of PCNA significantly (P<0.01). (3) Apoptosis index was not significantly difference among the different groups (P>0.05). (4) HIF-1α, iNOS and P-ERK1/2 expression was poorly positive in normoxic group, positive after hypoxia for 4h (P<0.01), reaching its peak at 8 h hypoxia (P<0.01), HIF-1α, P-ERK1/2 expression declined after 24 h hypoxia. L-NAME promoted the expression of HIF-1α, PD98059 inhibited the expression of HIF-1α, iNOS and P-ERK1/2 partly. ADM inhibited the expression of HIF-1α partly, promoted the expression of iNOS. CONCLUSION: (1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) HIF-1 plays an importent role in the proliferation of hypoxic PASMC.     

Effects of taurine on expression of glucose transporters in rat brain with diffused brain injury

AIM: To investigate the effect of taurine on the expression of glucose transporter 1 (GLUT1) and transporter 3 (GLUT3) in rat brain with diffused brain injury (DBI).METHODS: Sixty-four male Sprague-Dawley rats were randomly divided into 4 groups: sham-operated group, DBI group, low-dose taurine group (200 mg/kg, ig) and high-dose taurine group (300 mg/kg, ig).After fed with the corresponding drugs for 7 days, the animal model of DBI was made, and the rats were executed 24 h after DBI.The expression of GLUT1 and GLUT3 in the brain was detected by the methods of immunohistochemistry and Western blotting.The pathomorphological changes of the cerebral cortex were observed under electron microscope.RESULTS: The expression of GLUT1 was detected in capillary vascular endothelial cells in each group, and cytoplasm-positive cells or the cells with buffy membrane were observed.No significant difference of the GLUT1 expression in brain tissues between DBI group and sham-operated group was detected.Compared with DBI group, the expression of GLUT1 in the brain tissues were significantly increased in low-and high-dose taurine groups (P<0.01).The expression of GLUT1 in the brain tissues in low-dose taurine group were significantly higher than that in high-dose taurine group (P<0.05).The positive staining of GLUT3 only appeared in the periphery of the third ventricle in each group in the cells with buffy membrane or positive cytoplasm.The expression of GLUT3 in the brain tissues in DBI group was significantly higher than that in sham-operated group (P<0.01).The expression of GLUT3 in the brain tissues in low-and high-dose taurine groups was significantly higher than that in DBI group (P<0.01).Compared with low dose taurine group, the expression of GLUT3 in the brain tissues were significantly increased in high-dose taurine group (P<0.01).The pathological damage of cerebral cortex in low-dose taurine group was obviously alleviated.CONCLUSION: Taurine may take part in the neuroprotective mechanisms in DBI by increasing the expression of GLUT1 and GLUT3 at protein level to maintain the energy supply in brain tissues.     

Deoxyribozyme inhibits the proliferation of human umbilical artery smooth muscle cells

AIM: To observe the inhibition effects of 10-23 deoxyribozyme (DNAzyme) specific to human proliferating cell nuclear antigen (PCNA) gene on the proliferation of human umbilical artery smooth muscle cells (HUASMC) in vitro. METHODS: Using lipofectamine (Lip)-mediated method, the DNAzyme specific to PCNA was introduced into HUASMC. ［3H］-TdR incorporation was determined. The cell proliferation was examined by MTT assay and cell cycle was detected by flow cytometry. RESULTS: ［3H］-TdR incorporation in 1.0 μmol/L DNAzyme group was lower than that in control group (P<0.05) at 2 days after transfection. Compared with control group, the A value of MTT cholorimetric analysis decreased in 1.0 μmol/L DNAzyme group and antisense oligodeoxynucleotide (ASODN) group, at 2, 3 and 5 days after transfection significantly (P<0.01). The decrease in A value showed a dose-dependent manner within the experimental range at 5 days. After 2 days, the percentage of quiescence cells (G0/G1) was 73.8%, 54.7% and 41.1% in DNAzyme, ASODN and control groups, respectively. CONCLUSION: The DNAzyme specific to PCNA suppresses the proliferation of HUASMC effectively in vitro.     

Role of asiatic acid in treatment of insulin resistance in mouse adipocytes

AIM：To investigate the effects of asiatic acid, one of triterpenoids from Psidium guajava leaves, on the proliferation and differentiation of 3T3-L1 preadipocytes, and glucose and lipid metabolism of insulin-resistant adipocytes. METHODS：The proliferation of 3T3-L1 preadipocytes was tested by MTT assay, and the accumulation of lipid droplets in differentiated preadipocytes was measured by oil red O staining. The insulin-resistant cell model was established by exposure of the cells to dexamethasone. The cellular glucose uptake was determined by glucose oxidase-peroxidase assay. The free fat acid (FFA) concentration was detected by colorimetric method. Secreted adiponectin were measured by ELISA. The protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and protein tyrosine phosphatase 1B (PTP1B) in insulin-resistant adipocytes were analyzed by Western blotting. RESULTS：Compared with medium group, asiatic acid increased the proliferation of 3T3-L1 preadipocytes and inhibited their differentiation at a concentration range of 10~100 μmol/L (P<0.05 or P<0.01). At concentrations of 30 μmol/L and 100 μmol/L, asiatic acid enhanced cellular glucose uptake in the insulin-resistant adipocytes both in basic and insulin-stimulation states. Asiatic acid decreased FFA production (P<0.05), and down-regulated the protein expression of PTP1B (P<0.05, or P<0.01). However, no effect on the secretion of adiponectin and the protein expression of PPARγ was observed (P>0.05). CONCLUSION：Asiatic acid enhances glucose uptake and inhibits FFA production in insulin-resistant adipocytes via down-regulating the protein expression of PTP1B, all of which play the roles of increasing insulin signaling sensitivity to improve insulin resistance.     

Effect of γ radioactive stent on proliferation and apoptosis of smooth muscle cells

AIM: To observe effect of γ radioactive ［103Pd］ stent on the proliferation and apoptosis of smooth muscle cells, the mechanism of radioactive stent preventing in-stent restenosis was explored. METHODS: Fifty male New Zealand rabbits were randomized into stent group and ［103Pd］ stent group. Control group was set up. The materials were harvested on 3, 7, 14, 28, 56 days after operation and the following investigation were carried out, including pathomorphology, immunohistochemistry, apoptosis (TUNEL) and in situ hybridization studies.RESULTS: ① The severity of the stenosis in ［103Pd］ stent group was less severe than that in stent group. It was most obvious on 56 th day (P<0.01). ② The expression of proliferating cell nuclear antigen(PCNA) of ［103Pd］ stent group was lower than that in stent group on 3 to 28 days. It was most obvious on 7th day, 16.35%±0.79% vs 24.36±0.55% (P<0.01). ③ TUNEL method showed that the ［103Pd］ stent group had much more apoptosis of VSMC than that in stent group. The highest rate of apoptosis appeared on day 7, 14.72%±0.53% vs 12.42%±1.13% (P<0.01). ④ By calculating the ratio of PCNA/apoptosis (P〖KG*6〗∶〖KG-*2〗A), a much lower ratio was seen in ［103Pd］-stent groups than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05). ⑤ For bcl-2/bax ratio, the result in ［103Pd］-stent group was lower than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05).CONCLUSION: γ radioactive stent inhibits the proliferation and accelerates apoptosis of injured media vascular smooth muscle cells. It decreases the ratio of proliferation to apoptosis and relieves the severity of restenosis.     

Effect of di-indolyl thiozoline on proliferation of lung cancer A549 cells

AIM: To investigate the effect of di-indolyl thiozoline (DIIT) on the proliferation of human lung cancer A549 cells. METHODS: The effects of DIIT on the proliferation of human lung cancer A549 cell line were determined by CCK-8 assay and EdU assay. The effects of DIIT on the expression of cyclin-dependent kinase 4 (CDK4), cyclin D1, and the phosphorylation of Akt and mTOR were determined by Western blot. RESULTS: After the A549 cells were treated with DIIT at 12.5, 25, 50 and 100 mg/L, the cell viability detected by CCK-8 assay was decreased by 12%, 27% (P<0.01), 33% (P<0.01) and 52% (P<0.01), respectively, compared with DMSO control group. The EdU positive cell number determined by EdU assay was decreased by 10%, 21% (P<0.05), 26% (P<0.05) and 34% (P<0.01), respectively, compared with DMSO control group. Compared with DMSO control group, DIIT inhibited the phosphorylation of Akt and mTOR and the expression of cyclin CDK4 and cyclin D1 (P<0.05). CONCLUSION: Di-indolyl thiozoline inhibits the proliferation of A549 cells, which may be related to the decreases in phosphorylation levels of Akt and mTOR and the inhibition of cell cycle-related protein expression.     

Effect of proliferating cell nuclear antigen specific antisense oligonucleotide on differentiation of cord blood CD34+ cells

AIM:To study the effect of proliferating cell nucler antigen antisense oligonucleotide on ex vivo expansion of cord blood CD34+ hematopoietic stem/progenitor cells. METHODS:CD34+ cells were purified from fresh cord blood by immunomagnetic beads. CD34+ cells were incubated in liquid culture system with different concentrations of PCNA-ASODN. Using flow cytometry, the number of different kinds of stem/progenitor cells and PCNA expression were measured after CD34+ cell incubation. RESULTS:PCNA was lowly expressed in low experiential group, with a positive rate of (27.2±3.6)% and (19.0±1.5)%, the positive rate of control group was (53.8±8.3)% (P<0.01), a high significant difference was observed. Just in low concentration of PCNA-ASODN, the percentage of CD34+cells were increased to (33.4±3.2)%, CD34+CD38-cells expanded (57.8±9.9) folds, and the percentage of CD34+cells were (25.2±2.6)% (P<0.01), the CD34+CD38-cells expanded (43.5±7.4) folds (P<0.05), it has significant difference compared with the control group. CONCLUSION:Low concentration of PCNA-ASODN decreases PCNA expression effectively, slows down differentiation of CD34+cells during ex vivo expansion procedure, and improves the expansion efficiency.     

Baicalein inhibits proliferation and migration of gastric cancer MGC-803 cells

AIM: To investigate the effects of baicalein (BAI) on the proliferation and migration of gastric cancer MGC-803 cells and the mechanisms. METHODS: After MGC-803 cells were treated with BAI at different concentrations, the viability of the MGC-803 cells was tested by MTT assay. The cell colony formation ability were detected by plate colony formation assay. Wound-healing and Transwell cell migration assays were used to test the migration ability of the MGC-803 cells. The concentration of 12-hydroxyeicosatetraenoic acid (12-HETE) was measured by ELISA. The protein levels of platelet type 12-lipoxygenase (p12-LOX), vascular endothelial growth factor (VEGF), p-ezrin and epithelial-mesenchymal transition (EMT) markers in MGC-803 cells were determined by Western blot. RESULTS: BAI significantly inhibited the proliferation, plate colony formation and migration abilities of the MGC-803 cells (P<0.05 or P<0.01), down-regulated the concentration of p12-LOX metabolite 12-HETE significantly (P<0.05 or P<0.01), decreased the protein levels of p12-LOX, VEGF, p-ezrin, vimentin and Snail (P<0.05 or P<0.01), and increased the protein expression of E-cadherin (P<0.01). CONCLUSION: BAI suppresses the proliferation and migration abilities of gastric cancer MGC-803 cells effectively. These effects of BAI may be related to regulating the protein levels of p12-LOX, VEGF, p-ezrin and EMT-related proteins.