Papillary renal cell carcinoma with extensive spindle cell foci: mimicker of mucinous tubular and spindle cell carcinoma

ABSTRACT Papillary renal cell carcinoma (PRCC) is the second most common renal cell carcinoma (RCC), accounting for 10-15% of cases. Mucinous tubular and spindle cell carcinoma (MTSCC), on the other hand, accounts for only 1% of renal tumors and has a more favorable prognosis compared to PRCC. We report a 75-year-old female with a left upper pole solid renal mass displaying features of both papillary renal cell carcinoma (PRCC) and mucinous tubular and spindle cell carcinoma (MTSC). In this case, a shaggy luminal surface, multiple papillations, and psammoma bodies, absence of E-cadherin expression, and strong CD10 expression favored PRCC. Both immunohistochemistry and genomic analysis are critical to diagnose and differentiate tumors that may have overlapping features accurately.

Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells / 中华肿瘤杂志

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.

Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells / 中国实验方剂学杂志

ObjectiveTo study the inhibitory effect of polyphyllin Ⅰ （PPI） on the growth of colorectal cancer cells and its molecular mechanism. MethodRKO cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 0.6， 0.8， 1.0 μmol·L-1， respectively. HRT18 cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 1.2， 1.4， 1.6 μmol·L-1， respectively. The effects of PPI on the proliferation and morphology of colorectal cancer were detected by cell proliferation toxicity assay， trypan blue exclusion assay， plate clone formation assay， and confocal high-intension cell imaging analysis system. Flow cytometry was used to detect the apoptosis rate of colorectal cancer cells. The pQCXIP-GFP-LC3 plasmid transfection assay was used to detect the formation of autophagosomes in colorectal cancer cells after PPI treatment. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3， Caspase-8， and poly ADP ribose polymerase （PARP）， the expression of autophagy related protein LC3Ⅱ， and the expression and phosphorylation of Hippo signaling pathway proteins LATS1 and YAP. In the plvx-Flag-YAP plasmid transfection assay， YAP was overexpressed and treated with PPI， and the proliferation of colorectal cancer cells was detected by cytotoxicity assay. The expression of LC3Ⅱ and PARP in colorectal cancer cells was detected by Western blot. SwissADME predicted pharmacokinetic parameters of PPI. ResultAs compared with the blank group， the survival rate and clone formation ability of colorectal cancer cells in the PPI group were significantly decreased （P<0.01）， the cell area of colorectal cancer cells in the PPI group was significantly decreased， and the roundness of colorectal cancer cells was significantly increased （P<0.01）. As compared with the blank group， the apoptosis rate of colorectal cancer cells in PPI treatment groupw was significantly increased （P<0.01）， the expression of apoptotic proteins Caspase-3 and Caspase-8 protein precursor in PPI treatment groups was decreased， and the cleavage of PARP was increased （P<0.01）. As compared with the blank group， the expression level of autophagy-related protein LC3Ⅱ in colorectal cancer cells in PPI treatment groups was significantly increased， and the formation of autophagosomes was promoted （P<0.01）. As compared with the blank group， the expression of YAP protein in colorectal cancer cells in PPI treatment groups was significantly decreased， and the expressions of phosphorylated LATS1 and YAP were significantly increased （P<0.01）. As compared with the blank group， overexpression of YAP could significantly antagonize the effect of PPI on apoptosis， autophagy activation， and proliferation inhibition of colorectal cancer cells. SwissADME simulation results showed that PPI had good drug like activity. ConclusionPPI can induce apoptosis and autophagy of colorectal cancer cells through targeted activation of Hippo signaling pathway， thereby inhibiting their proliferation.

Research progress on the mechanism of Hippo signaling pathway in organ regeneration / 药学实践杂志

Hippo signal pathway is one of the main signal pathways regulating cell proliferation and apoptosis in multicellular animals, which plays a vital role in the maintenance of tissue homeostasis and the regulation of organ growth. Most mammals have limited regenerative potential, and recent studies have shown that Hippo signal pathway is critical in the regeneration of various tissues and organs. The role of Hippo signaling pathway in organ regeneration and the research progress of related targets were introduced, the mechanism of Hippo signaling pathway promoting regeneration analyzes were aralyzed in this review, which provide theoretical reference for the treatment of diseases related to organ regeneration.

Advances in the regulation of autosomal dominant polycystic kidney disease by Hippo signaling pathway / 中华肾脏病杂志

The Hippo signaling pathway is highly conserved in evolution, and participates in the regulation of cell proliferation, differentiation, and tissular dynamic balance, and plays an important role in regulating tissue, organ size, and cell number. Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and one of the most common causes of end-stage renal disease. Emerging studies have identified the Hippo signaling pathway is closely related to the occurrence and development of ADPKD. The abnormal activity and expression of the main members of the pathway affect the cilia and cell polarity of renal tubular epithelial cells and induce the formation of renal cysts. The review summarizes the potential mechanism of the Hippo pathway in the pathogenesis of ADPKD, the crosstalk with other signaling pathways, and the variances in different species, and discusses the strategies for the treatment of ADPKD based on the Hippo signaling pathway to provide new strategies for the treatment.

Based on the Hippo signaling pathway to explore the mechanism of autophagy in lung injury of acute respiratory distress syndrome induced by sepsis / 中华危重病急救医学

The systemic inflammatory response caused by various pathogenic factors is a key stage in the development of acute respiratory distress syndrome (ARDS). At present, suppression of the inflammatory response and symptomatic support are main methods for the treatment of ARDS. Alveolar epithelial autophagy has an important role in the regulation of the inflammatory response in ARDS. Autophagy is a normal immune mechanism in the body, and it is a metabolic process by which phagocytes degrade intracellular components with the help of lysosomes to maintain intracellular homeostasis. Current studies have shown that pathogenic factors both inside and outside the lung can cause alveolar epithelial cells to form an unfavorable internal environment of hypoxia, starvation, infection, and even apoptosis by triggering inflammatory responses, leading to autophagy dysfunction. Excessive autophagy activation can continue to aggravate inflammatory responses. Autophagy related proteins such as Beclin1, microtubule-associated protein 1 light chain 3 (LC3), mammalian target of rapamycin (mTOR), and p62 are common autophagic markers in current research, which play a crucial role in regulating the autophagic process and the development of lung injury. Therefore, the expression of cellular autophagy genes can be used as early markers and important mechanisms of lung injury in septic ARDS. The Hippo signaling pathway is derived from the protein kinase Hippo in Drosophila, and the Hippo and autophagy are two conserved pathways that are essential for the protection of homeostasis in vivo. The mutual regulation of Hippo signaling pathway and autophagy is currently a hot topic in the academic community. This paper reviews the relevant literature to explore whether the Hippo signaling pathway can regulate cellular autophagy to alleviate the inflammatory response in septic ARDS, so as to provide further research directions for the treatment of ARDS.

Recent research progress on the role of Hippo-Yap signaling pathway in retinal diseases / 中华眼底病杂志

The classical Hippo pathway leads to the phosphorylation of downstream effector molecules Hippo-Yes-associated protein (Yap) and transcriptional coactivator PDZ-binding motif (Taz) serine sites through a kinase response, thereby promoting cell proliferation, controlling cell polarity, changing cytoskeleton, it plays an important regulatory role in various pathophysiological processes such as epithelial-mesenchymal transition and inhibition of cell contact. Studies have shown that Yap/Taz can affect the progression of vitreoretinal diseases, opening up new prospects for the pathogenesis and clinical treatment of diabetic retinopathy, proliferative vitreoretinopathy, and retinal ischemia-reperfusion injury. Exploring the molecular mechanism of Yap/Taz provides a possible therapeutic target for future research in the treatment of retinal fibrosis diseases such as diabetic retinopathy and proliferative vitreoretinopathy. At the same time, regulating the activity of local Yap/Taz in the retina will also become an effective therapeutic target for damage-repair in retinal ischemia-reperfusion injury. However, Yap inhibitors have potential retinal toxicity and are still in the preclinical development stage. Further research on the mechanism of action and clinical safety of Yap inhibitors will provide new methods for the treatment of retinal diseases.

Scutellarin induces apoptosis of colorectal cancer HCT116 cells via Hippo signaling pathway / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of scutellarin on the apoptosis of human colorectal cancer cells via the Hippo signaling pathway in vitro. METHODS MTT colorimetric method was used to detect the influence of scutellar?in on the survival rate of HCT116 cells. And the effect of scutellarin at various concentrations on cell morphology was observed by microscopy. Cell scratch experiment was used to detect the influence of scutellarin on the migration of HCT116 cells. Hoechst33342/PI double staining method was used to detect the effect of scutellarin on the apoptosis of HCT116 cells. Western blotting method was used to assess the action of scutellarin on the expressions of Hippo signal?ing pathway-related proteins Mst1, Lats1, YAP1, p-YAP(Ser127), TAZ, and its downstream effector proteins c-Myc and cyclin D1, as well as apoptosis-related proteins Bcl-2 and Bax in HCT116 cells. RESULTS Scutellarin significantly affected the morphology of HCT116 cells and reduced the survival rate of HCT116 cells. Hoechst33342/PI double stain?ing showed that scutellarin effectively induced the apoptosis of HCT116 cells. Western blotting analysis showed that the expression levels of Hippo signaling pathway-related proteins Mst1, Lats1, YAP1, TAZ and its downstream effector pro?teins c-Myc, cyclin D1 were down-regulated in a concentration-dependent manner by scutellarin, and the expression of p-YAP (ser127) was up-regulated. Moreover, scutellarin substantially lessened the expression level of apoptosis-related protein Bcl-2, and promoted the protein level of Bax. CONCLUSION Scutellarin may inhibit the proliferation and migra?tion of HCT116 cells, while induce its apoptosis, potentially by activation of Hippo signaling pathway.

Research progress on the role of Hippo signaling pathway in posterior capsular opacification / 国际眼科杂志(Guoji Yanke Zazhi)

@#Posterior capsular opacification is the most common complication after cataract extraction, which seriously influences the quality of life of patients. At present, there is no effective measure to prevent posterior capsular opacification. Surgery or Nd:YAG laser is often used in clinical, and a new treatment is urgently needed. Hippo signaling pathway is involved in the steady-state regulation of many mammalian cells and organs. Recent studies have shown that Hippo signaling pathway can regulate the proliferation, apoptosis, differentiation and other behaviors of lens epithelial cells. Hippo signaling pathway may provide a new target in the treatment of posterior capsular opacification. This article reviews the composition, regulatory mechanism of Hippo signaling pathway and its application in posterior capsular opacification. In order to provide a broader idea for the prevention and treatment of posterior capsular opacification.

SHANK2 is a frequently amplified oncogene with evolutionarily conserved roles in regulating Hippo signaling

Dysfunction of the Hippo pathway enables cells to evade contact inhibition and provides advantages for cancerous overgrowth. However, for a significant portion of human cancer, how Hippo signaling is perturbed remains unknown. To answer this question, we performed a genome-wide screening for genes that affect the Hippo pathway in Drosophila and cross-referenced the hit genes with human cancer genome. In our screen, Prosap was identified as a novel regulator of the Hippo pathway that potently affects tissue growth. Interestingly, a mammalian homolog of Prosap, SHANK2, is the most frequently amplified gene on 11q13, a major tumor amplicon in human cancer. Gene amplification profile in this 11q13 amplicon clearly indicates selective pressure for SHANK2 amplification. More importantly, across the human cancer genome, SHANK2 is the most frequently amplified gene that is not located within the Myc amplicon. Further studies in multiple human cell lines confirmed that SHANK2 overexpression causes deregulation of Hippo signaling through competitive binding for a LATS1 activator, and as a potential oncogene, SHANK2 promotes cellular transformation and tumor formation in vivo. In cancer cell lines with deregulated Hippo pathway, depletion of SHANK2 restores Hippo signaling and ceases cellular proliferation. Taken together, these results suggest that SHANK2 is an evolutionarily conserved Hippo pathway regulator, commonly amplified in human cancer and potently promotes cancer. Our study for the first time illustrated oncogenic function of SHANK2, one of the most frequently amplified gene in human cancer. Furthermore, given that in normal adult tissues, SHANK2's expression is largely restricted to the nervous system, SHANK2 may represent an interesting target for anticancer therapy.

Study on Hippo signaling pathway in mouse liver metastasis semi-spleen model of colorectal cancer / 肿瘤

Objective: To study the role of Hippo signaling pathway using mouse liver metastasis and semi-spleen model of colorectal cancer in vivo. Methods: Twenty BALB/c mice were randomly divided into sham operated group and experimental group. In the sham operation group, the abdomen of mouse was opened and closed. In the experimental group, the mouse colonic adenocarcinoma CT26 cells were injected into the lower pole of spleen, and excised to establish a liver metastasis model. After 7 days, the mice were sacrificed and dissected. The expression levels of epinephrine (EPI), glucagon (GC), lysobisphosphatidic acids (LPA) and sphingosine-1 phosphate (S1P) in plasma collected from the eyeballs of mice were detected by ELISA. The successful semi-spleen liver metastasis modeling of colorectal cancer was determined by HE staining. The yes-associated protein 1 (YAP1) and phospho-YAP1 (p-YAP1) were localizated by immunohistochemistry. The expression levels of YAP1 and p-YAP1 proteins in the liver tissues of mice were detected by Western blotting. Results: The liver metastasis rate was 100% after 7 days of spleen hemisection model establishment. The levels of plasma EPI and GC in the sham operated group were significantly higher than those in the experimental group (both P< 0.01). The levels of plasma LPA and S1P in the experimental group were significantly higher than those in the sham operated group (both P< 0.01). The HE staining result of experimental group showed typical features of poorly differentiated adenocarcinoma with mitotic division. In the normal liver tissues of sham operated group, YAP1 protein expressed in the cytoplasm [nuclear YAP (-) and cytoplasmic YAP (+)], p-YAP1 protein expressed in the cytoplasm and nuclei [nuclear p-YAP1 (+) and cytoplasmic p-YAP1 (+)]; In the tumor liver tissues of experimental group, YAP1 protein expressed in the cytoplasm and nuclei [nuclear YAP1 (+) and cytoplasmic YAP1 (+)], p-YAP1 protein was not expressed in the cytoplasm or nuclei [nuclear p-YAP1 (-) and cytoplasmic p-YAP1 (-)]. The expression level of YAP1 protein in the liver of experimental group was significantly higher than that in the sham operated group (P< 0.01), while the expression level of p-YAP1 in the liver of experimental group was significantly lower than that in the sham operated group (P < 0.01). Conclusion: The liver metastasis model of colorectal cancer is successfully established. The expressions of EPI and GC are negatively correlated with YAP1, and positively correlated with p-YAP1. The expressions of LPA and S1P are positively correlated with YAP1, and negatively correlated with p-YAP1. It is expected to develop a targeted therapeutic study for Hippo signaling pathway-related blood factor modulators.

Downregulation of Herg1 suppresses osteosarcoma proliferation and invasion by targeting Hippo signaling pathway / 中华肿瘤杂志

Objective@#To detect the effect and regulatory mechanism of human ether à go-go related gene 1 (Herg 1) knockdown on the proliferation and invasion of osteosarcoma (OS).@*Methods@#We constructed a recombinant adenovirus vector (Ad5-Herg1-shRNA) expressing short hair RNA (shRNA) against Herg1 and tested the knockdown efficiency. Then, the effects of Herg 1 knockdown on the proliferation, growth and invasion of osteosarcoma were measured by using cell counting kit-8 (CCK-8), wound healing assay, Transwell assay and xenograft model of nude mice, respectively. Tandem affinity purification, mass spectrometry and dual luciferase reporter assay were used to find out the molecules interacted with Herg1. Western blot was used to detect the expressions of large tumor suppressor gene (LATS1), p-LATS1, Yes-associated protein (YAP) and p-YAP in cells after infection of Ad5-Herg1-shRNA.@*Results@#Compared to Ad5-control-shRNA, Ad5-Herg1-shRNA dramatically inhibited the expression of Herg1 in OS cells. The result of CCK8 array demonstrated that 143B cell vitalities of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (65.47±3.90)% and (79.90±1.52)%, significantly lower than (100.00±6.14)% of Ad5-control-shRNA group. Meanwhile, U2OS cell vitality of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (69.69±1.36)% and (76.72±2.75)%, significantly lower than (100.00±3.01)% of Ad5-control-shRNA group (all P<0.001). The results of wound healing array showed that 143B cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (33.03±2.88)% and (36.47±4.16)%, significantly lower than (97.78±2.28)% of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (68.07±0.90)% and (73.97±1.25)%, significantly lower than (96.50±1.12)% of Ad5-control-shRNA group (all P<0.001). The results of Transwell showed that 143B cell invasion numbers of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 36.50±12.15 and 44.83±7.62, significantly lower than 195.33±19.68 of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 21.83±7.99 and 22.85±7.08, significantly lower than 83.33±12.36 of Ad5-control-shRNA group (all P<0.001). The results of xenograft model of OS showed that the tumor volume and weight of Ad5-Herg1-shRNA group were significantly smaller than of Ad5-control-shRNA group after 14 days and 5 weeks of inoculation, respectively (P<0.001). Moreover, knockdown of Herg1 inhibited the metastasis of OS cells. In mechanism, Herg1 protein interacted with NF2 protein. Knockdown of Herg1 significantly suppressed the expression levels of LATS1 and YAP protein, and promoted the phosphorylation of LATS1 and YAP in OS cells (all P<0.001).@*Conclusion@#Our findings suggest that Herg1 participates in the proliferation and motility of OS cells and may serve as a potential therapeutic target for osteosarcoma patients.

Effects of Hippo signaling pathway on lung injury repair by mesenchymal stem cells in acute respiratory distress syndrome / 中华危重病急救医学

Objective To investigate the effects of Hippo signaling pathway on lung injury repair of mesenchymal stem cells (MSC) in acute respiratory distress syndrome (ARDS) and its mechanism. Methods Mouse bone marrow-derived MSC (mMSCs) cell lines with low expression of large tumor suppressor 2 (LATS2) were constructed by lentiviral vector transfection. Male C57BL/6 mice aging 6-8 weeks old were divided into four groups according to random number table (n = 36). The ARDS animal model (ARDS group) was reproduced by intratracheally injection of 2 g/L lipopolysaccharide (LPS) 50 μL, the normal saline (NS) control group was injected with an equal volume of NS. After 4 hours of model reproduction, 5×104 mMSCs transfected with blank lentivirus vector (MSC-shcontrol group) or shLATS2 lentivirus vector (MSC-shLATS2 group) were transplanted intratracheally, while NS control group and ARDS group were injected with equal volume of phosphate buffered saline (PBS). Mice were sacrificed at 3, 7, and 14 days after modeling, and lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. Near-infrared fluorescence imaging, immunofluorescence staining and Western Blot were used to track mMSCs in lung tissue. Retension and proportion of mMSC differentiation into type Ⅱ alveolar epithelial cells (AECⅡ) were evaluated. Lung tissue wet weight/body weight ratio (LWW/BW) and total protein (TP) and albumin (ALB) in BALF were determined to reflect pulmonary edema. The expression of Occludin protein in lung epithelium was tested by Western Blot to reflect permeability of epithelium. The levels of interleukins (IL-1β, IL-6, IL-10) in BALF were assessed by enzyme-linked immunosorbent assay (ELISA) to reflect lung inflammation. Hematoxylin-eosin (HE) staining and modified Masson staining were carried out, and the scores were assessed to reflect lung injury and evaluate pulmonary fibrosis. Results The signal intensity of isolated lung fluorescence images at 3 days in the MSC-shLATS2 group was significantly higher than that in the MSC-shcontrol group (fluorescence intensity: 0.039±0.005 vs. 0.017±0.002, P < 0.05), the number of green fluorescent protein (GFP)-positive cells in lung tissue was also significantly higher than that in the MSC-shcontrol group (cells/HP:29.65±6.98 vs. 17.50±4.58, P < 0.05), but they all decreased with time; and the proportion of mMSCs differentiated into AECⅡ was significantly increased [(64.12±15.29)% vs. (19.64±3.71)%, P < 0.05]. Compared with the NS control group, the levels of surface active protein C (SPC) and Occludin protein in the ARDS group were significantly decreased, LWW/BW ratio and TP, ALB and inflammatory factors levels in BALF were significantly increased; extensive alveolar and interstitial edema, hemorrhage and diffuse inflammatory cell infiltration were found in lung tissue, and the lung injury score was significantly increased; collagen fibers were deposited in alveolar septum and alveolar cavity, and pulmonary fibrosis score was also increased significantly. Compared with the ARDS group, the expression levels of SPC and Occludin at 14 days in the MSC-shcontrol group and the MSC-shLATS2 group were significantly increased (SPC/β-actin: 0.51±0.12, 0.68±0.10 vs. 0.27±0.08, Occludin/β-actin: 0.49±0.19, 0.79±0.11 vs. 0.25±0.08, all P < 0.05), TP, ALB, IL-1β, IL-6 levels in BALF at 3 days were significantly decreased [TP (g/L): 8.08±1.72, 5.12±0.87 vs. 12.55±2.09; ALB (g/L): 0.71±0.21, 0.44±0.18 vs. 1.18±0.29, IL-1β(ng/L): 99.26±14.32, 60.11±8.58 vs. 161.86±25.17, IL-6 (ng/L): 145.54±13.29, 101.74±11.55 vs. 258.79±27.88, all P < 0.05], and IL-10 was significantly increased (ng/L: 190.83±22.61, 316.65±37.88, both P < 0.05). Furthermore, all the above parameters in the MSC-shLATS2 group were significantly improved as compared with those in the MSC-shcontrol group (all P < 0.05). LWW/BW ratio in the MSC-shLATS2 group was significantly lower than that in the ARDS group and the MSC-shcontrol group (mg/g: 9.85±1.51 vs. 16.78±1.92, 14.88±1.74, both P < 0.05). Conclusion Inhibiting Hippo signaling pathway by low expression of LATS2 could promote the retention of mMSCs in lung tissue and differentiation into AECⅡcells of ARDS mice, improve pulmonary edema and alveolar epithelial permeability, regulate pulmonary inflammatory response, and alleviate pathological damage and fibrosis of lung tissue.

Modulation of Hippo signaling pathway facilitates mesenchymal stem cells to repair lung injury in murine lipopolysaccharide induced acute respiratory distress syndrome / 中华急诊医学杂志

Objectives To determine the effect of Hippo signaling pathway on lung injury repair of bone-marrow derived mesenchymal stem cells (mMSCs) in murine lipopolysaccharide (LPS) induced acute respiratory distress syndrome (ARDS).Methods C57BL/6 mouse bone marrow-derived MSC (mMSCs) cell lines with low expression of large tumor suppressor 1 (LATS 1) were constructed by lentiviral vector transfection.ARDS was modeled by intratracheally injection of 2 mg/mL lipopolysaccharide (LPS)50 μL.C57BL/6 mice were randomly(random number) divided into four groups (n=36):normal control group,ARDS group,ARDS mice transplanted with mMSCs transfected with blank lentivirus vector (MSC-shcontrol group) or sh-LATS 1 lentivirus vector (MSC-shLATS 1 group).Mice were sacrificed at 3,7,and 14 d after modeling,and lung tissue and bronchoalveolar lavage fluid (BALF) were collected.Near-infrared fluorescence imaging,immunofluorescence staining and Western blot were used to evaluate retention and differentiation of mMSCs in lung tissue.Lung tissue wet weight/body weight ratio (LWW/BW) and total protein (TP) and albumin (ALB) in BALF were determined to reflect pulmonary edema.The expression of Occludin protein in lung epithelium was tested by Western blot.The levels of interleukins (IL-1β,IL-6,and IL-10) in BALF were assessed by enzyme-linked immunosorbent assay (ELISA).Lung injury score and pulmonary fibrosis score in lung tissue were assessed.Results The retention ofmMSCs at 3,7 and 14 d in the MSC-shLATS1 group were significantly higher than those in the MSC-shcontrol group [(26.25±4.58) vs (12.13±3.75) cells/HP,(20.49±3.86) vs (9.97±2.76) cells/HP,and (13.77±3.55) vs (6.89±2.10) cells/HP.all P＜0.05],so was the differentiation of mMSCs into type Ⅱ alveolar epithelial cells at 14 d [(64.12±15.29)％ vs (19.64±3.71)％,P＜0.05].LWW/BW and TP and ALB in BALF at 3 and 14 d in the MSC-shLATS1 group [(9.85±1.51),(6.11±0.83) (mg/g) and (5.12±0.87),(3.05±0.87) (mg/mL)and (0.44±0.18),(0.33±0.04) (mg/mL)] were higher than those in the MSC-shcontrol group [(14.88± 1.74),(8.04±l.70)(mg/g) and (8.08±1.72),(5.94±1.20) (mg/mL) and (0.71±0.21),(1.07±0.29) (mg/mL)] (all P＜0.05),so was the relative expression of Occludin protein[(0.79±0.11) vs (0.49±0).19),(P＜0.05)].The levels of IL-1β and IL-6(pg/mL) in BALF in the MSC-shLATS1 group [(60.11±8.58),(101.74±11.55)] was lower than those in the MSC-shcontrol group [(99.26±14.32),(145.54±13.29)] (all P＜0.05),but the levels of IL-10 in BALF in the MSC-shLATS 1 group (316.65±37.88)pg/mL was higher than those in the MSC-shcontrol group (190.83±22.61)pg/mL (P＜0.05).Lung injury scores at 3 and 14 d in the MSC-shLATS1 group [(7.18±1.12),(3.33±0.49)] was lower than those in the MSC-shcontrol group [(9.72±1.45),(5.11±0.86)] (all P＜0.05),so was pulmonary fibrosis score at 14 d [(0.68±0.12) vs (1.47±0.18),P＜0.05].Conclusion Inhibition of Hippo signaling pathway through underexpression of LATS1 could improve the therapeutic effects of mMSCs in murine LPS-induced ARDS.

Hippo signaling pathway and colorectal cancer / 国际肿瘤学杂志

Hippo signaling pathway plays a significant role in the development of colorectal cancer,and acts as an important regulatory pathway which regulates cell proliferation and cell differentiation.Multiple proteins and genes in Hippo signaling pathway,especially the downstream YAP protein,play important roles in the occurrence,metastasis,drug resistance and recurrence of colorectal cancer.YAP and other related genes can be used as targeted markers for predicting drug resistance to colorectal cancer.Hippo signaling pathway gene network can be used as targeted therapy or combination therapy,thus providing new ideas for the treatment of colorectal cancer.

Expression and role of Hippo signaling pathway in non-small cell lung cancer / 国际肿瘤学杂志

Hippo signaling pathway plays an important role in the growth and regeneration of animal organs. Studies have shown that the misexpression of Hippo signaling pathway composed of yes-associated protein and trascriptional co-activator with PDZ-binding motif is involved in the development of non-small cell lung cancer(NSCLC)and has synergistic effect with mutant p53. In addition,its overexpression leads to drug resistance in lung cancer treatment and inhibition of its expression may benefit cancer patients. The expression and role of Hippo signaling pathway in NSCLC are described in detail,which is expected to provide a new direction for targeted therapy of lung cancer.

Effects of Hippo signaling on anti-oxidative stress of mesenchymal stem cells in vitro / 中华危重病急救医学

Objective To explore the effects of Hippo signaling on anti-oxidative stress of mouse marrow mesenchymal stem cells (mMSCs) in vitro. Methods mMSCs derived from C57BL/6 mice were identified using fluorescence-activated cell sorting analysis and the capabilities of osteogenic, chondrogenic and adipogenic differentiation were evaluated. 2-deoxy-D-glucose (2-DG) or XMU-MP-1 was used to modulate Hippo signaling. Oxidative stress was induced by H2O2treatment and the effect of oxidative stress induced by H2O2on survival of mMSCs was evaluated using methyl thiazolyl tetrazolium (MTT) assay. The effect of oxidative stress induced by H2O2on Hippo signaling and the effect of Hippo signaling on capability of anti-oxidative stress of mMSCs were analyzed through apoptosis-regulated proteins (Bcl-2 and Bax) using Western Blot. Results Hippo signaling was activated by 2-DG in a concentration-dependent manner and the effect was most prominent by 5 mmol/L of 2-DG [compared with the blank control group, large tumor suppressor 1 (LATS1) protein (grey value): 2.33±0.25 vs. 0.98±0.03, phosphorylated Yes-associated protein (p-YAP)/YAP protein ratio (grey value): 2.30±0.35 vs. 1.01±0.05, 14-3-3 protein (grey value):2.19±0.40 vs. 0.99±0.04, all P < 0.05]; Hippo signaling was inhibited by 100 nmol/L of XMU-MP-1 [compared with the blank control group, LATS1 protein (grey value): 0.69±0.10 vs. 0.98±0.03, p-YAP/YAP protein ratio (grey value):0.65±0.06 vs. 1.01±0.05, 14-3-3 protein (grey value): 0.75±0.11 vs. 0.99±0.04, all P < 0.05]. Death of mMSCs was induced by H2O2in a concentration-dependent manner and the minimal effective concentration was 0.1 mmol/L [compared with the blank control group, survival rate of mMSCs: (81.25±11.85)% vs. (100.44±12.39)%, P < 0.05]. Inhibition of Hippo signaling was induced by H2O2in a concentration-dependent manner and the minimal effective concentration was also 0.1 mmol/L [compared with the blank control group, LATS1 protein (grey value): 0.75±0.06 vs. 1.01±0.09, p-YAP/YAP protein ratio (grey value): 0.69±0.05 vs. 0.98±0.05, both P < 0.05], those effects might associate with reduction of Bcl-2/Bax ratio (grey value: 0.48±0.18 vs. 1.06±0.09, P < 0.05). Compared with the treatment of 0.1 mmol/L of H2O2, activation of Hippo signaling by 5 mmol/L of 2-DG [ LATS1 protein (grey value):0.95±0.05 vs. 0.64±0.06, p-YAP/YAP protein ratio (grey value): 0.87±0.03 vs. 0.45±0.16, both P < 0.05] improved survival of mMSCs [(92.80±9.43)% vs. (75.47±9.43)%, P < 0.05] through an increase of Bcl-2/Bax ratio (grey value:1.14±0.16 vs. 0.77±0.12, P < 0.05); however, inhibition of Hippo signaling by 100 nmol/L of XMU-MP-1 [ LATS1 protein (grey value): 0.39±0.03 vs. 0.64±0.06, p-YAP/YAP protein ratio (grey value): 0.28±0.04 vs. 0.45±0.16, both P < 0.05] decreased survival of mMSCs [(57.54±4.59)% vs. (75.47±9.43)%, P < 0.05] through an decrease of Bcl-2/Bax ratio (grey value: 0.63±0.20 vs. 0.77±0.12, P < 0.05). Compared with normal lung tissue, acute respiratory distress syndrome (ARDS) lung tissue markedly activate Hippo signaling in mMSCs [LATS1 protein (grey value): 1.71± 0.08 vs. 1.00±0.10, p-YAP/YAP protein ratio (grey value): 2.46±0.39 vs. 1.01±0.04, 14-3-3 protein (grey value):2.27±0.52 vs. 1.01±0.08, all P < 0.05]. Conclusion Hippo signaling could affect survival and capability of anti-oxidative stress of mMSCs via modulation of Bcl-2/Bax ratio in vitro.

Galectin-3 induces differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of galectin-3 in inducing the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocyte- like cells and explore the involvement of the signaling pathways in the induced cell differentiation.</p><p><b>METHODS</b>The third passage of cultured rat femoral BMSCs were treated with 0.5 μg/mL galectin-3, 20 ng/mL hepatocyte growth factor (HGF) or both to induce their differentiation, with untreated rat BMSCs and hepatocytes as controls. At 7, 14, 21 and 28 days of induction, the cells were examined for morphological changes followed by glycogen staining, quantitative real-time PCR and Western blotting. Gene microarray technique was used to examine the mRNA expression profile of the BMSCs induced with galectin-3. The BMSCs were also induced with galectin-3 in combination with XMU-MP-1, a Hippo signaling pathway inhibitor, after which Western blotting was performed to detect the expressions of YAP, P-YAP, ALB, AFP and CK-18 in the cells.</p><p><b>RESULTS</b>The cells isolated from the femoral bone marrow of SD rats showed a consistent surface marker phenotype with the BMSCs. Induction with galectin-3, HGF, or both all resulted in gradual morphological changes of the BMSCs into hepatocyte-like cells, and the cells with a combined induction for 28 days showed the highest morphological similarity with hepatocytes. The cells induced with galectin-3, HGF, or their combination for 28 days all showed increased positivity rate of glycogen staining, which was the highest in the cells with combined induction ( < 0.05) without significant difference between the cells induced with galectin-3 and HGF alone ( > 0.05). Induction with galectin-3 and HGF alone both increased the expressions of AFP, ALB and CK-18 mRNAs in the cells, and their expression levels were similar between the cells at 28 days ( > 0.05). Galectin-3 and HGF did not show an interactive effect on the mRNA expressions of AFP (=0.236, =0.640) or ALB (=50.639, =0.000), but had a synergistic effect on CK-18 mRNA expression (=50.639, =0.000). The protein expressions of AFP, ALB and CK18 were also increased in the induced cells but not detected in the cells without induction. Gene microarray results revealed 27 up-regulated genes and 62 down-regulated genes in galectin-3-induced BMSCs involving TGF-β, PI3K-Akt and Hippo signal pathways. Induction with galectin-3 and galectin-3+XMU-MP-1 increased YAP expression in the cells, and galectin-3+XMU-MP-1 was more efficient to induce the differentiation of the BMSCs.</p><p><b>CONCLUSIONS</b>Galectin-3 can induce the differentiation of rat BMSCs into hepatocyte-like cells, and the combination with HGF increases the efficiency of induced differentiation of the cells. TGF-β, PI3K-Akt and Hippo pathways are involved in the induced differentiation of the BMSCs, and inhibiting Hippo pathway can improve the induction efficiency.</p>

Under-expression of LATS1 promotes the differentiation, proliferation and migration of mesenchymal stem cells by inhibition the Hippo signaling pathwayin vitro / 中华危重病急救医学

Objective To explore the effects of under-expression of large tumor suppressor 1 (LATS1) on activation of Hippo signaling pathway and differentiation, proliferation, migration of bone marrow mesenchymal stem cells (mMSCs) of micein vitro.Methods mMSCs of C57BL/6 mice were divided into normal control (MSC) group, empty vector control (MSC-GFP) group, LATS1-over-expressing (MSC-LATS1) group, empty vector without LATS1 shRNA control (MSC-shControl) group and LATS1-under-expressing (MSC-shLATS1) group. Lentiviral vectors with activated,inactivated LATS1 (the key molecule of Hippo signaling pathway) modifications and empty vectors were constructed and were used to infect mMSCsin vitro. The transduction efficiencies mediated by the lentiviral vectors were evaluated by fluorescence microscopy and flow cytometry. The mRNA expression of LATS1 was quantified by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expressions of LATS1, YAP (p-YAP), 14-3-3 were quantified by Western Blot to evaluate the activation of Hippo signaling pathway. Osteogenic and adipogenic differentiation of mMSCs were evaluated through measurement of Runx2, OSX and C/EBPα, PPAR-γ mRNA by qRT-PCR, as well as Alizarin Red S and Oil red O staining. Proliferation of mMSCs was evaluated using methy thiazdyl tetrazolium (MTT) assay. The scratch test and Transwell chamber test were used to analyze the horizontal and vertical migration ability of mMSCs.Results The transduction efficiencies mediated by the lentiviral vectors were 94.74%-96.10%. Compared with MSC-GFP group, the activation of Hippo signaling pathway was promoted in MSC-LATS1 group [LATS1 mRNA (2-ΔΔCT): 4.37±0.21 vs. 1.20±0.04, LATS1 protein (gray value): 2.21±0.06 vs. 1.09±0.10, p-YAP/YAP protein (gray value): 1.51±0.13 vs. 0.98±0.05, 14-3-3 protein (gray value): 1.92±0.18 vs. 1.10±0.09, allP < 0.05], osteogenic and adipogenic differentiation of mMSCs were decreased in MSC-LATS1 group [mineralization (A value):0.13±0.02 vs. 0.40±0.03, Runx2 mRNA (2-ΔΔCT): 0.51±0.02 vs. 0.98±0.09, OSX mRNA (2-ΔΔCT): 0.41±0.04 vs. 1.04±0.09, lipid accumulation (A value): 0.10±0.02 vs. 0.25±0.03, C/EBPα mRNA (2-ΔΔCT): 0.33±0.03 vs. 1.11±0.09, PPAR-γ mRNA (2-ΔΔCT): 0.29±0.02 vs. 1.04±0.10, allP < 0.05], the proliferation rate of mMSCs at 4-7 days was decreased in MSC-LATS1 group and so were the horizontal and vertical migration of mMSCs [wound healing rate: (18.65±3.53)% vs. (40.29±1.87)%, migrated cells (cells/MP): 35.99±6.18 vs. 103.67±17.77, bothP <0.05]. Compared with MSC-shControl group, the activation of Hippo signaling pathway was inhibited in MSC-shLATS1 group [LATS1 mRNA (2-ΔΔCT): 0.16±0.01 vs. 0.98±0.03, LATS1 protein (gray value): 0.38±0.03 vs. 1.04±0.07, p-YAP/YAP protein (gray value): 0.58±0.04 vs. 1.05±0.06, 14-3-3 protein (gray value): 0.14±0.02 vs. 1.02±0.09, allP < 0.05], osteogenic and adipogenic differentiation of mMSCs were increased in MSC-shLATS1 group [mineralization (A value): 0.93±0.13 vs. 0.44±0.05, Runx2 mRNA (2-ΔΔCT): 1.44±0.12 vs. 0.95±0.04, OSX mRNA (2-ΔΔCT):1.67±0.06 vs. 1.10±0.11, lipid accumulation (A value): 0.47±0.06 vs. 0.28±0.04, C/EBPα mRNA (2-ΔΔCT):3.98±0.61 vs. 0.99±0.10, PPAR-γ mRNA (2-ΔΔCT): 3.05±0.36 vs. 0.98±0.14, allP < 0.05], the proliferation rate of mMSCs at 3-7 days was increased in MSC-shLATS1 group and so were the horizontal and vertical migration of mMSCs [wound healing rate: (80.18±6.98)% vs. (46.18±1.01)%, migrated cells (cells/MP): 212.69±41.21 vs. 115.87±35.15, bothP < 0.05].Conclusions Under-expression of LATS1 promotes the differentiation, proliferation, migration of mMSCs by inhibition of Hippo signaling pathwayin vitro.

Research progress of relation between Hippo-YAP signaling pathway and tumor / 肿瘤研究与临床

The Hippo-YAP signaling pathway,an important signaling pathway of tumor suppressor,is featured by functions of adjusting the organ size and maintaining the dynamic balance between cell proliferation and apoptosis.Several researches have revealed that the Hippo-YAP signaling pathway can affect cell proliferation and apoptosis so as to participate in the development of tumor.This paper will review the effect and mechanism of Hippo-YAP signaling pathway in tumor.