Smad7 Protein Expression Research Articles

(E)-SIS3 suppressed osteosarcoma progression via promoting cell apoptosis, arresting cell cycle, and regulating the tumor immune microenvironment.

Osteosarcoma is a prevalent malignant bone tumor with a poor prognosis. Mothers against decapentaplegic homolog 3 (Smad3) present as a therapeutic target in antitumor treatment, whereas its functions in the osteosarcoma have not been well explored. In the current study, we aimed to investigate the effects of Smad3 in the progression of osteosarcoma. The tumor immune single-cell hub 2 website was used for graph-based visualization of Smad3 status in osteosarcoma single-cell database. Western Blot was applied to detect the expression of Smad3 protein in cell lines. Colony formation and cell counting kit-8 assays were used to evaluate cell proliferation. Transwell and wound healing assays were used to detect the migration and invasion abilities of cells. Cell apoptosis rates and cell cycle changes were explored by using flow cytometry analysis. The xenograft tumor growth model was applied to explore the effect in tumor growth after Smad3 blockage in vivo. Moreover, to confirm the potential mechanism of Smad3's effects on osteosarcoma, bioinformatics analysis was performed in TARGET-Osteosarcoma and GSE19276 databases. Our study found that the Smad3 protein is overexpressed in 143B and U2OS cells, suppressing the expression of Smad3 protein in osteosarcoma cells by Smad3 target inhibitor (E)-SIS3 or lentivirus can inhibit the proliferation, migration, invasion, promote cell apoptosis, arrest cell G1 cycle in osteosarcoma cells in vitro, and suppress tumor growth in vivo. Furthermore, the bioinformatics analysis demonstrated that high expression of Smad3 is closely associated with low immune status in TARGET-Osteosarcoma and GSE19276 databases. Our study suggested that Smad3 could contribute positively to osteosarcoma progression via the regulation of tumor immune microenvironment, and Smad3 may represent as an valuable potential therapeutic target in osteosarcoma therapy.

A negative feedback loop centered on SMAD3 expression in transforming growth factor β1-induced corneal myofibroblast differentiation

SMAD3 downregulation is documented in transforming growth factor β1 (TGF-β1)-induced corneal fibroblasts differentiation to myofibroblasts (“fibroTOmyoDiff”) or corneal wound healing. However, the exact regulatory mechanism of TGF-β1/SMAD3 pathway in this context remains unclear. Here, we investigated the role and related mechanism of SMAD3 down-regulation in TGF-β1-induced human corneal fibroTOmyoDiff. By detecting expression changes of SMAD family during this process, we demonstrated that SMAD3 protein expression was dramatically decreased in the process and the decrease occurred mainly in SMAD3 gene transcription. Furthermore, SMAD3 overexpression using lentivirus infection and knockdown using sgRNA lentivirus infection or siRNAs revealed that SMAD3 overexpression enhanced TGF-β1-induced corneal fibroTOmyoDiff and vice versa. In addition, specific siRNAs and inhibitors targeting particular signaling pathway were used to figure out the intracellular signaling pathway regulating SMAD3, and the result showed that the decease of SMAD3 induced by TGF-β1 stimulation in human corneal fibroblasts (HCFs) was strikingly prevented by SMAD4 knockdown or p38 signaling inhibitor SB203580 treatment. Collectively, these results demonstrate that, in TGF-β1 induced corneal fibroTOmyoDiff, down-regulation of SMAD3 expression regulated by SMAD4 and p38 signaling pathways forms a negative feedback loop of TGFβ signaling to avoid excessive activation of the signaling, which suggest that SMAD3 may be a key target for corneal fibrosis treatment.

Genetic analysis of low-grade adenosquamous carcinoma of the breast progressing to high-grade metaplastic carcinoma

PurposeLow-grade adenosquamous carcinoma (LGASC) is a rare type of metaplastic carcinoma of the breast (MBC) with an indolent clinical course. A few LGASC cases with high-grade transformation have been reported; however, the genetics underlying malignant progression of LGASC remain unclear.MethodsWe performed whole-genome sequencing analysis on five MBCs from four patients, including one case with matching primary LGASC and a lymph node metastatic tumor consisting of high-grade MBC with a predominant metaplastic squamous cell carcinoma component (MSC) that progressed from LGASC and three cases of independent de novo MSC.ResultsUnlike de novo MSC, LGASC and its associated MSC showed no TP53 mutation and tended to contain fewer structural variants than de novo MSC. Both LGASC and its associated MSC harbored the common GNAS c.C2530T:p.Arg844Cys mutation, which was more frequently detected in the cancer cell fraction of MSC. MSC associated with LGASC showed additional pathogenic deletions of multiple tumor-suppressor genes, such as KMT2D and BTG1. Copy number analysis revealed potential 18q loss of heterozygosity in both LGASC and associated MSC. The frequency of SMAD4::DCC fusion due to deletions increased with progression to MSC; however, chimeric proteins were not detected. SMAD4 protein expression was already decreased at the LGASC stage due to unknown mechanisms.ConclusionNot only LGASC but also its associated high-grade MBC may be genetically different from de novo high-grade MBC. Progression from LGASC to high-grade MBC may involve the concentration of driver mutations caused by clonal selection and inactivation of tumor-suppressor genes.

Methylation status, mRNA and protein expression of the SMAD4 gene in patients with non-melanocytic skin cancers.

SMAD4 is a potent tumor suppressor. SMAD4 loss increases genomic instability and plays a critical role in the DNA damage response that leads to skin cancer development. We aimed to investigate SMAD4 methylation effects on mRNA and protein expression of SMAD4 in cancer and healthy tissues from patients with basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), and basosquamous skin cancer (BSC). The study included 17 BCC, 24 cSCC and nine BSC patients. DNA and RNA were isolated from cancerous and healthy tissues following punch biopsy. Methylation-specific polymerase chain reaction (PCR) and real-time quantitative PCR methods were used to examine SMAD4 promoter methylation and SMAD4 mRNA levels, respectively. The percentage and intensity of staining of the SMAD4 protein were determined by immunohistochemistry. The percentage of SMAD4 methylation was increased in the patients with BCC (p = 0.007), cSCC (p = 0.004), and BSC (p = 0.018) compared to the healthy tissue. SMAD4 mRNA expression was decreased in the patients with BCC (p˂0.001), cSCC (p˂0.001), and BSC (p = 0.008). The staining characteristic of SMAD4 protein was negative in the cancer tissues of the patients with cSCC (p = 0.00). Lower SMAD4 mRNA levels were observed in the poorly differentiated cSCC patients (p = 0.001). The staining characteristics of the SMAD4 protein were related to age and chronic sun exposure. Hypermethylation of SMAD4 and reduced SMAD4 mRNA expression were found to play a role in the pathogenesis of BCC, cSCC, and BSC. A decrease in SMAD4 protein expression level was observed only in cSCC patients. This suggests that epigenetic alterations to the SMAD4 gene are associated with cSCC. The name of the trial register: SMAD4 Methylation and Expression Levels in Non-melanocytic Skin Cancers; SMAD4 Protein Positivity. The registration number: NCT04759261 ( https://clinicaltrials.gov/ct2/results?term=NCT04759261 ).

Effect and Mechanism of CircPTPN4 Expression in Carboxylated Single-Walled Carbon Nanotubes on Renal Fibrosis

This research was aimed to investigate the effect of circPTPN4 overexpression in carboxylated single-walled carbon nanotubes (SWCNT-COOH) on renal fibrosis (RF) in mice. SWCNT was oxidized to SWCNT-COOH by concentrated sulfuric acid and nitric acid (3:1, v/v). After characterization and analysis, the effect of SWCNT-COOH on erythrocyte status was detected. The RF mice (RFM) model was induced by SWCNT-COOH, normal mice as normal control (NC), and 1×108 TU/mL lenti-NC was injected into the tail vein as negative control, and 1×108 TU/mL lenti-circPTPN4 was injected for circPTPN4 overexpression. The expression of circPTPN4 was tested by qRT-PCR. Serum creatinine (Cr), blood urea nitrogen (BUN), interleukin 1β (IL-1β), IL-6, transforming growth factor β1 (TGF-β1), kidney injury molecule 1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) were tested by ELISA. HE, Masson, and immunohistochemistry were used to detect the pathological changes of kidney tissue. The protein expressions of collagen I (COL1), fibronectin (Fn), α-SMA, TGF-β1, Smad3, p-Smad3, and Smad7 were tested by Western blot (WB). It presented SWCNT-COOH was successfully prepared by chemical oxidation method, with an average particle size of 98.1 nm and an average Zeta potential of −38.91 mV. SWCNT-COOH had good hydrophilicity, and did not cause aggregation and morphology changes of red blood cells. As against NC, the expression of circPTPN4 in RFM and lenti-NC groups was decreased; the kidney coefficient was increased, and the normal structure of the kidney was abnormal, accompanied by inflammatory cell infiltration and collagen microformation; serum Cr, BUN, IL-1β, IL-6, and TGF-β1 were increased, and KIM-1 and NGAL in renal cortex were decreased; the protein expressions of COL1, Fn, α-SMA, TGF-β1, and p-Smad3 in the kidney were increased, and the protein expression of Smad7 was decreased (P <0.05). As against RFM, the expression of circPTPN4 in lenti-circPTPN4 group was increased; the kidney coefficient decreased and the kidney structure returned to normal; COL1, Fn, α-SMA, TGF-β1, and p-Smad3 in the kidney were decreased, and that of Smad7 was increased (P <0.05). SWCNT-COOH has good blood compatibility characteristics and can induce RF in mice. The expression of circPTPN4 was down-regulated in the RFM. Overexpression of circPTPN4 can restore the normal renal function of RFM, inhibiting the inflammatory response and RF process.

Effect mechanism of acupuncture for anti-asthmatic airway remodeling based on TGF-β1 / Smad3 signaling pathway

To observe the effect of acupuncture at "Feishu" (BL 13) + "Dingchuan" (EX-B 1) and "Kongzui" (LU 6) + "Yuji" (LU 10) for the airway remodeling in asthma rats based on the transforming growth factor-β1 (TGF-β1)/ Smad family member 3 (Smad3) signaling pathway; and explore the efficacy difference between the two acupoint combinations. Forty SPF male SD rats, aged 4 weeks, were randomly divided into a blank group (n = 10) and a modeling group (n = 30). The ovalbumin (OVA) sensitization method was used to establish asthma model in the modeling group. After successful model preparation, the rats of the modeling group were randomized into a model group, an acupuncture at "Feishu" (BL 13) + "Dingchuan" (EX-B 1) (AAF) group, and acupuncture at "Kongzui" (LU 6)+"Yuji" (LU 10) (AAK) group, with 10 rats in each one. Starting from day 15 of the experiment, 5 min after motivating, acupuncture was applied to "Feishu" (BL 13) + "Dingchuan" (EX-B 1) and "Kongzui" (LU 6)+"Yuji" (LU 10) in the AAF group and the AAK group respectively. The intervention was delivered for 30 min each time, once daily, lasting 3 weeks consecutively. Using lung function detector, the airway resistance (RL) and dynamic compliance (Cdyn) of the lungs were detected. The histomorphology of lung tissues was detected with HE staining and Masson staining, and the mRNA and protein expression of TGF-β1 and Smad3 in lung tissues was detected with the real-time PCR and Western blot methods. Compared with the blank group, RL was increased and Cdyn was decreased in the rats of the model group (P<0.01); and RL was reduced and Cdyn was increased in the AAF group and the AAK group when compared with those in the model group (P<0.01, P<0.05). The rats of the model group had bronchial lumen stenosis, inflammatory cell infiltration, collagen fibre hyperplasia and thickened smooth muscle in the lung tissues when compared with those in the blank group; and in comparison with the model group, all of the above morphological changes were attenuated in the AAF group and the AAK group. Besides, these morphological changes of the lung tissues were more alleviated in the AAF group when compared with those in the AAK group. In comparison with the blank group, the mRNA and protein expression of TGF-β1 and Smad3 of the lung tissues was increased in the model group (P<0.01), and it was reduced in the AAF group and the AAK group when compared with that in the model group (P<0.05, P<0.01). The mRNA expression of TGF-β1 and Smad3 was lower in the AAF group when compared with that in the AAK group (P<0.05). Acupuncture at either "Feishu" (BL 13)+"Dingchuan" (EX-B 1) or "Kongzui" (LU 6)+"Yuji" (LU 10) reduces the airway remodeling in the rats with asthma, which may be related to the down-regulation of mRNA and protein expression of TGF-β1 and Smad3. The better efficacy is obtained with acupuncture at "Feishu" (BL 13)+"Dingchuan" (EX-B 1).

BSHXF-medicated serum combined with ADSCs regulates the TGF-β1/Smad pathway to repair oxidatively damaged NPCs and its component analysis

Ethnopharmacological relevanceLower back pain (LBP) is a common and frequent clinical condition, and intervertebral disc degeneration (IDD) is recognized as the leading cause of LBP, typically manifested by increased nucleus pulposus cell (NPC) senescence and death. In recent years, the treatment of IDD with stem cell injections has had great potential compared to surgical treatment. Combining the two may achieve better results, as BuShenHuoXueFang (BSHXF) is an herbal formula that improves the survival rate of transplanted stem cells and enhances their efficacy. Aim of the studyWe aimed to qualitatively and quantitatively analyze BSHXF-medicated serum and investigate the molecular mechanism of BSHXF-mediated serum in promoting the differentiation of adipose mesenchymal stem cells (ADSCs) into NPCs and delaying the senescence of NPCs by regulating the TGF-β1/Smad pathway. Materials and methodsIn this study, an ultrahigh-performance liquid chromatography-quadrupole-time-of-flight mass spectrometer (UPLC-Q-TOF-MS) was used to establish a method for the analysis of rat serum samples to track the active components in vivo; the oxidative damage model of NPCs was induced by T-BHP, and a Transwell chamber was used to construct a coculture system of ADSCs and NPCs. Flow cytometry was used to determine the cell cycle; SA-β-Gal staining was used to assess cell senescence; ELISA was used to detect IL-1β, IL-6 inflammatory factors, CXCL-1, CXCL-3, CXCL-10 chemokines, and TGF-β1 in the supernatants of ADSCs and NPCs. WB was used to detect COL2A1, COL1A1, and Aggrecan in ADSCs to assess the manifestation of NP differentiation in ADSCs, and the WB method was used to detect COL2A1, COL1A1, Aggrecan, p16, p21, p53, and p-p53 protein expression in NPCs to reflect the cellular senescence status and to detect TGF-β1, Smad2, Smad3, p- Smad2, and p- Smad3 protein expression in NPCs to reflect the pathway condition. ResultsWe finally identified 70 blood components and their metabolites, including 38 prototypes, from the BSHXF-medicated serum. Compared with that in the nonmedicated serum group, the TGF-β1/Smad pathway was activated in the medicated serum group, ADSCs moved toward NPC characteristics, the number of NPCs in the S/G2M phase increased, the number of senescent NPCs decreased, IL-1β and IL-6 inflammatory factors in the Transwell decreased, CXCL-1, CXCL-3, and CXCL-10 chemokines decreased, and the expression of p16, p21, p53 and p-p53 proteins in NPCs was inhibited. ConclusionBy regulating the TGF-β1/Smad pathway, BSHXF-medicated serum promoted ADSCs to NPCs, effectively alleviated the cycle blockage of NPCs after oxidative damage, encouraged the growth and proliferation of NPCs, delayed the aging of NPCs, improved the deteriorating microenvironment around NPCs, and repaired oxidatively damaged NPCs. The combination of BSHXF or its compounds with ADSCs has great potential for the treatment of IDD in the future.

Antitumor Activity of Luteolin Against Ehrlich Solid Carcinoma in Rats via Blocking Wnt/β-Catenin/SMAD4 Pathway.

Background Ehrlich solid carcinoma (ESC) is characterized by rapid proliferation and a short survival time. Because Ehrlich ascites carcinoma (EAC) resembles human cancer cells, Ehrlich solid and ascetic forms are commonly used to determine the anticancer effects of various compounds. Luteolin is a flavonoid compound found in many dietary sources, including carrots, peppers, celery, olive oil, peppermint, and oregano. Luteolin has potent anti-inflammatory, antidiabetic, antitumor, and antiapoptotic activities. Aims This study aims to investigate the antitumor activity of luteolin against ESC in rats by affecting the Wnt/β-catenin/SMAD4 pathway. Methods We introduced 0.15 ml of Ehrlich cells (2 × 106) ESC into the left hind thighs of rats. After eight days of inoculation, the rats orally received 25 mg/kg of luteolin daily. We stained sections of tumor tissues with Masson's trichrome. We used another part of the tumor tissue to assess gene and protein expression of Wnt, β-catenin, E-cadherin, and SMAD4. Results Treatment of carcinoma rats with luteolin increased the mean survival time and reduced tumor volume and weight. In addition, examination of tumor tissue stained with Masson's trichrome showed loosely to densely packed collagen fibers in between neoplastic cells and scattered papillary expansion of a loose blue band of collagen expression along the covering adipose connective tissue and extending in a fine strand in between muscle fibers, which was ameliorated by treating rats with luteolin. Finally, treating ESC in rats with luteolin overexpressed E-cadherin and downregulated Wnt, β-catenin, and SMAD4. Conclusions We found luteolin has antineoplastic activity against ESC by reducing tumor size and weight while improving the structure of muscle cells. It works by suppressing Wnt, β-catenin, and SMAD4, resulting in decreased tumor cell proliferation and differentiation. Additionally, luteolin overexpresses E-cadherin, leading to reduced tumor cell invasion and metastasis.

Mediator1 involved in functional integration of Smad3 and Notch1 promoting enamel mineralization

Enamel hypoplasia is a tooth development defection due to the disruption of enamel matrix mineralization, manifesting as chalky white phenotype. Multiple genes may be involved in this tooth agenesis. It has been proved that ablation of coactivator Mediator1 (Med1) switches the cell fate of dental epithelia, resulting in abnormal tooth development via Notch1 signaling. Smad3 (−/−) mice displays the similar chalky white incisors. However, the expression of Smad3 in Med1 ablation mice and the impact of Med1 on functional integration between Smad3 and Notch1 remains unclear. Cre-loxP-based C57/BL6 mice with epithelial-specific Med1 knockout (Med1 KO) backgrounds were generated. Mandibles and dental epithelial stem cells (DE-SCs) from incisors cervical loop (CL) were isolated from wild-type (CON) mice and Med1 KO mice. Transcriptome sequencing was used to analyze the differences of CL tissue between KO and CON mice. The results revealed the enrichment of TGF-β signaling pathway. qRT-PCR and western blot were performed to show the gene and protein expression of Smad3, pSmad3, Notch1 and NICD, the key regulators of TGF-β and Notch1 signaling pathway. Expression of Notch1 and Smad3 was confirmed to be down-regulated in Med1 KO cells. Using activators of Smad3 and Notch1 on Med1 KO cells, both pSmad3 and NICD were rescued. Moreover, adding inhibitors and activators of Smad3 and Notch1 to cells of CON groups respectively, the protein expressions of Smad3, pSmad3, Notch1 and NICD were synergistically affected. In summary, Med1 participates in the functional integration of Smad3 and Notch1, thus promoting enamel mineralization.

Loss of function SMAD4 nonstop mutations in human cancer.

SMAD4 is a tumour suppressor gene that is mutated in a variety of cancers. SMAD4 nonstop mutations, which convert stop codons to sense codons that extend transcription, have been identified in genomic databases but have not been characterised in human pathology samples. The frequency of SMAD4 nonstop mutations and the consequences of nonstop mutations on SMAD4 protein expression are unknown. We retrospectively analysed our cancer sequencing database of 38,002 tumour specimens and evaluated the spectrum of SMAD4 mutations. SMAD4 protein expression was evaluated by immunohistochemistry in tumours with SMAD4 nonstop mutations. In total, 1956 SMAD4 mutations were identified in 1822 tumours. SMAD4 mutations were most common in tumours of the gastrointestinal tract and included nonsense variants (n=344), frameshift indels (n=258), splice site variants (n=104), and missense variants at codon R361 (n=245). In a subset of cases with immunohistochemistry, SMAD4 expression was lost in 23 of 25 tumours (92%) with protein truncating variants and in 7 of 27 tumours (26%) with missense variants. Four cases harboured SMAD4 nonstop mutations. SMAD4 nonstop mutations were identified in two pancreatic adenocarcinomas, one colonic adenocarcinoma, and one non-small cell lung carcinoma. Immunohistochemistry demonstrated loss of SMAD4 protein expression in each of the four tumours with SMAD4 nonstop mutations. SMAD4 nonstop mutations are associated with loss of SMAD4 protein expression in multiple tumour types. SMAD4 nonstop mutations should be clinically interpreted as pathogenic loss of function alterations when identified in cancer sequencing panels.

MiR-21 regulated hair follicle cycle development in Cashmere goats by targeting FGF18 and SMAD7

Increasing Cashmere production can add value because it is the primary product of Cashmere goats. Recent years, peoples find miRNAs are crucial in regulating the development of hair follicle. Following Solexa sequencing, many miRNAs were distinguishingly expressed in telogen skin samples of goats and sheep in earlier study. But the method through which miR-21 controls the growth of hair follicles is still ambiguous. Bioinformatics analysis was used to predict the target genes of miR-21. The mRNA level of miR-21 in telogen Cashmere goat skins was higher than in anagen, according to the results of qRT-PCR, and the target genes expressed similarly with miR-21. Western blot showed similar trend, the protein expression of FGF18 and SMAD7 were lower in anagen samples. The Dual-Luciferase reporter assay confirmed miRNA-21's relationship with its target gene, and the consequences indicated found FGF18 and SMAD7 have positive correlations with miR-21. Western blot and qRT-PCR distinguished the expression of protein and mRNA in miR-21 and its target genes. According to the consequence, we found that target genes expression was increased by miR-21 in HaCaT cells. This study identified that miR-21 might take part in the development of Cashmere goat’s hair follicles by targeting FGF18 and SMAD7.

TGFβ carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma.

Contributions of TGFβ to cancer progression are well documented. However, plasma TGFβ levels often do not correlate with clinicopathological data. We examine the role of TGFβ carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC). The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFβ expression levels during oral carcinogenesis. In human HNSCC, TGFβ and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFβ levels were evaluated by ELISA and TGFβ bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFβ content was quantified using bioassays and bioprinted microarrays. During 4-NQO carcinogenesis, TGFβ levels in tumour tissues and in serum increased as the tumour progressed. The TGFβ content of circulating exosomes also increased. In HNSCC patients, TGFβ, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFβ levels. Neither TGFβ expression in tumours nor levels of soluble TGFβ correlated with clinicopathological data or survival. Only exosome-associated TGFβ reflected tumour progression and correlated with tumour size. Circulating TGFβ+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

FA-HA-Amygdalin@Fe2O3 and/or γ-Rays Affecting SIRT1 Regulation of YAP/TAZ-p53 Signaling and Modulates Tumorigenicity of MDA-MB231 or MCF-7 Cancer Cells.

Breast cancer (BC) has a complex and heterogeneous etiology, and the emergence of resistance to conventional chemo-and radiotherapy results in unsatisfactory outcomes during BC treatment. Targeted nanomedicines have tremendous therapeutic potential in BC treatment over their free drug counterparts. Hence, this study aimed to evaluate the newly fabricated pH-sensitive multifunctional FAHA- Amygdalin@Fe2O3 nano-core-shell composite (AF nanocomposite) and/or γ-radiation for effective localized BC therapy. The physicochemical properties of nanoparticles were examined, including stability, selectivity, responsive release to pH, cellular uptake, and anticancer efficacy. MCF-7 and MDA-MB-231 cells were treated with AF at the determined IC50 doses and/or exposed to γ-irradiation (RT) or were kept untreated as controls. The antitumor efficacy of AF was proposed via assessing anti-proliferative effects, cell cycle distribution, apoptosis, and determination of the oncogenic effectors. In a bio-relevant medium, AF nanoparticles demonstrated extended-release characteristics that were amenable to acidic pH and showed apparent selectivity towards BC cells. The bioassays revealed that the HA and FA-functionalized AF markedly hindered cancer cell growth and enhanced radiotherapy (RT) through inducing cell cycle arrest (pre-G1 and G2/M) and increasing apoptosis, as well as reducing the tumorigenicity of BCs by inhibiting Silent information regulation factor 1 (SIRT1) and restoring p53 expression, deactivating the Yes-associated protein (YAP)/ Transcriptional coactivator with PDZ-binding motif (TAZ) signaling axis, and interfering with the tumor growth factor- β(TGF- β)/SMAD3 and HIF-1α/VEGF signaling hub while up-regulating SMAD7 protein expression. Collectively, the novel AF alone or prior RT abrogated BC tumorigenicity.

Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro

To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1. rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

KNTC1, regulated by HPV E7, inhibits cervical carcinogenesis partially through Smad2

Cervical cancer is the second most common malignancy of the female reproductive tract worldwide. Although cervical cancer is caused by human papillomavirus (HPV) infection, its underlying pathogenesis requires further investigation. The present study investigated the role of kinetochore associated protein 1 (KNTC1) in cervical cancer and its association with the key virus oncoprotein, HPV E7. A series of bioinformatic analyses revealed that KNTC1 might be involved in the tumorigenesis of multiple human malignancies, including cervical cancer. Tissue microarray analysis showed that in vivo KNTC1 expression was higher in high-grade squamous intraepithelial lesions (HSILs) than in normal cervix and even higher in cervical cancer. In vitro silencing of KNTC1 increased the proliferation, invasion and migration of cervical cancer cell lines. Although not affecting apoptosis, KNTC1 silencing significantly promoted G1/S phase transition of the cell cycle. High-throughput analysis of mRNA expression showed that KNTC1 could regulate its downstream target protein Smad2 at the transcriptional level. Moreover, as the key oncoprotein of the virus, HPV E7 could inhibit the expression of KNTC1 protein, and decrease Smad2 protein expression with or without the aid of KNTC1. These results indicated that KNTC1 is a novel tumor suppressor that can impede the initiation and progression of cervical carcinoma, providing insight into the molecular mechanism by which HPV induces cervical cancer.

Monocyte-derived immature dendritic cells negatively regulate hepatic stellate cells in vitro by secreting IL-10

The development of liver fibrosis is associated with inflammatory responses resulting from chronic liver disease. Immature dendritic cells (imDCs) play an important role in modulating the inflammatory environment of the liver. This study investigated the effects of imDCs on the regulation of hepatic stellate cells (HSCs) during liver fibrosis. We isolated and induced imDCs from monocytes of healthy volunteers, activated LX-2 cells with TGF-β to establish in vivo liver fibrosis HSCs model, and then set up a cell co-culture system with transwell membranes. imDC surface markers and apoptosis rates of LX-2 cells were detected by flow cytometry. The concentration of IL-10 secreted by imDC was measured through ELISA. The expression of α-SMA in LX-2 after co-culture was examined by qRT‑PCR. Proliferation of LX-2 cells were detected by CCK-8. The western blot was used to illustrate the LX-2 activation-related proteins such as Smad3/7 and TGF-β1. The imDCs co-culture group and the interleukin-10 (IL-10) treatment group had similar results, as they were both able to increase apoptosis, inhibit proliferation, downregulate α-SMA mRNA, and reduce TGF-β1 and Smad3 protein expression in LX-2 cells. Additionally, the Smad7 protein level was increased after treatment with imDC and IL-10. However, the results in the IL-10 antagonist group showed the opposite trend to that of imDCs and IL-10 groups. Thus, these results suggest that imDC secretion of IL-10 negatively regulates activated LX-2 cells, probably via inhibition of the TGF-β1/Smad3 pathway and increased expression of Smad7 protein. This may be a potential therapeutic target for liver fibrosis.

Endosulfan induced kidney cell injury by modulating ACE2 through up-regulating miR-429 in HK-2 cells

Endosulfan, a typical organochlorine pesticide, is widely used in agricultural countries and was detected in blood samples from the general population. Studies have shown a positive correlation between chronic kidney disease of unknown aetiology (CKDu) and endosulfan. CKDu has become endemic in agricultural countries, with clinical manifestations of tubulointerstitial fibrosis.The goal of this study was to investigate the effects of endosulfan in kidney cell injury in human renal tubular epithelial cells (HK-2), focusing on apoptosis, inflammatory response, and epithelial-mesenchymal transition (EMT). We found that endosulfan induced apoptosis in HK-2 cells by up-regulating the expression of BAX, APAF-1, Caspase-3 and mitochondrial Cytochrome c was released into the cytosol. Endosulfan caused an inflammatory response, showing the increase in the secretion and mRNA expression levels of IL-6/IL-8. Endosulfan triggered EMT, characterized by downregulation of E-cadherin and upregulation of Vimentin. Western blot results showed that p-Smad3 and Smad3 protein expression were elevated while the expression of Smad7 were decreased in endosulfan-exposed groups. Dual luciferase reporter assay confirmed the potential binding capacity of miR-429 to 3'-UTR of ACE2. Endosulfan causes upregulation of miR-429 and downregulation of ACE2 in HK-2 cells. Overexpression of miR-429 or silencing of ACE2 in HK-2 cells caused apoptosis, inflammation and EMT through TGF signaling pathway. These findings suggest that endosulfan can lead to kidney cell injury by modulating ACE2 through up-regulating miR-429, providing new evidence for the pathogenesis of CKDu.

Molecular Mechanisms Linking Empagliflozin to Renal Protection in the LLC-PK1 Model of Diabetic Nephropathy.

Chronic diabetes complications, including diabetic nephropathy (DN), frequently result in end-stage renal failure. This study investigated empagliflozin (SGLT2i) effects on collagen synthesis, oxidative stress, cell survival, and protein expression in an LLC-PK1 model of DN. Combinations of high glucose (HG) and increasing empagliflozin concentrations (100 nM and 500 nM), as well as combinations of HG, H2O2, and empagliflozin, were used for cell culture treatment. The cell viability, glutathione (tGSH), ECM expression, and TGF-β1 concentration were measured. In addition, the protein expression of Akt, pAkt, GSK3, pGSK3, pSTAT3, and SMAD7 was determined. The addition of both concentrations of empagliflozin to cells previously exposed to glucose and oxidative stress generally improved cell viability and increased GSH levels (p &lt; 0.001, p &lt; 0.05). In HG30/H2O2/Empa500-treated cells, significant increase in pSTAT3, pGSK3β, GSK3β, SMAD7, and pAKT levels (p &lt; 0.001, p &lt; 0.001, p &lt; 0.05) was observed except for AKT. Lower drug concentrations did not affect the protein expression levels. Furthermore, empagliflozin treatment (100 nM and 500 nM) of HG30/H2O2-injured cells led to a decrease in TGF-β1 levels (p &lt; 0.001). In cells exposed to oxidative stress and hyperglycemia, collagen production remained unchanged. Renoprotective effects of empagliflozin, in this LLC-PK1 cell model of DN, are mediated via activation of the Akt/GSK-3 signalling pathway, thus reducing oxidative stress-induced damage, as well as enhanced SMAD7 expression leading to downregulation of TGF-β1, one of the key mediators of inflammation and fibrosis.

Effect of macrophage-derived exosomes on promoting hepatic stellate cell activation and platelet-derived growth factor expression

Objective: To explore the effect of macrophage-derived exosomes on the activation of hepatic stellate cells and its possible mechanism. Methods: Differential ultracentrifugation was used to extract macrophage exosomes. The exosomes were co-cultured with the mouse hepatic stellate cell line JS1, and a control group was established with phosphate buffered saline (PBS). Cell immunofluorescence was used to observe the expressional conditions of F-actin. Cell counting kit-8 (CCK8) was used to detect the survival rate of JS1 cells in the two groups. The activation indices of JS1 cells [collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA)] and its key signal pathway activation index expression level [transforming growth factor (TGF)-β1/Smads, platelet-derived growth factor (PDGF)] in the two groups were determined using Western blot and RT-PCR. Data comparison between two groups was performed using an independent sample t-test. Results: The membrane structure of exosomes was clearly observed by transmission electron microscopy. The expression of exosome marker proteins CD63 and CD81 was positive, suggesting that exosomes were successfully extracted. Exosomes were co-cultured with JS1 cells. Compared with the PBS control group, there was no statistically significant difference in the proliferation rate of JS1 cells in the exosomes group (P＞0.05). The expression of F-actin was significantly increased in the exosome group. The mRNA and protein expression levels of α-SMA and ColⅠwere significantly increased in exosome group JS1 cells (all P＜0.05). The mRNA relative expression levels of α-SMA in PBS and exosome group were 0.25±0.07 and 1.43±0.19, respectively, while that of ColⅠ was 1.03±0.04 and 1.57±0.06, respectively. The mRNA and protein expressions of PDGF were significantly increased in exosome group JS1 cells (P＜0.05). The mRNA relative expression levels of PDGF in the PBS group and exosome group were 0.27±0.04 and 1.65±0.12, respectively. There were no statistically significant differences in the mRNA and protein expressions of TGF-β1, Smad2 and Smad3 between the two groups (P＞0.05). Conclusion: Macrophage-derived exosomes significantly promote the activation of hepatic stellate cells. JS1 cells may be the underlying mechanism for the up-regulation of PDGF expression.

Abstract A081: Molecular phenotyping and risk stratification of intraductal papillary mucinous neoplasms

Abstract Objective: Morphological subtyping of intraductal papillary mucinous neoplasms of the pancreas (IPMNs) into pancreatobiliary-type, intestinal-type, or gastric-type is a prognostic factor, however, molecular foundations explaining the difference of clinical behaviors are unknown. Our research aimed to elucidate the molecular phenotypes associated with subtypes of IPMNs and establish a risk stratification model. Design: In 171 IPMNs with high-grade dysplasia (HGD) and invasive carcinoma, we analyzed protein expressions of p16, p53, SMAD4, STK11, and β-catenin by immunohistochemistry, and mutations and copy number variations of KRAS and GNAS by droplet digital PCR, and analyzed the association with clinicopathological factors. Results: In HGD components of IPMNs of pancreatobiliary- (n=66), intestinal- (n=70), and gastric-type (n=35), alterations were detected respectively as follows: KRAS mutation; 98%, 62%, and 83% (P&amp;lt;0.05), GNAS mutation; 11%, 82%, and 51% (P&amp;lt;0.05), p53; 59%, 33%, and 20% (P&amp;lt;0.05), SMAD4; 67%, 1%, 37% (P&amp;lt;0.05), STK11; 43%, 3%, and 6% (P&amp;lt;0.05), β-catenin; 48%, 68%, and 23% (P&amp;lt;0.05), and p16; 65%, 52%, and 40% (P=0.105). Aberrations of 4 driver genes, p16, p53, SMAD4, and STK11, were succeeded from components of low-grade dysplasia to invasive carcinoma and associated with the risk of invasion of intraductal neoplasms and the patients’ outcomes. The combinatorial system by the number of accumulated gene alterations well stratified the risk of invasion (0–1 vs 2; odds ratio (OR) 4.62, 0–1 vs 3–4; OR 12.90) and patients’ outcome (0–1 vs 2; hazard ratio (HR) 4.01, 2 vs 3–4; HR 3.98, 0–1 vs 3–4; HR 15.99 in disease-free survival, 0–1 vs 2; HR 6.62, 2 vs 3–4; HR 4.43, 0–1 vs 3–4; HR 29.32 in overall survival). Moreover, GNAS amplification was an independent risk factor of invasion (OR 7.79) and showed poor prognosis (overall survival, log-rank P&amp;lt;0.05) in intestinal-type IPMNs, while sole GNAS activating mutation was an independent low risk factor for invasion (OR 0.33). KRAS amplification might be a poor prognostic factor in pancreatobiliary-type IPMNs (HR 2.7, P=0.07). Conclusion: The molecular patterns involving the malignant progression are different among epithelial subtypes of IPMNs. The molecular phenotypes of combinatorial genomic alterations of tumor suppressor genes with signature of KRAS and GNAS of intraductal components may stratify the risk of invasion and survival in IPMN patients. Molecular phenotyping using liquid biopsy of pancreatic juice during the surveillance might be additional value of decision making for operative timing in IPMNs showing equivocal radiological findings. Citation Format: Yuko Omori, Yusuke Ono, Takashi Kokumai, Fumiko Date, Ryota Higuchi, Goro Honda, Yusuke Mizukami, Takanori Morikawa, Michiaki Unno, Toru Furukawa. Molecular phenotyping and risk stratification of intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A081.