Research on Function of Exosome of miR-328-3p Secreted by Bone Marrow Mesenchymal Stem Cells (BMSCs) on Restraining the Gastric Cancer Through Being Down-Regulated with Trefoil Factor 3

Certain progress has been made in the therapeutic method against gastric cancer such as surgical operation combined with chemotherapy and radiation therapy in recent years. But the therapeutic efficacy and prognosis on gastric cancer was still not satisfactory. The function of exosome of miR-328–3p secreted by bone marrow stromal cells (BMSCs) on restraining the gastric cancer was studied in the present study. The BMSCs with highly-expressed miR-328-3p was established. The exosome in cell supernatant was collected. The exosome of BMSCs and MSCs with highlyexpressed miR-328-3p was added into SGC-7901 cells followed by analysis of miR-328-3p level by Real-time PCR and TFF3 (Trefoil Factor 3) level in exosome by Western blot, cell proliferation, expression of E-cadherin, Vimentin and Caspase-3. miR-328-39 expression was reduced and TFF3 was elevated in gastric cancer tissue (P < 0.05). miR-328-3p was upregulated and TFF3 was downregulated after addition of BMSCs exosomes along with increased cell proliferation and reduced E-cadherin and Caspase3 expression (P < 0.05). In conclusion, exosome of BMSCs could be regulated by miR-328-3p and TFF3 expression is restrained so as to regulate the biological behaviors of gastric cancer cell.

miR-16 Inhibits Extracellular Signal-Regulated Kinases (ERK) Mitogen-Activated Protein Kinases (MAPK) Signaling to Affect Epithelial-Mesenchymal Transition (EMT) and Invasion of Glioma Cells

Abnormal MEK1 expression is associated with tumor cell EMT, invasion and metastasis. Decreased miR-16 level is associated with glioma. Bioinformatics analysis showed a relationship between miR-16 and MEK1. This study assessed whether miR-16 regulates MEK1 expression and affects glioma cell EMT and invasion. The tumor tissues and adjacent glioma tissues were collected to measure miR-16 and MEK1 mRNA. The dual luciferase assay validated the relation of miR-16 with MEK1. U251 cells were cultured and assigned into NC group and mimic group, followed by analysis of cell biological behaviors, and MEK1, p-ERK1/2, E-cadherin, N-Cadherin expression. Compared with adjacent tissues, miR-16 expression was significantly decreased and MEK1 was elevated in glioma tissues. Compared with HEB, miR-16 in glioma U251 and SHG44 cells was decreased and MEK1 was increased. Dual luciferase reporter gene experiments confirmed the relation of miR-16 with MEK1. Transfection of miR-16 mimic significantly down-regulated MEK1, p-ERK1/2 and N-cadherin in U251 cells, upregulated E-cadherin, inhibited cell proliferation, promoted apoptosis, and attenuated EMT and invasion of glioma cells. In conclusion, decreased miR-16 expression and increased MEK1 expression is related to glioma pathogenesis. Overexpression of miR-16 can inhibit MEK1 expression, ERK/MAPK signaling, glioma cell proliferation, promote apoptosis, and attenuate EMT and invasion.

Exosomal miRNA-455 from Bone Marrow Stromal Cells (BMSCs) Promotes Macrophage Phagocytosis and Restrains Progression of Gastric Cancer (GC)

Multiple miRNAs are differentially expressed in gastric cancer (GC). Herein, this study aims to investigate miR-455’s role in GC and its mechanism. Exosomes (exo) separated from BMSCs after transfection were co-cultured with either phagocytes, GC cells (NCI-N87 cell), or macrophages combined with NCI-N87cells (mixed group) followed by analysis of the expression of PTEN, N-cadherin, E-cadherin, and PI3K, and AKT by RT-qPCR and Western blot. Increased miR-455 expression was observed in GC cells upon transfection. GC cells in the mixed group relative to NCI-N87 group exhibited a lower cell migration and invasion and impaired proliferative capacity (p < 0.05), accompanied with higher expressions of N-cadherin, E-cadherin, PI3K, and AKT, and decreased level of PTEN (p < 0.05). The combined treatment resulted in a higher phagocytic rate (12.38±0.21%) and phagocytic index (14.29±2.11%) compared to treatment with only phagocytes (p < 0.05). In conclusion, BMSC-derived exosomal miR-455 inhibits the growth of GC cells and promotes the phagocytosis through inactivating PI3K/AKT signaling pathway.

The Effects and Mechanisms of Improvement of Hydroxysafflor Yellow A in Pulmonary Fibrosis

Purpose: To discuss the effects and mechanisms of improvement of Hydroxysafflor yellow A in pulmonary fibrosis by in vivo study. Material and Methods: In this study, dividing the C57BL/6 mice as 4 group, there were 10 mice in every group. Collecting the serum of difference groups and measuring the Hyp, SOD, MDA, TNF-α and IL-6 levels. Lung tissues were taken out and evaluating the pathology by HE staining and fibrosis degree by Masson staining. The relative proteins (α-SMA and E-cadherin) were measured by IHC and WB in lung tissues of difference groups. Results: With HSYA or DXM supplement, the Hyp, MDA, TNF-α and IL-6 concentrations significantly suppressed and SOD concentration significantly enhanced (P < 0.05, respectively). Compared with Sham group, the pathology injury and fibrosis degree of Model group were significantly up-regulation (P < 0.001, respectively); With HSYA or DXM treatment, the pathology injury and fibrosis degree of HSYA and DXM groups were significantly improved (P < 0.05, respectively). By IHC and WB assay, the α-SMA and E-cadherin proteins expressions of Model group were significantly differences (P < 0.001, respectively); however, the α-SMA and E-cadherin proteins expressions of HSYA and DXM groups were significantly improved with HSYA or DXM supplement (P < 0.05, respectively). Conclusion: HSYA improves pulmonary fibrosis by regulation α-SMA and E-cadherin in vivo study.

The Impacts of Bone Marrow Mesenchymal Stem Cells (BMSCs)-Derived Periostin on Epithelial-Mesenchymal Transition (EMT) of Cervical Cancer Cells

Epithelial-mesenchymal transition (EMT) is closely related to the migrating and invading behaviors of cells. Periostin is one of the essential components in the extracellular matrix and can induce EMT of cells and their sequential metastasis. But its underlying mechanism is unclear. The Hela and BMSC cell lines were assigned into Periostin-mimic group, Periostin-Inhibitor group and Periostin-NC group followed by analysis of cell migration and invasion, expression of E-Cadherin, Vimentin, β-Catenin, Snail, MMP-2, MMP-9, PTEN, and p-PTEN. Cells in Periostin-mimic group exhibited lowest migration, least number of invaded cells, as well as lowest levels of Vimentin, β-Catenin, Snail, MMP-2, MMP-9, p-PTEN, Akt, p-Akt, p-GSK-3β, p-PDK1 and p-cRcf, along with highest levels of E-cadherin and PTEN. Moreover, cells in Periostin-NC group had intermediate levels of these above indicators, while, the Periostin-Inhibitor group exhibited the highest migration rate, the most number of invaded cells, and the highest levels of these proteins (P < 0.05). In conclusion, BMSCs-derived Periostin can influence the EMT of cervical cancer cells possibly through restraining the activity of the PI3K/AKT signal transduction pathway, indicating that Periostin might be a target of chemotherapy in clinics for the treatment of cervical cancer.

Slug and Vimentin Downregulation at the Metastatic Site Is Associated With Skip-N2 Metastasis of Lung Adenocarcinoma

Objective: Lung cancer displays heterogeneity both in the tumor itself and in its metastatic regions. One interesting behavior of the tumor is known as Skip N2 metastasis, which N2 lymph nodes contain tumor cells while N1 are clean. In this study, mRNA levels of epithelial mesenchymal transition (EMT) related genes in skip N2 and normal N2 involvements of non-small cell lung cancer tissues were investigated to evaluate the possible molecular background that may contribute to the pathogenesis of Skip N2 metastasis. Materials and Methods: Eighty-three surgically resected and paraffin embedded lymph node samples of lung cancer patients were analyzed in this study, which 40 of them were Skip N2. N2 tissues were sampled from 50% tumor containing areas and total RNA was extracted. mRNA levels for 18S, E-cadherin, Vimentin, ZEB1 and SLUG were analyzed via qPCR and E-cadherin and vimentin protein levels via immunohistochemistry (IHC). Bioinformatic analysis were adopted using online datasets to evaluate significantly co-expressed genes with SLUG in lung cancer tissue samples.Results: Skip-N2 patients who had adenocarcinoma subtype had better survival rates. Comparative analysis of PCR results indicated that Skip N2 tumor tissues had increased E-Cadherin/Vimentin ratio and ZEB1 mRNA expression, and significantly decreased levels of SLUG. E-cadherin IHC staining were higher in Skip N2 and Vimentin were in Non-Skip N2. TP63 had a strong correlation with SLUG expression in the bioinformatics analyses.Conclusion: The results indicate that, at molecular level, Skip N2 pathogenesis has different molecular background and regulation of SLUG expression may orchestrate the process.

Effect of miR-384-targeting LINC00491 on proliferation, migration and invasion of tongue squamous cell carcinoma cells

Purpose: To investigate the effect of long-chain non-coding RNA LINC00491 (LncRNA LINC00491) on the proliferation, migration and invasion of tongue squamous cell carcinoma (TSCC) cells, and the underlying mechanism. Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) was applied to determine the expressions of LINC00491 and micro-RNA-384 (miR-384). Furthermore, LINC00491 and miR-384 were transfected into CAL-27 cells, while cell cycle was analyzed using flow cytometry. Cell proliferation was determined by methyl thiazolyl diphenyl-tetrazolium (MTT) assay. Cell migration and invasion were evaluated using Transwell experiments. The relationship between LINC00491 and miR-384 was confirmed using double luciferase reporting assay, while protein expression levels of P21, Ki67, E- cadherin, N-cadherin, and vimentin were assayed with Western blotting. Results: The expression of LINC00491 increased in TSCC tissues (p < 0.05). The proportion of cells in G1-phase increased, while the proportion of cells in S-phase decreased (p < 0.05). There was decrease in cell survival, cell migration and cell invasion (p < 0.05). The protein expression levels of Ki67, N- cadherin, and vimentin were lowered, while those of P21, E-cadherin protein were increased (p < 0.05). Transfection of LINC00491 and miR- 384 reduced the proportion of cells in G1 phase, but increased the proportion of cells in S-phase (p < 0.05). Moreover, cell survival, migration and invasion were increased. The protein expressions of Ki67, N-cadherin, and vimentin rose, while those of P21 and E-cadherin decreased (p < 0.05). Conclusion: LINC00491 promotes the proliferation, migration and invasion of TSCC cells by inhibiting miR-384. This finding provides a potential target for the treatment of TSCC.

Assessment of the Molecular Heterogeneity of E-Cadherin Expression in Invasive Lobular Breast Cancer

Mutations and loss of E-cadherin protein expression define the vast majority of invasive lobular carcinomas. In a subset of these cases, the heterogeneous expression of E-cadherin is observed either as wild-type (strong membranous) expression or aberrant expression (cytoplasmic expression). However, it is unclear as to whether the two components would be driven by distinct genetic or epigenetic alterations. Here, we used whole genome DNA sequencing and methylation array profiling of two separately dissected components of nine invasive lobular carcinomas with heterogeneous E-cadherin expression. E-cadherin negative and aberrant/positive components of E-cadherin heterogeneous tumours showed a similar mutational, copy number and promoter methylation repertoire, suggesting they arise from a common ancestor, as opposed to the collision of two independent tumours. We found that the majority of E-cadherin heterogeneous tumours harboured CDH1 mutations in both the E-cadherin negative and aberrant/positive components together with somatic mutations in additional driver genes known to be enriched in both pure invasive carcinomas of no special type and invasive lobular breast cancers, whereas these were less commonly observed in CDH1 wild-type tumours. CDH1 mutant tumours also exhibited a higher mutation burden as well as increased presence of APOBEC-dependent mutational signatures 2 and 13 compared to CDH1 wild-type tumours. Together, our results suggest that regardless of E-cadherin protein expression, tumours showing heterogeneous expression of E-cadherin should be considered as part of the spectrum of invasive lobular breast cancers.

Upregulation of CD36, a Fatty Acid Translocase, Promotes Colorectal Cancer Metastasis by Increasing MMP28 and Decreasing E-Cadherin Expression

Altered fatty acid metabolism continues to be an attractive target for therapeutic intervention in cancer. We previously found that colorectal cancer (CRC) cells with a higher metastatic potential express a higher level of fatty acid translocase (CD36). However, the role of CD36 in CRC metastasis has not been studied. Here, we demonstrate that high expression of CD36 promotes invasion of CRC cells. Consistently, CD36 promoted lung metastasis in the tail vein model and GI metastasis in the cecum injection model. RNA-Seq analysis of CRC cells with altered expression of CD36 revealed an association between high expression of CD36 and upregulation of MMP28, a novel member of the metallopeptidase family of proteins. Using shRNA-mediated knockdown and overexpression of CD36, we confirmed that CD36 regulates MMP28 expression in CRC cells. siRNA-mediated knockdown of MMP28 decreases invasion of CRC cells, suggesting that MMP28 regulates the metastatic properties of cells downstream of CD36. Importantly, high expression of MMP28 leads to a significant decrease in active E-cadherin and an increase in the products of E-cadherin cleavage, CTF1 and CTF2. In summary, upregulation of CD36 expression promotes the metastatic properties of CRC via upregulation of MMP28 and an increase in E-cadherin cleavage, suggesting that targeting the CD36–MMP28 axis may be an effective therapeutic strategy for CRC metastasis.