miR-29b Derived from Bone Marrow Stromal Cell (BMSC) Exosomes Improves Laryngeal Carcinoma by Inhibiting Forkhead Box Protein P1 (FOXP1) to Decrease Cyclin E2 Transcription

This study intends to investigate whether miR-29b derived from BMSC exosomes (BMSC-exos) affects laryngeal cancer progression. RT-qPCR detected miR-29b level in BMSCs and BMSC-exos. After miR-29b was overexpressed in BMSCs, exos were extracted from BMSCs and used to treat laryngeal cancer cells, followed by CCK-8 assay and soft agar assay. When cells were treated with FOXP1 inhibitor or cyclin E2 vector, Western blot analyzed the expression of related proteins and flow cytometry assessed cell cycle distribution. In vivo experiment was conducted to assess miR-29b’s effect on tumor growth. miR-29b was upregulated in BMSC-exos, but lowly expressed in cancer cells. miR-29b upregulation inhibited the proliferation of laryngeal cancer cells and delayed tumor progression In vivo by inducing cell cycle arrest. Importantly, miR-29b bound 3′UTR of FXOP1 to inhibit its expression, and further reduced cyclin E2 level. sh-FXOP1 or cyclin E2 vector can restore the cell cycle and proliferation caused by miR-29b. In conclusion, miR-29b enriched in BMSC-exo can down-regulate cyclin E2 expression through targeted inhibition of FXOP1, thereby inhibiting the progression of laryngeal cancer.

USP13 Regulates the Breast Tumor Proliferation and Migration through the Stabilization of PDCD4 Protein

Background: Programmed cell death protein 4 (PDCD4), which serves as a tumor suppressor protein, plays a important role in cell proliferation,apoptosis, cell migration and DNA-damage response.However, the exact mechanism for the deubiquitination of PDCD4 remain unclear.Methods: Western blotting was used to detect the expression of PDCD4 in the breast cancer tissues and BC cell lines. We identified the potential PDCD4 associated deubiquitinase by RNAi screening. GST-Pull down and domain-mapping analysis were used to reveal that USP13 and PDCD4 directly interact with each other.Flow cytometry was used to detect the changes of G1 to S phase. Soft agar assay was used to measure the changes of the cell proliferation efficiency.Results: The expression of PDCD4 was decreased in the breast cancer tissues and BC cell lines. USP13 as a potential PDCD4 associated deubiquitinase. USP13 physically interacted with PDCD4 and greatly increased the steady state of PDCD4 through the ubiquitin-proteasome pathway.Importantly, silencing of the USP13 facilitated cell cycle from G1 to Sphase, promoted breast tumor cells proliferation and migration through downregulation of PDCD4. Conclusions: Together, these results suggest that USP13 plays an important role in the breast tumor proliferation and migration through modulating PDCD4 stability.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs’ pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

Effects of formula composed of active monomers of removing heat and phlegm prescription on malignant phenotypes and growth signaling in esophageal carcinoma cells.

e16051 Background: Esophageal carcinoma (EC) ranks seventh in the incidence of malignant tumors and the sixth mortality worldwide. Removing heat and phlegm prescription(RHPP, composed of Rhizoma Dioscoreae Nipponicae, Rhizoma Paridis, Saponin, Rhizoma Bolbostemmae) was an effective formula screened from 531 Chinese herbs for treating EC in our previous study. The purpose of this study is to further track, isolate, purify and identify the active monomers of RHPP, study the effect of each active monomer on Eca109, EC9706, EC-1, TE-1 four EC cells and the signaling mechanism, then optimize and compose new active monomers formula and study its mechanism on EC cells. Methods: The active monomer of RHPP was extracted, separated and identified by ethanol extractioncolumn, chromatography, recrystallization, HPLC, spectroscopic method,etc. The effects of four kinds of active monomers and their composed formula on the proliferation, migration, clone formation, cell cycle and signal pathway proteins expression of Eca109 cell, EC9706 cell and TE-1 cell were investigated by MTT assay, RTCA assay, soft agar assay, flow cytometry and western blot analysis. Results: Four active monomers were identified from Rhizoma Dioscoreae Nipponicae (CSL), Saponin (ZJ), Rhizoma Bolbostemmae (TBM), Rhizoma Paridis (CL) respectively with molecular weights of 868, 1003, 1365, 854.5, and corresponding molecular formulas of C45H72O16, C50H82O20, C64H100O31 and C44H70O16(China patient No. ZL202010136431.6). Four active monomers significantly affected the cell morphology, proliferation, migration, cloning ability, cell cycle of four EC cells. The IC50 values of the active monomer of CSL, ZJ, TBM and CL for Eca109 were 1.29±0.12, 2.42±0.09, 1.93±0.09, 3.04±0.28μg/ml respectively; for EC9706 were 1.41±0.02, 5.94±0.45, 1.97±0.09, 0.63±0.04μg/ml respectively; for TE-1 were 3.72±0.28, 35.58±0.58, 7.90±0.41, 1.85±0.09μg/ml respectively; for EC-1 were 1.11±0.14, 3.56±0.28, 1.23±0.02, 0.61±0.02μg/ml respectively. Four active monomers could downregulate EGFR, PLC-γ1, and PKCα protein expression. Using the baseline proportional increase and decrease design method to compose new removing heat prescription(RH), removing phlegm prescription(RP) and removing heat and phlegm prescription (RHPP), the ratio of each drug monomer in RH, RP and RHPP were mCSL: mCL = 6: 4, mZJ: mTBM = 3: 7, mCSL: mCL: mZJ: mTBM = 48: 32: 6: 14. RH, RP and RHPP all could inhibit the proliferation, migration, cloning ability, cell cycle of four EC cells. Conclusions: The four active monomers and new composed RHPP all can inhibit the proliferation of EC9706, Eca109, TE-1, EC-1 four EC cells, which have close relationship with EGFR, PLC-γ1, and PKCαproteins expression. This study provides new formula and basis for the development of anti-esophageal carcinoma drugs in traditional Chinese medicine.

miR-190 promotes malignant transformation and progression of human urothelial cells through CDKN1B/p27 inhibition

Background Although miR-190 has been reported to be related to human diseases, especially in the development and progression of cancer, its expression in human bladder cancer (BC) and potential contribution to BC remain unexplored. Methods RT-qPCR was used to verify the expression level of miR-190 and CDKN1B. Flow cytometry (FCM) assays were performed to detect cell cycle. Soft agar assay was used to measure anchorage-independent growth ability. Methylation-Specific PCR, Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved. Results Our studies revealed that downregulation of the p27 (encoded by CDKN1B gene) protein is an important event related to miR-190, promoting the malignant transformation of bladder epithelial cells. miR-190 binds directly to CDKN1B 3’-UTR and destabilizes CDKN1B mRNA. Moreover, miR-190 downregulates TET1 by binding to the TET1 CDS region, which mediates hypermethylation of the CDKN1B promoter, thereby resulting in the downregulation of CDKN1B mRNA. These two aspects led to miR-190 inhibition of p27 protein expression in human BC cells. A more in-depth mechanistic study showed that c-Jun promotes the transcription of Talin2, the host gene of miR-190, thus upregulating the expression of miR-190 in human BC cells. Conclusions In this study, we found that miR-190 plays an important role in the development of BC. Taken together, these findings indicate that miR-190 may promote the malignant transformation of human urothelial cells by downregulating CDKN1B, which strengthens our understanding of miR-190 in regulating BC cell transformation.

Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

Background Lung adenocarcinoma (LUAD) metastasis significantly reduces patient survival; hence inhibiting the metastatic ability of lung cancer cells will greatly prolong patient survival. Alkaline phosphatase (ALPL), a homodimeric cell surface phosphohydrolase, is reported to play a controversial role in prostate cancer and ovarian cancer cell migration; however, the function of ALPL in LUAD and the related mechanisms remain unclear. Methods TCGA database was used to analysis the expression of ALPL, and further verification was performed in a cohort of 36 LUAD samples by qPCR and western blot. Soft-agar assay, transwell assay and lung metastasis assay were employed to detect the function of ALPL in LUAD progression. The qPCR, luciferase promoter reporter assay and western blot were used to clarify the molecular mechanisms of ALPL in promoting metastasis in LUAD. Results ALPL was downregulated in LUAD, and the disease-free survival rate of patients with low ALPL was significantly reduced. Further studies showed that overexpression of ALPL in LUAD cell lines did not significantly affect cell proliferation, but it did significantly attenuate lung metastasis in a mouse model. ALPL downregulation in LUAD led to a decrease in the amount of phosphorylated (p)-ERK. Because p-ERK promotes the classical c-Myc degradation pathway, the decrease in p-ERK led to the accumulation of c-Myc and therefore to an increase in RhoA transcription, which enhanced LUAD cell metastasis. Conclusion ALPL specially inhibits the metastasis of LUAD cells by affecting the p-ERK/c-Myc/RhoA axis, providing a theoretical basis for the targeted therapy of clinical LUAD.

Validation of a Novel Xeno-Free Method for Human Endometrial Mesenchymal Stromal Cells (E-MSCs) Isolation and Culture

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of Endometrial Mesenchymal Stromal Cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, the surgical curettage and the Vacuum Aspiration Biopsy Random Assay, and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell lines were isolated after a mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential and multilineage differentiation. The expression of mesenchymal and stemness markers was tested by FACS analysis and Real-Time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed to efficiently isolate and expand E-MSCs using a xeno-free method preserving their mesenchymal and stemness phenotype, proliferative potential and multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting Vabra endometrial sampling as alternative to surgical curettage.

MiR-190 Promotes Malignant Transformation and Progression of Human Urothelial Cells through CDKN1B Inhibition

BackgroundAlthough miR-190 has been reported to be related to human diseases, especially in the development and progression of cancer, its expression in human bladder cancer (BC) and potential contribution to BC remain unexplored. MethodsqRT-PCR was used to verify the expression level of miR-190 and CDKN1B. Flow cytometry (FCM) assays were performed to detect cell cycle. Soft agar assay was used to measure anchorage-independent growth ability. Methylation-Specific PCR, Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved. ResultsOur studies revealed that downregulation of the CDKN1B/p27 protein is an important event related to miR-190, promoting the malignant transformation of bladder epithelial cells. miR-190 binds directly to CDKN1B 3’-UTR and destabilizes CDKN1B mRNA. Moreover, miR-190 downregulates TET1 by binding to the TET1 CDS region, which mediates hypermethylation of the CDKN1B promoter, thereby resulting in the downregulation of CDKN1B mRNA. These two aspects led to miR-190 inhibition of p27 protein expression in human BC cells. However, the increased expression of miR-190 in BC and its upstream regulatory mechanism remain unclear. A more in-depth mechanistic study showed that c-Jun promotes the transcription of Talin2, the host gene of miR-190, thus upregulating the expression of miR-190 in human BC cells. ConclusionIn this study, we found that miR-190 plays an important role in the development of BC. Taken together, these findings indicate that miR-190 may promote the malignant transformation of human urothelial cells by downregulating CDKN1B, which strengthens our understanding of miR-190 in regulating BC cell transformation.

Alkaline Phosphatase Downregulation Promotes Lung Adenocarcinoma Metastasis via the C-Myc/RhoA Axis

Background: Lung adenocarcinoma (LUAD) metastasis significantly reduces patient survival; hence inhibiting the metastatic ability of lung cancer cells will greatly prolong patient survival. Alkaline phosphatase (ALPL), a homodimeric cell surface phosphohydrolase, is reported to play a controversial role in prostate cancer and ovarian cancer cell migration; however, the function of ALPL in LUAD and the related mechanisms remain unclear.Methods: TCGA database was used to analysis the expression of ALPL, and further verification was performed in a cohort of 36 LUAD samples by qPCR and western blot. Soft-agar assay, transwell assay and lung metastasis assay were employed to detect the function of ALPL in LUAD progression. The qPCR, luciferase promoter reporter assay and western blot were used to clarify the molecular mechanisms of ALPL in promoting metastasis in LUAD.Results: ALPL was downregulated in LUAD, and the disease-free survival rate of patients with low ALPL was significantly reduced. Further studies showed that overexpression of ALPL in LUAD cell lines did not significantly affect cell proliferation, but it did significantly attenuate lung metastasis in a mouse model. ALPL downregulation in LUAD led to a decrease in the amount of phosphorylated (p)-ERK. Because p-ERK promotes the classical c-Myc degradation pathway, the decrease in p-ERK led to the accumulation of c-Myc and therefore to an increase in RhoA transcription, which enhanced LUAD cell metastasis.Conclusion: ALPL specially inhibits the metastasis of LUAD cells by affecting the p-ERK/c-Myc/RhoA axis, providing a theoretical basis for the targeted therapy of clinical LUAD.

MiR-190 Promotes Malignant Transformation and Progression of Human Urothelial Cells through CDKN1B Inhibition

BackgroundAlthough miR-190 has been reported to be related to human diseases, especially in the development and progression of cancer, its expression in human bladder cancer (BC) and potential contribution to BC remain unexplored.MethodsqRT-PCR was used to verify the expression level of miR-190 and CDKN1B. Flow cytometry (FCM) assays were performed to detect cell cycle. Soft agar assay was used to measure anchorage-independent growth ability. Methylation-Specific PCR, Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved.ResultsOur studies revealed that downregulation of the CDKN1B/p27 protein is an important event related to miR-190, promoting the malignant transformation of bladder epithelial cells. miR-190 binds directly to CDKN1B 3’-UTR and destabilizes CDKN1B mRNA. Moreover, miR-190 downregulates TET1 by binding to the TET1 CDS region, which mediates hypermethylation of the CDKN1B promoter, thereby resulting in the downregulation of CDKN1B mRNA. These two aspects led to miR-190 inhibition of p27 protein expression in human BC cells. However, the increased expression of miR-190 in BC and its upstream regulatory mechanism remain unclear. A more in-depth mechanistic study showed that c-Jun promotes the transcription of Talin2, the host gene of miR-190, thus upregulating the expression of miR-190 in human BC cells.ConclusionIn this study, we found that miR-190 plays an important role in the development of BC. Taken together, these findings indicate that miR-190 may promote the malignant transformation of human urothelial cells by downregulating CDKN1B, which strengthens our understanding of miR-190 in regulating BC cell transformation.