Impact of lncRNA FGD5-AS1 on the malignant biological behavior of bladder cancer cells by regulating miR-129-5p/CDK6 axis / 现代泌尿外科杂志

【Objective】 To investigate the impact of long non-coding RNA (lncRNA) FGD5-AS1 on the malignant biolo-goical behavior of bladder cancer (BC) cells by regulating micro RNA (miR)-129-5p/cyclin dependent kinase 6 (CDK6) axis. 【Methods】 Human BC cell line T24 was cultured from tumor tissue and paracancerous tissue of 105 patients with confirmed BC. The expressions of FGD5-AS1, miR-129-5p and CDK6 mRNA in tissue samples and T24 cells were detected with RT-qPCR. T24 cells were randomly divided into control group, si-NC group, si-FGD5-AS1 group, si-FGD5-AS1+inhibitor NC group and si-FGD5-AS1+miR-129-5p inhibitor group. The cell viability, migration, invasion andapoptosis were detected with CCK-8, Wound healing test, Transwell assay and flow cytometry, respectively. The expressions of Bax, Bcl-2, Caspase3 and CDK6 were detected with Western blot. The relationship between FGD5-AS1 and miR-129-5p, between miR-129-5p and CDK6 were verified with double luciferase reporter gene experiment. 【Results】 FGD5-AS1 and CDK6 mRNA were highly expressed in BC tissue, while miR-129-5p was lowly expressed (P<0.05). After FGD5-AS1 silencing, the expression of FGD5-AS1,A450 value, cell scratch healing rate, cell invasion number, and expressions of Bcl-2 and CDK6 were significantly lower, while the apoptosis rate and expressions of miR-129-5p, Bax and Caspase3 were significantly higher (P<0.05). Inhibition of miR-129-5p expression reversed the effects of FGD5-AS1 silencing on various indexes of BC cells (P<0.05). FGD5-AS1 negatively regulated the expression of miR-129-5p, and miR-129-5p negatively regulated the expression of CDK6. 【Conclusion】 Silencing FGD5-AS1 may inhibit the expression of CDK6 protein by up-regulating miR-129-5p, thus inhibiting the proliferation, migration and invasion of BC cells and promoting cell apoptosis.

Influence of Limonin on Malignant Biological Behavior of Non-small Cell Lung Cancer Cells by Regulating JAK2/STAT3 Signaling Pathway / 肿瘤防治研究

Objective To investigate the influence of limonin on the malignant biological behavior of non-small cell lung cancer (NSCLC) cells by regulating the protein tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods CCK-8 method was applied to detect the survival rate of A549 cells treated with different concentrations of limonin (0, 5, 10, 25, 50, 75, 100 μmol/L). A549 cells were separated into normal culture (NC) group, low-dose limonin group (treatment with 10 μmol/L limonin for 24 h), medium-dose limonin group (treatment with 25 μmol/L limonin for 24 h), high-dose limonin group (treatment with 50 μmol/L limonin for 24 h), coumermycin A1 group (treatment with 10 μmol/L JAK2 activator coumermycin A1+50 μmol/L limonin for 24 h), and AG490 group (treatment with 10 μmol/L JAK2 inhibitor AG490+50 μmol/L limonin for 24 h). Clone formation assay was applied to detect the clones of each group of cells. Transwell assay was applied to detect cell migration and invasion, and flow cytometry was applied to detect apoptosis. Western blot analysis was applied to detect the protein expression levels of JAK2, p-JAK2, STAT3, p-STAT3, E-cadherin, N-cadherin, and vimentin in each group. Results The viability of A549 cells decreased significantly in a limonin concentration-dependent manner (P < 0.05), with IC50 of 45.16±1.66 μmol/L. Concentrations of 10, 25, and 50 μmol/L were selected for subsequent experiments. The numbers of clones, migration, and invasion of A549 cells and the protein expression levels of IL-6, p-JAK2, p-STAT3, N-cadherin, and vimentin in the low-, medium-, and high-dose limonin groups significantly decreased, compared with those in the NC group, and the apoptosis rate and E-cadherin protein expression significantly increased (P < 0.05). The JAK2 activator coumermycin A1 attenuated the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and attenuated the apoptosis ability. The JAK2 inhibitor AG490 enhanced the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and enhanced the apoptosis ability. Conclusion Limonin can inhibit the malignant biological behavior of NSCLC cells, such as proliferation, migration, and invasion, by inhibiting the JAK2/STAT3 pathway.

Experimental Study on the Mechanism of Mangiferin Inhibiting Malignant Biological Characteristics of Multiple Myeloma and Exerting Anticancer Effect / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of pure Chinese herbal extract Mangiferin on the malignant biological behaviors of multiple myeloma (MM) cells, and to analyze the molecular mechanism of the anti-myeloma effect of Mangiferin, so as to provide experimental basis for MM replacement therapy.@*METHODS@#U266 and RPMI8226 of human MM cell lines were intervened with different concentrations of Mangiferin. Cell proliferation was detected by CCK-8 method. Annexin V/PI double staining flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of apoptosis and related signaling pathway proteins, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of matrix metalloproteinase (MMP) and CXC chemokine receptor (CXCR) family.@*RESULTS@#Mangiferin could inhibit the proliferation activity of U266 and RPMI8226 cells and induce cells apoptosis. After Mangiferin intervened in U266, RPMI8226 cells for 48 h, the expression of Bcl-2 family pro-apoptotic protein Bax was up-regulated, while the expression of survivin and Bcl-xL proteins was down-regulated and caspase-3 was hydrolyzed and activated to promote cell apoptosis, besides, the expression of Bcl-2 protein in U266 cells was also significantly down-regulated to induce apoptosis (P<0.05). After Mangiferin intervenes in MM cells, it can not only increase the expression level of tumor suppressor p53, but also induce programmed cell death of MM cells by inhibiting the expression of anti-apoptotic molecules and down-regulating the phosphorylation levels of AKT and NF-κB. In addition, after the intervention of Mangiferin, the expressions of CXCR4, MMP2 and MMP9 in U266 cells were down-regulated (P<0.05), while there is no effect on the expressions of CXCR2, CXCR7 and MMP13 (P>0.05). However, the expressions of CXCR4, MMP9, and MMP13 in RPMI8226 cells were down-regulated (P<0.01), the expression of MMP2 was weakly affected, and the expression of CXCR2 and CXCR7 was basically not affected (P>0.05).@*CONCLUSION@#Mangiferin can inhibit the proliferation and induce apoptosis of MM cells, and its mechanism may be related to inhibiting the activation of NF-κB signaling pathway, affecting the expression of Bcl-2 family proteins, and inhibiting the expression of core members of MMP and CXCR family.

The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML.@*METHODS@#Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05).@*CONCLUSION@#Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.

Influence of LncRNA NKILA on Biological Behavior of Malignant Tumors and Related Mechanism / 肿瘤防治研究

NKILA is a kind of newly-discovered lncRNA whose expression is aberrant in diverse malignant tumors. The existing researches have confirmed that NKILA participates in the occurrence and development of tumors mainly by regulating the NF-κB signaling pathway, and has significance to the cancer diagnosis, treatment and prognostic evaluation of patients. This article reviews the abnormal expressions and biological effects of NKILA, and the up- and down-stream mechanisms of NKILA regulating malignant biological behavior in different cancers.

Lidocaine inhibits the malignant proliferation, invasion and regulates the mitochondrial respiration of osteosarcoma MG-63 cells / 中国临床药理学与治疗学

AIM: To investigate the effects of lidocaine on malignant proliferation, invasion and mitochondrial respiration of osteosarcoma MG-63 cells. METHODS: MG-63 cells were treated with 25, 50 and 100 μmol/L of lidocaine and were randomly divided into four groups: lidocaine 0 μmol/L, lidocaine 25 μmol/L, lidocaine 50 μmol/L and lidocaine 100 μmol/L for subsequent experiments. BrdU staining was used to detect cell proliferation. Transwell for cell invasion. Protein expression levels of Ki67, Survivin, VEGF and Vimentin were detected by Western blot. Mitochondrial membrane potential was detected by flow separator. Activity of mitochondrial respiratory complex was detected by Clark oxygen electrode method. The kit detected the content of ATP, SOD and MDA.RESULTS: Results showed that compared with lidocaine 0 μmol/L group, BrdU positive cells in lidocaine 50, 100 μmol/L group was significantly reduced (P<0.05), invasive cells was significantly reduced (P<0.05), Ki67, Survivin, VEGF, Vimentin protein levels decreased significantly (P<0.05), mitochondrial membrane potential decreased significantly, compound I, II, IV activity decreased significantly (P<0.05), ATP, SOD content decreased significantly (P<0.05), MDA content was significantly increased (P<0.05). CONCLUSION: Lidocaine can inhibit the malignant proliferation, invasion and improve the mitochondrial function of osteosarcoma MG-63 cells.

Yiqi Huoxue decoction inhibits malignant biological behavior of human lung cancer cells via the miR-21/PTEN signaling pathway / 中国临床药理学与治疗学

AIM: To investigate the inhibitory effect of Yiqi Huoxue decoction on the malignant biological behavior of lung cancer cells and its mechanism. METHODS: Human lung cancer A549 cells were treated with different doses of Yiqi Huoxue Decoction serum (5%, 10%, 15%). CCK-8 assay, transwell chamber experiment, flow cytometry, Western blot and qRT-PCR method were used to study the effect of different doses of Yiqi Huoxue Decoction-containing serum to cell proliferation, cell migration and invasion, cell apoptosis, PTEN protein and miR-21 expression. RESULTS:Compared with the drug-free serum group, survival rate, migration and invasion ability of A549 cells decreased after treatment with different doses of drug-containing serum. The apoptosis rate of A549 cells increased, PTEN mRNA and the expression of its protein increased, the expression of miR-21 decreased, and the medium-dose (10%) drug-containing serum group had the best effect. After the transfection of miR-21 mimics, miR-21 expression was up-regulated, while PTEN protein expression was down-regulated in cells. PTEN protein expression was up-regulated after treatment with medium-dose (10%) drug-containing serum. CONCLUSION: Yiqi Huoxue Decoction can effectively inhibit the malignant cell biological behavior of human lung cancer A549 cells and may be related to the regulation of the miR-21/PTEN signaling pathway.

Exosome-mediated Let-7a inhibits malignant biological behaviors of triple-negative breast cancer cells by down-regulating MYC expression / 中国肿瘤生物治疗杂志

@#Objective: To investigate the role of exosome (EXO) transporting Let-7a to regulate MYC gene in the malignant biological behaviors of triple negative breast cancer (TNBC) cell, and to explore the underlying mechanism. Methods: After the completion of cell culture, the gene and protein expressions of MYC and Let-7a in TNBC MDA-MB-231cells were detected by qPCR and WB, respectively. Recombinant lenti-virus vector carrying Let-7a and Crisper/Cas-9 system with MYC knockdown were transfected into MDA-MB-231 cells; MTT assay, Transwell assay and Scratch healing assay were performed to examine the proliferation, invasion and migration of MDA-MB-231 cells. Luciferase activity assay was performed to validate the binding between MYC and Let-7a. EXO was isolated and identified by transmission electron microscopy and WB assay in wild-type and Let-7a over-expressed MDA-MB-231 cells, respectively. After co-incubation of two types of EXO and MDA-MB-231 cells, the effects of Let-7a on biological behaviors of MDAMB-231 cells via EXO were detected by qPCR, WB, MTT and Transwell etc. Results: Let-7a was negatively correlated with MYC in breast cancer tissues and cell lines (all P<0.05); MYC promoted while Let-7a inhibited the proliferation, migration and invasion of breast cancer cells (all P<0.01); Let-7a silenced MYC by acting on 3'UTR of MYC gene, thereby reducing the expression of MYC protein (P<0.05); Let-7a was enveloped by EXO and transported to cancer cells, there by inhibiting the proliferation, migration and invasion of MDA-MB-231 cells. Conclusion: EXO some mediated Let-7a silences MYC gene by acting on its 3'UTR region, thus inhibiting the proliferation, migration and invasion of MDA-MB-231 cells.

Effect of shRNA interfering BAMBI on malignant biological behaviors of human colon cancer SW480 cell / 中国肿瘤生物治疗杂志

@# Objective: To explore the effect of shRNA interfering BAMBI (bone morphogenetic protein and activin membrane bound inhibitor) on the proliferation, apoptosis, invasion and migration of human colon cancer SW480 cells and the possible mechanisms. Methods: After successful transfection with sh-BAMBI in SW480 cells, the mRNA and protein epxressions of BAMBI were detected by qRT-PCR and Western blotting, respectively. Cell proliferation was measured by MTT; apoptosis was tested by Hoechst33258 staining; cell invasion was detected by transwell assay; and cell migration was measured by wound healing assay. The expressions of TGF-β/ Smad/2 signaling pathway related proteins were detected by Western blotting. Results: The mRNA and protein levels of BAMBI in shBAMBI group were lower than those of control group (P<0.05). Compared with control group, cell proliferation in sh-BAMBI group was obviously decreased (P<0.05), while apoptosis was obviously increased (P<0.01); in the meanwhile, cell invasion and migration in sh-BAMBI group were significantly reduced (P<0.05). In addition, the protein level of TGF-β and the ratio of p-Smad/2/ Smad/2 in shBAMBI group were significantly higher than those in control group (P<0.05). Conclusion: Interference of BAMBI by shRNA inhibits proliferation, invasion and migration but induces apoptosis of human colon cancer SW480 cells and activates TGF-β/Smad/2 pathway.