Hsa-miR-557 Inhibits Osteosarcoma Growth Through Targeting KRAS

Objective: Osteosarcoma is the most common malignancy in the skeletal system; studies showed an important role of miRNAs in tumorigenesis, indicating miRNAs as possible therapeutic molecules. This study found abnormal hsa-miR-557 expression levels in osteosarcoma and tried to explore the potential function and the mechanism.Methods: Differential expression genes of osteosarcoma were analyzed using GSE28423 from the GEO database. Survival analysis of miRNAs was conducted with data obtained from the TARGET-OS database. STRING and miRDIP were used to predict target genes of hsa-miR-557; KRAS was then verified using dual-luciferase reporter assay. Expression of genes was detected by qPCR, and levels of proteins were detected by Western blot. The proliferation ability of cells was detected by CCK-8 and cell cycle analysis. Tumor formation assay in nude mice was used to detect the influence of osteosarcoma by hsa-miR-557 in vivo.Results: Analysis from the GEO and TARGET databases found 12 miRNAs that are significantly related to the osteosarcoma prognosis, 7 downregulated (hsa-miR-140-3p, hsa-miR-564, hsa-miR-765, hsa-miR-1224-5p, hsa-miR-95, hsa-miR-940, and hsa-miR-557) and 5 upregulated (hsa-miR-362-3p, hsa-miR-149, hsa-miR-96, hsa-miR-744, and hsa-miR-769-5p). CCK-8 analysis and cell cycle analysis found that hsa-miR-557 could significantly inhibit the proliferation of osteosarcoma cells. The tumor formation assay in nude mice showed that tumor sizes and weights were inhibited by hsa-miR-557 transfection. Further studies also proved that hsa-miR-557 could target the 3′UTR of KRAS and modulate phosphorylation of downstream proteins.Conclusion: This study showed that hsa-miR-557 could inhibit osteosarcoma growth both in vivo and in vitro, by modulating KRAS expression.

Tumor-derived extracellular vesicles inhibit HGF/c-Met and EGF/EGFR pathways to accelerate the radiosensitivity of nasopharyngeal carcinoma cells via microRNA-142-5p delivery

Radioresistance prevails as one of the largest obstacles in the clinical treatment of nasopharyngeal carcinoma (NPC). Meanwhile, tumor-derived extracellular vesicles (TEVs) possess the ability to manipulate radioresistance in NPC. However, its mechanism remains to be further explored. Therefore, the current study set out to explore the mechanism of microRNA (miR)-142-5p delivered by TEVs in regard to the radiosensitivity of NPC. Firstly, peripheral blood samples were collected from patients with radioresistance and radiosensitivity, followed by RT-qPCR detection of miR-142-5p expression. A dual-luciferase reporter assay was carried out to elucidate the targeting relationship of miR-142-5p with HGF and EGF. In addition, radiotherapy-resistant NPC cell models were established by screening NPC cells with gradient increasing radiation exposure, and co-incubated with EVs isolated from miR-142-5p mimic-transfected NPC cells, followed by overexpression of HGF and EGF. Moreover, cell viability was detected by means of MTS, cell proliferation with a colony formation assay, cell apoptosis with flow cytometry, and expression patterns of related genes with the help of Western blot analysis. NPC xenotransplantation models in nude mice were also established by subcutaneous injection of 5-8FR cells to determine apoptosis, tumorigenicity, and radiosensitivity in nude mice. It was found that miR-142-5p was poorly expressed in peripheral blood from NPC patients with radioresistance. Mechanistic experimentation illustrated that miR-142-5p inversely targeted HGF and EGF to inactivate the HGF/c-Met and EGF/EGFR pathways, respectively. NPC cell apoptosis was observed to be augmented, while their radioresistance and proliferation were restricted by EVs-miR-142-5p or HGF silencing, or EGF silencing. Furthermore, EVs-miR-142-5p inhibited growth and radioresistance and accelerated the apoptosis of radiotherapy-resistant NPC cells in nude mice by inhibiting the HGF/c-Met and EGF/EGFR pathways. Collectively, our findings indicated that TEVs might inhibit the HGF/c-Met and EGF/EGFR pathways by delivering miR-142-5p into radiotherapy-resistant NPC cells to enhance radiosensitivity in NPC.

Regulation of NSCLC Cell Proliferation By MARCH7 Via the NF-κB and Wnt/β-Catenin Signaling Pathways

The aim of the study was to explore the role of the E3 ubiquitin ligase MARCH7 in the development of non-small-cell lung cancer (NSCLC) and to explore the underlying molecular mechanism.Western blot and immunohistochemistry results showed that the expression of MARCH7 in NSCLC cancer tissues was higher than that in paracancerous tissues. Tissue microarray staining results and clinicopathological parameters of NSCLC patients revealed that MARCH7 expression was closely related to TNM stage, degree of tumor differentiation and lymph node metastasis of NSCLC patients. Furthermore, univariate and multivariate analyses and survival curve analysis showed that high expression of MARCH7 was associated with poor prognosis.In vitro, siRNA was constructed and transfected into A549 cells to inhibit the expression of MARCH7. The CCK-8 assay indicated that the growth rate of tumor cells in the interference group was reduced. The number of colonies and cells in the interference group decreased in the plate clone formation experiment. Flow cytometry showed that G0/G1 phase cells were predominantly increased after blocking endogenous MARCH7 expression, and G0/G1 phase arrest occurred in A549 cells. The reporter gene activity of the NF-κB signaling pathway and Wnt/β-catenin signaling pathway was reduced, as validated by a double luciferase reporter gene assay. Western blot analysis showed that the expression of NF-κB P50, NF-κB P65 and β-catenin was decreased, while the expression of E-cadherin was elevated.In vivo, MARCH7-overexpressing virus was constructed and transfected into A549 cells and then subcutaneously injected into nude mice. It was demonstrated that the tumor volume was significantly larger in the MARCH7 overexpression group than in the control nude mice during the same period. Elevated expression of PCNA and Ki-67 was observed in the tumor mass of the MARCH7 overexpression group, as measured by immunohistochemical analysis, accompanied by enhanced levels of NF-κB P50, NF-κB P65 and β-catenin, as detected by Western blot. These results provide a new idea for the experimental basis for the treatment of NSCLC in the future.

Identification of SRXN1 and KRT6A as Key Genes in Smoking-Related Non-Small-Cell Lung Cancer Through Bioinformatics and Functional Analyses

BackgroundLung cancer is the leading cause of cancer-related mortality worldwide. Although cigarette smoking is an established risk factor for lung cancer, few reliable smoking-related biomarkers for non-small-cell lung cancer (NSCLC) are available. An improved understanding of these biomarkers would further the development of new biomarker-targeted therapies and lead to improvements in overall patient survival.MethodsWe performed bioinformatic analysis to screened potential target genes, then quantitative PCR, western, siRNA, CCK-8, flow cytometry, tumorigenicity assays in nude mice were performed to validated the function.ResultsIn this study, we identified 83 smoking-related genes (SRGs) based on an integration analysis of two Gene Expression Omnibus (GEO) datasets, and 27 hub SRGs with potential carcinogenic effects by analyzing a dataset of smokers with NSCLC in The Cancer Genome Atlas (TCGA) database. A survival analysis revealed three genes with potential prognostic value, namely SRXN1, KRT6A and JAKMIP3. A univariate Cox analysis revealed significant associations of elevated SRXN1 and KRT6A expression with prognosis. A receiver operating characteristic (ROC) curve analysis indicated the high diagnostic value of SRXN1 and KRT6A for smoking and cancer. Quantitative PCR and western blotting validated the increased expression of SRXN1 and KRT6A mRNA and protein, respectively, in lung cancer cell lines and NSCLC tissues. In patients with NSCLC, SRXN1 and KRT6A expression was associated with the tumor–node–metastasis (TNM) stage, presence of metastasis, history of smoking and daily smoking consumption. Furthermore, inhibition of SRXN1 or KRT6A suppressed viability and enhanced apoptosis in the A549 human lung carcinoma cell line. Tumorigenicity assays in nude mice confirmed that the siRNA-mediated downregulation of SRXN1 and KRT6A expression inhibited tumor growth in vivo.ConclusionsIn summary, SRXN1 and KRT6A act as oncogenes in NSCLC and might be potential biomarkers of smoking exposure and the early diagnosis and prognosis of NSCLC in smokers, which is vital for lung cancer therapy.

Hedgehog−Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1

BackgroundCisplatin (DDP) resistance remains a key challenge in improving the clinical outcome of patients with ovarian cancer (OC). Gli2 overexpression can lead to DDP resistance in OC cells, but the specific underlying regulatory mechanism remains unclear. The membrane transporter encoding gene MDR1 positively regulates chemotherapy resistance in various cancer types. We evaluated MDR1 as a potential Gli2 downstream target and the contribution of the Gli2/MDR1 axis in promoting DDP resistance in OC cells.MethodsTo generate drug-resistant SKOV3/DDP cells, SKOV3 cells were grown for six months under continuous induction wherein the DDP concentration was steadily increased. Gli2 expression in OC cells with varying DDP sensitivities was detected using western blot. Cell counting kit-8 assays were used to assess the DDP sensitivity of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells and reversal of DDP resistance in SKOV3/DDP and A2780/DDP cells. Cell proliferation was analyzed using 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays. The transcriptional regulation of MDR1 by Gli2 was determined using luciferase reporter assays. Finally, xenograft OC tumors were generated in nude mice, which were then treated with intraperitoneal DDP or phosphate-buffered saline (PBS) injections to investigate if Gli2 affected DDP resistance in OC in vivo.ResultsDDP-resistant SKOV3/DDP and A2780/DDP cells showed higher expression of Gli2 and MDR1 as compared with that in DDP-sensitive OC cells. Gli2 knockdown in SKOV3/DDP cells significantly reduced MDR1 expression, whereas it increased DNA damage, thereby sensitizing OC cells to DDP. Similar results were obtained after targeting Gli2 expression with the Gli-antagonist 61 inhibitor (GANT61) in SKOV3/DDP and A2780/DDP cells. In cells stably overexpressing Gli2, treatment with gradient concentrations of verapamil, an MDR1 inhibitor, significantly inhibited MDR1 expression. Our findings indicate that downregulation of MDR1 expression may reverse OC cell resistance to DDP. Moreover, dual-luciferase reporter gene assays confirmed that MDR1 is a direct downstream target of Gli2, with Gli2 positively regulating MDR1 expression. Finally, subcutaneous xenotransplantation in nude mice demonstrated that Gli2 plays a key role in regulating OC drug resistance.ConclusionsWe identified a mechanism by which Hedgehog-Gli signaling regulates OC chemoresistance by modulating MDR1 expression. Hence, Gli2 and MDR1 are potential biomarkers and therapeutic targets in patients with chemoresistant OC.

LncRNA HCG18 upregulates TRAF4/TRAF5 to facilitate proliferation, migration and EMT of epithelial ovarian cancer by targeting miR-29a/b

Background Although long noncoding RNA HLA complex group 18 (lncRNA HCG18) has been suggested to regulate cell growth in several tumours, the function of HCG18 in epithelial ovarian cancer (EOC) and its mechanism are still unclear. Methods shRNAs were applied to reduce HCG18 and related genes. For overexpression of miRNA, a miRNA mimic was transfected into cells. Quantitative real-time PCR (qRT–PCR) was used to detect levels of HCG18, miR-29a/b, and mRNAs. MTT, colony formation, wound healing and Transwell assays were used to evaluate cell proliferation, migration and invasion, respectively. A luciferase reporter assay was utilized to evaluate NF-κB activity and the binding of miRNAs with HCG18 or TRAF4/5. BALB nude mice injected with cells stably expressing shHCG18 or shNC were used for in vivo modelling. Subcutaneous tumour growth was monitored in nude mice, and immunohistochemistry (IHC) was used to determine expression of the proliferation marker Ki67. Results Abnormal expression of HCG18 and miR-29a/b was observed in EOC tissues. Knockdown of HCG18 using shRNA inhibited proliferation, migration, EMT and the proinflammatory pathway in EOC cells. miR-29a/b mimics and TRAF4/5 knockdown exhibited effects similar to HCG18 knockdown. Further experiments suggested that HCG18 directly targets miR-29a/b and upregulates TRAF4/5 expression, which are inhibited by targeting miR-29a/b. Moreover, overexpression of TRAF4/5 antagonized the inhibitory effect of HCG18 knockdown, suggesting that they are involved in HCG18-mediated oncogenic effects. Silencing HCG18 reduced tumour size and levels of Ki67 and TRAF4/5 while increasing miR-29a/b levels in vivo. Conclusions Taken together, our data revealed an oncogenic signalling pathway mediated by HCG18 in ovarian cell lines, which functions as a ceRNA of miR-29a/b and thus derepresses expression levels of TRAF4/5, facilitating NF-κB pathway-mediated promotion of EOC cell proliferation and migration.

Exosome-Transmitted miR-128 Targets CCL18 to Inhibit the Proliferation and Metastasis of Urothelial Carcinoma

Objective: To investigate the regulatory function of exosome-transmitted miR-128 and chemokine (C-C motif) ligand 18 (CCL18) on urothelial carcinomas (UCs).Methods: Tumor tissues, paracancerous tissues, and serum were collected from 20 patients with UCs (diagnosed at Beijing Friendship Hospital, Capital Medical University). CCL18 was detected by immunohistochemistry and ELISA. PCR was used to measure the expression levels of CCL18 and mir-183, miR-128, mir-33a in UCs. We acquired exosomes from mesenchymal stem cells and synthesized exosomes overexpressing miR-128 (HMSC-128-EV). The effects of miR-128 on the migration and invasion abilities, apoptosis and epithelial-mesenchymal transition of BUC T24 cells were investigated by co-culturing HMSC-128-EV. The therapeutic potential of miR-128 on disease models was explored by injecting HMSC-128-EV into nude mice.Results: The expression of CCL18 in UCs was significantly higher than that in normal tissues (p < 0.05), and the serum level of CCL18 in patients with UC was significantly increased compared with those in healthy controls (p < 0.05). CCL18 overexpression or downregulation enhanced or suppressed the proliferation, migration and invasion of BUC T24 cells, resectively (p < 0.05). The exosome-transmitted miR-128 can inhibit cell proliferation (p < 0.05), invasion (p < 0.05), and migration (p < 0.05) in UCs, and these effects can be reversed by CCL18. In terms of apoptosis, miR-128 was able to promote the occurrence of BUC T24 apoptosis (p < 0.05), which can also be reversed by CCL18. In addition, miR-128 can inhibit the proliferation (p < 0.05) and metastasis (p < 0.05) of UCs in nude mice.Conclusion: The miR-128 inhibits the proliferation, invasion, migration of UCs, and promotes its apoptosis by regulating CCL18 secretion.

Bisdemethoxycurcumin Induces Cell Apoptosis and Inhibits Human Brain Glioblastoma GBM 8401/Luc2 Cell Xenograft Tumor in Subcutaneous Nude Mice In Vivo

Bisdemethoxycurcumin (BDMC) has biological activities, including anticancer effects in vitro; however, its anticancer effects in human glioblastoma (GBM) cells have not been examined yet. This study aimed to evaluate the tumor inhibitory effect and molecular mechanism of BDMC on human GBM 8401/luc2 cells in vitro and in vivo. In vitro studies have shown that BDMC significantly reduced cell viability and induced cell apoptosis in GBM 8401/luc2 cells. Furthermore, BDMC induced apoptosis via inhibited Bcl-2 (anti-apoptotic protein) and increased Bax (pro-apoptotic proteins) and cytochrome c release in GBM 8401/luc2 cells in vitro. Then, twelve BALB/c-nude mice were xenografted with human glioblastoma GBM 8401/luc2 cancer cells subcutaneously, and the xenograft nude mice were treated without and with BDMC (30 and 60 mg/kg of BDMC treatment) every 3 days. GBM 8401/luc2 cell xenografts experiment showed that the growth of the tumors was significantly suppressed by BDMC administration at both doses based on the reduction of tumor size and weights. BDMC did not change the body weight and the H&E histopathology analysis of liver samples, indicating that BDMC did not induce systemic toxicity. Meanwhile, treatment with BDMC up-regulated the expressions of BAX and cleaved caspase-3, while it down-regulated the protein expressions of Bcl-2 and XIAP in the tumor tissues compared with the control group. This study has demonstrated that BDMC presents potent anticancer activity on the human glioblastoma GBM 8401/luc2 cell xenograft model by inducing apoptosis and inhibiting tumor cell proliferation and shows the potential for further development to the anti-GBM cancer drug.

Hsa_Circular RNA_0001013 Exerts Oncogenice Effects in Gastric Cancer Via the microRNA-136/TWSG1 Axis

Background：Gastric cancer (GC) is one of the most principle malignant cancers in the digestive system. Moreover, the critical role of circular RNAs (circRNAs) has been identified in GC development. Methods：In this context, the purpose of research was to explore the regulatory mechanism circ_0001013, a novel circRNAs predicted by our research, in GC. The differential circRNAs and related mechanism in GC were predicted by microarray analysis. Circ_0001013, miR-136, and TWSG1 expression in GC clinical samples and cells was detected by RT-qPCR. The relationship among circ_0001013, miR-136, and TWSG was assessed by dual-luciferase reporter assay, biotin coupled probe pull-down assay, and biotin coupled miRNA capture. After gain- and loss-of-function assays in GC cells, cell proliferation, migration, invasion, and cell cycle and apoptosis were measured by EdU assay, scratch test, Transwell assay, and flow cytometry respectively. The effect of circ_0001013 on tumor growth was detected by xenograft tumor in nude mice. Results ：Microarray analysis predicted a novel circRNA, circ_0001013, was upregulated in GC, which was confirmed by RT-qPCR detection in GC tissues and cells. Besides, miR-136 was downregulated but TWSG1 was highly expressed in GC tissues. Mechanically, circ_0001013 could bind to miR-136, and miR-136 negatively targeted TWSG1 in GC cells. Silencing circ_0001013 or TWSG1 or overexpressing miR-136 decreased GC cell proliferation, migration, invasion, and cell cycle arrest and accelerated cell apoptosis. Circ_0001013 silencing decreased TWSG1 expression and inhibited transplanted tumor growth in nude mice. Conclusion：Circ_0001013 elevated TWSG1 expression by binding to miR-136, thereby exerting oncogenic effect in GC.

Ginkgolic Acid (GA) Inhibits the Growth of OCa by Inhibiting lncRNA MALAT1/JAK2 Axis

Objective. We aimed to observe the impact of ginkgolic acid (GA) on the proliferation and metastasis ability of ovarian cancer (OCa) cells and to further explore whether GA affects the malignant progress of OCa via regulating the lncRNA MALAT1/JAK2 axis. Methods. OCa cells SKOV3 and CAOV3 were administered with 1 ng/ml GA, 5 ng/ml GA, 10 ng/ml GA, 20 ng/ml GA, and DSMO as control, respectively. The cell proliferation and migration ability of the abovementioned cells in each group were measured by CCK-8 test and Transwell experiments. The expression levels of lncRNA MALAT1 and JAK2 protein were examined by qRT-PCR and western blot, respectively. Subsequently, in OCa cells treated with GA, lncRNA MALAT1 overexpression vector was transfected to continue to detect the proliferation activity and migration ability of each treatment group. Finally, the regulation of GA on activity of lncRNA MALAT1/JAK2 axis in OCa cells was further explored in nude mice. Results. Our data showed that the proliferation inhibition rate of cells at each ginkgolic acid concentration was higher than that of the control group ( P < 0.05 ), suggesting that GA has an inhibitory influence on the proliferation of OCa cells, in a dose-dependent way. GA was able to inhibit the proliferation rate and migration ability of OCa cells. Administration of ginkgolic acid downregulated the levels of lncRNA MALAT1 and JAK2 protein. Overexpression of lncRNA MALAT1 partially reversed the inhibited OCa proliferative capacity caused by GA treatment. Consistent with the results observed in vitro, we also found that the OCa tumor weight and volume of nude mice injected with lncRNA MALAT1 overexpression vector were enhanced and JAK2 protein level increased remarkably in comparison to the ginkgolic acid group. Conclusions. In summary, GA may exert its inhibitory effect on the proliferative and migratory capacities of OCa cells through suppressing the activity of lncRNA MALAT1/JAK2 axis.