Qiaoyun Chen

Decreased expression of microRNA-21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis

The imbalance of Th17/Treg cell populations has been suggested to be involved in the regulation of rheumatoid arthritis (RA) pathogenesis; however, the mechanism behind this phenomenon remains unclear. Recent studies have shown how microRNAs (miRNAs) are important regulators of immune responses and are involved in the development of a variety of inflammatory diseases, including RA. In this study, we demonstrated that the frequencies of CD3+CD4+IL‐17+Th17 cells were significantly higher, and CD4+CD25+FOXP3+ Treg cells significantly lower in peripheral blood mononuclear cells from RA patients. Detection of cytokines from RA patients revealed an elevated panel of pro‐inflammatory cytokines, including IL‐17, IL‐6, IL‐1β, TNF‐α and IL‐22, which carry the inflammatory signature of RA and are crucial in the differentiation and maintenance of pathogenic Th17 cells and dysfunction of Treg cells. However, the level of miR‐21 was significantly lower in RA patients, accompanied by the increase in STAT3 expression and activation, and decrease in STAT5/pSTAT5 protein and Foxp3 mRNA levels. Furthermore, lipopolysaccharide stimulation up‐regulated miR‐21 expression from healthy controls, but down‐regulated miR‐21 expression from RA patients. Therefore, we speculate that miR‐21 may be part of a negative feedback loop in the normal setting. However, miR‐21 levels decrease significantly in RA patients, suggesting that this feedback loop is dysregulated and may contribute to the imbalance of Th17 and Treg cells. MiR‐21 may thus serve as a novel regulator in T‐cell differentiation and homoeostasis, and provides a new therapeutic target for the treatment of RA.

Upregulation of mdr1 gene is related to activation of the MAPK/ERK signal transduction pathway and YB-1 nuclear translocation in B-cell lymphoma

OBJECTIVE Multidrug resistance (MDR) in human B-cell lymphoma constitutes a major obstacle to the effectiveness of chemotherapy. The aim of this study was to investigate the molecular mechanism of MDR in B-cell lymphoma. MATERIALS AND METHODS The B-cell lymphoma MDR sublines were developed by exposing the parental Daudi cells to stepwise increasing concentrations of doxorubicin. Interaction of Y-box binding protein-1 (YB-1) with the Y-box motif of the mdr1 gene promoters was studied by electrophoretic mobility shift assay. The effects of YB-1 on mdr1 promoter activity were examined by luciferase assay. After silencing of YB-1 gene by shRNA, the role of YB-1 nuclear translocation in the formation of induced MDR was examined. Expression of mdr1 and YB-1 was examined further after Daudi cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitor PD98059 for 1 hour. RESULTS Doxorubicin-resistant sublines was generated from the Daudi cell line by stepwise selection in doxorubicin. We found that acquisition of MDR is associated with enhanced YB-1 nuclear translocation and MAPK/extracellular signal-regulated kinase (ERK) activity. Electrophoretic mobility shift assay revealed that doxorubicin increased binding of YB-1 to the Y-box of mdr1 promoter. Luciferase reporter assays demonstrated that the Y-box region is essential for YB-1 regulation of mdr1 expression. The introduction of exogenous YB-1 shRNA into Daudi cells resulted in decreased levels of the expression of mdr1 gene and P-glycoprotein induced by doxorubicin. When Daudi cells were pretreated with MAPK inhibitor PD98059, the phosphorylation of ERK was effectively inhibited as well as the nuclear translocation of YB-1 and the expression of mdr1 gene. CONCLUSION Doxorubicin can increase expression of mdr1/P-glycoprotein through activating MAPK/ERK transduction pathway, then increasing expression of YB-1, inducing YB-1 nuclear translocation, and enhancing DNA-binding activity of YB-1.

miR-449a Regulates Proliferation and Chemosensitivity to Cisplatin by Targeting Cyclin D1 and BCL2 in SGC7901 Cells

BackgroundRecently, several miRNAs have been determined as tumor suppressors in various cancers, such as microRNA-449a. However, the exact molecular mechanisms underlying miR-449a regulated cell proliferation and chemosensitivity in gastric cancer cells have not been well documented.AimThe present study was designed to test whether miR-449a mediates cell proliferation and chemosensitivity in gastric cancer cells via regulating cyclin D1 and BCL2.MethodsIn vitro, the ability of cell proliferation and cell viability were measured by MTT assay; cell cycle and cell apoptosis was detected by FCM. qRT-PCR was used to measure the expression of miR-449a. Western blot and real-time PCR assays were used to detect the expression of cyclin D1 and BCL2 in gastric cancer cell line SGC7901.ResultsmiR-449a expression was downregulated in gastric cancer cell line SGC7901 and human gastric cancer tissues, compared to the gastric epithelial cell line GES-1 and matched non-tumor associated tissues. Upregulation of miR-449a reduced the proliferation of SGC7901 cells. Ectopic expression of miR-449a decreased the percentage of S phase cells, increased the percentage of G1/G0 phase cells and increased the apoptosis induced by cisplatin. Moreover, miR-449a inhibited SGC7901 cells proliferation and enhanced cisplatin chemosensitivity by downregulating expression of BCL2 and cyclin D1, respectively, via directly targeting the 3′-untranslated regions of BCL2 and cyclin D1 mRNA.ConclusionsThis is the first report to provide evidence that miR-449a could modulate cell cycle and apoptosis through regulating cyclin D1 and BCL2 expression in SGC7901 cells.

Berberine Sensitizes Human Ovarian Cancer Cells to Cisplatin Through miR-93/PTEN/Akt Signaling Pathway

Background: Berberine, a well-known component of the Chinese herbal medicine Huanglian, has wide range of biochemical and pharmacological effects, including antineoplastic effect, but the exact mechanisms remain unclear. The aim of the present study was to evaluate the potential chemo-sensitization effect of berberine in ovarian cancer cell line A2780. Methods: The expression of miR-93 was measure by RT-PCR. The target of miR-93 was confirmed by luciferase activity assay. Hoechst 33258 staining, Annexin V and PI double staining were used for apoptosis analysis. Results: In this study, we found A2780/DDP cells that were incubated with berberine combined with cisplatin had a significantly lower survival than the control group. Berberine enhanced cisplatin induced apoptosis and induced G0/G1 cell cycle arrest in A2780 cells. Next, we observed that the miR-93 levels in cisplatin resistant cell lines were higher than that in cisplatin sensitive cell lines. Furthermore, our study found berberine could inhibit miR-93 expression and function in ovarian cancer, as shown by an increase of its target PTEN, an important tumor suppressor in ovarian cancer. A2780 cells that were treated with PTEN siRNA had increased survival compared to NC group and this could be partly alleviated by the AKT inhibitor Triciribine. More importantly, A2780 cells that were treated with PTEN siRNA had a survival pattern that is similar to cells with miR-93 overexpression. Conclusion: The results suggested that berberine modulated the sensitivity of cisplatin through miR-93/PTEN/AKT signaling pathway in the ovarian cancer cells.

Tetrandrine Induces Mitochondria-Mediated Apoptosis in Human Gastric Cancer BGC-823 Cells

Tetrandrine, a bis-benzylisoquinoline alkaloid isolated from the dried root of Hang-Fang-Chi ( Stephania tetrandra S. Moore), has been reported to possess anti-cancer effects on many tumors. In this study, we investigated tetrandrine-induced apoptosis on human gastric cancer BGC-823 cells in vitro and in vivo. The results showed that tetrandrine significantly inhibited cell viability in a dose- and time-dependent manner and induced apoptosis. It increased the apoptosis; upregulation of Bax, Bak, and Bad; and downregulation of Bcl-2 and Bcl-xl in BGC-823 cells. Moreover, tetrandrine increased the activation of caspase-3 and -9, release of cytochrome c, and upregulation of apaf-1, suggesting that tetrandrine-induced apoptosis was related to the mitochondrial pathway. Meanwhile, pretreatment with the pan-caspase inhibitor z-VAD-fmk in BGC-823 cells reduced tetrandrine-induced apoptosis by blocking activation of caspases. Furthermore, tetrandrine effectively inhibited tumor growth via apoptosis induction, which was verified by immunohistochemical analysis in a nude mouse xenograft model. Taken together, we concluded that tetrandrine significantly inhibited the proliferation of gastric cancer BGC-823 cells through mitochondria-dependent apoptosis, which may play a promising role in gastric cancer therapy.

MicroRNA-449a reduces cell survival and enhances cisplatin-induced cytotoxicity via downregulation of NOTCH1 in ovarian cancer cells.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies. Platinum-based chemotherapy is the first-line treatment for the advanced ovarian cancer, but resistance to cisplatin remains a major obstacle to successful treatment. MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in disease processes, including the development of drug resistance. In this study, we found miR-449a were significantly downregulated in the cisplatin-resistant ovarian cell lines SKOV3/DDP and A2780/DDP, compared with their sensitive parent line SKOV3 and A2780, respectively. The overexpression of miR-449a increased cisplatin sensitivity of SKOV3/DDP and A2780/DDP cells by inhibiting proliferation and promoting apoptosis. The luciferase assay confirmed that miR-449a functioned through suppressing NOTCH1 directly. Concordantly, BALB/c nude mice that were injected intraperitoneally with SKOV3/DDP cells transfected with miR-449a mimics exhibited enhanced cisplatin sensitivity in vivo. Taken together, these results suggest that the ectopic expression of miR-449a may be a promising therapeutic strategy for the management of cisplatin resistance in ovarian cancer.

Enforced expression of miR-101 enhances cisplatin sensitivity in human bladder cancer cells by modulating the cyclooxygenase-2 pathway.

Alterations in microRNA (miRNA) expression have been shown to be involved in the tumor response to chemotherapy. However, the possible role of miR‑101 in cisplatin sensitivity in human bladder cancer cells remains unclear. In this study, quantitative polymerase chain reaction and western blotting were utilized to determine the expression profiles of miR‑101 and cyclooxygenase‑2 (COX‑2) in human bladder cancer cells. The effect of miR‑101 and small interfering RNA (siRNA) against COX‑2 on cell viability was evaluated using MTT assays, and apoptosis levels were determined using fluorescence‑activated cell sorting analysis of Annexin V/propidium iodide‑stained cells. Luciferase reporter plasmids were constructed to confirm direct targeting. This study found that the expression of miR‑101 was downregulated in the cisplatin‑resistant cell line T24/CDDP as compared with that in the parental line, T24. Furthermore, overexpression of miR‑101 significantly increased the anti‑proliferative effects and apoptosis induced by cisplatin, whereas knockdown of miR‑101 significantly decreased the anti‑proliferative effects and apoptosis induced by cisplatin. In addition, downregulation of miR‑101 induced cell survival and cisplatin resistance through the upregulation of COX‑2 expression. Luciferase gene reporter assays confirmed that COX‑2 was a direct target gene of miR‑101. Inhibition of COX‑2 using COX‑2 siRNA abrogated the cisplatin resistance induced by miR‑101 downregulation. These results suggest that miR‑101 may provide a novel mechanism for understanding cisplatin resistance in bladder cancer by modulating the COX‑2 pathway.

Triptolide modulates the sensitivity of K562/A02 cells to adriamycin by regulating miR-21 expression

Context: Multidrug-resistance is a serious obstacle encountered in leukemia treatment. Recent studies have shown microRNA-21 (miR-21) is overexpressed in several types of cancer and contributes to tumor resistance to chemotherapy. In our previous studies, we found triptolide (TPL) could enhance adriamycin-induced cytotoxicity and apoptosis in K562/A02 cells. Objective: In the present study, we investigated the mechanism of TPL on the sensitivity of K562/A02 cells to adriamycin. Materials and methods: Cell viability was assessed by methyl thiazolyl tetrazolium (MTT) assay. Expression of mature miR-21 was determined by SYBER green PCR. The miR-21 mimics and inhibitors were chemically synthesized and transfected into K562 cells or K562/A02 cells. PTEN protein levels was determined by western blots. PTEN promoter activity was measured by luciferase assays. Results: TPL (5 nmol/L) increased the sensitivity of K562/A02 to adriamycin. When adriamycin was combined with 5 nmol/L TPL, the mean apoptotic population of K562/A02 cells was increased from 4.3 to 18.5%, respectively. K562/A02 cells showed a significant reduction in miR-21 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) expressions after TPL treatment. K562/A02 cells that were transfected with the miR-21 inhibitor had a significantly higher PTEN protein level than the control. K562 cells that were pre-treated with PTEN siRNA had increased survival rate compared to the control group. Discussion and conclusion: Our findings indicated that triptolide modulates the sensitivity of K562/A02 cells to adriamycin by regulating miR-21 expression. Triptolide inhibited miR-21 expression and enhanced PTEN levels in K562/A02 cells.

Involvement of miR-30c in resistance to doxorubicin by regulating YWHAZ in breast cancer cells

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression implicated in cancer, which play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. The aim of this study was to investigate whether miR-30c mediated the resistance of breast cancer cells to the chemotherapeutic agent doxorubicin (ADR) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). miR-30c was downregulated in the doxorubicin-resistant human breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR compared with their parental MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, we observed that transfection of an miR-30c mimic significantly suppressed the ability of MCF-7/ADR to resist doxorubicin. Moreover, the anti-apoptotic gene YWHAZ was confirmed as a target of miR-30c by luciferase reporter assay, and further studies indicated that the mechanism for miR-30c on the sensitivity of breast cancer cells involved YWHAZ and its downstream p38 mitogen-activated protein kinase (p38MAPK) pathway. Together, our findings provided evidence that miR-30c was one of the important miRNAs in doxorubicin resistance by regulating YWHAZ in the breast cancer cell line MCF-7/ADR.

A Functional Variant at miR-520a Binding Site in PIK3CA Alters Susceptibility to Colorectal Cancer in a Chinese Han Population

An increasing body of evidence has indicated that polymorphisms in the miRNA binding site of target gene can alter the ability of miRNAs to bind their target genes and modulate the risk of cancer. We aimed to investigate the association between a miR-520a binding site polymorphism rs141178472 in the PIK3CA 3′-UTR and the risk of colorectal cancer (CRC) in a Chinese Han population. The polymorphism rs141178472 was analyzed in a case-control study, including 386 CRC patients and 394 age- and sex-matched controls; the relationship between the polymorphism and the risk of colorectal cancer was examined. Individuals carrying the rs141178472 CC genotype or C allele had an increased risk of developing CRC (CC versus TT, OR (95% CI): 1.716 (1.084–2.716), P = 0.022; C versus T, OR (95% CI): 1.258 (1.021–1.551), P = 0.033). Furthermore, the expression of PIK3CA was detected in the peripheral blood mononucleated cell of CRC patients, suggesting that mRNA levels of PIK3CA might be associated with SNP rs141178472. These findings provide evidence that a miR-520a binding site polymorphism rs141178472 in the PIK3CA 3′-UTR may play a role in the etiology of CRC.