Peiguo Cao

miR-33a functions as a tumor suppressor in melanoma by targeting HIF-1α

Background: Our previous findings showed that miR-33 expressed abnormally in clinical specimens of melanoma, but the exact molecular mechanism has not been elucidated. Object: To determine miR-33s roles in melanoma and confirm whether HIF-1α is a direct target gene of miR-33a. Methods: First miR-33a/b expression levels were detected in HM, WM35, WM451, A375 and SK-MEL-1. Then lentiviral vectors were constructed to intervene miR-33a expression in melanoma cells. Cell proliferation, invasion and metastasis were detected. A375 cells mice model was performed to test the tumorigenesis of melanoma in vivo. Finally the dual reporter gene assay was carried out to confirm whether HIF-1α is a direct target gene of miR-33a. Results: MiR-33a/b exhibited a lower expression in WM35, WM451, A375 and SK-MEL-1 of the metastatic skin melanoma cell lines than that in HM. Then inhibition of miR-33a expression in WM35 and WM451 cell lines could promote cell proliferation, invasion and metastasis. Conversely, increased expression of miR-33a in A375 cells could inhibit cellproliferation, invasion and metastasis. In vivo tests also confirmed that overexpression of miR-33a in A375 cells significantly inhibited melanoma tumorigenesis. Finally, we confirmed that HIF-1α is a direct target gene of miR-33a. Conclusion: The newly identified miR-33a/HIF-1α axis might provide a new strategy for the treatment of melanoma.

microRNA-195 functions as a tumor suppressor by inhibiting CBX4 in hepatocellular carcinoma

MicroRNA-195 (miR-195) plays important roles in tumor metastasis and angiogenesis, yet its function and mechanism of action in hepatocellular carcinoma (HCC) remain to be elucidated. In this study, we aimed to confirm whether chromobox homolog 4 (CBX4) is a direct target gene of miR-195 and determine the functions of miR-195 through the CBX4 pathway. miR-195 expression was slightly lower in the HCC tissues compared with that in the adjacent normal tissues. In addition, western blotting and qRT-RCR results showed that both CBX4 mRNA and protein levels were downregulated upon miR-195 overexpression. Luciferase reporter assays revealed that CBX4 is a direct target gene of miR-195. Furthermore, overexpression of CBX4 significantly restored the proliferative, invasive and migratory capacities of the HepG2 cells. Finally, in vivo experiments confirmed that high expression of CBX4 in HepG2 cells promoted tumor growth. In conclusion, our study demonstrated that miR-195 acts as a tumor suppressor by directly targeting CBX4 in HCC. This finding suggests a potential novel strategy for therapeutic interventions of this disease.

SiRNA-mediated flotillin-2 (Flot2) downregulation inhibits cell proliferation, migration, and invasion in gastric carcinoma cells.

The flotillin (Flot) protein family has been demonstrated to be involved in the development and progression of various cancers. However, the role of Flot2 in gastric carcinomas remains unknown. The present study aimed to investigate the clinical significance and the role of Flot2 in gastric carcinomas. Data of tissue microarray including 90 cases of gastric carcinoma samples and their matched adjacent tissues showed that, among 90 cases of adjacent tissues, 65 cases showed no Flot2 expression, and 25 cases showed low expression of Flot2, and its positive expression rate was only 38.5% (25/90); however, among 90 cases of gastric carcinomas, 6 cases showed no Flot2 expression, 26 cases showed low Flot2 expression, 28 cases showed moderate expression of Flot2, and 30 cases showed high expression of Flot2, and its positive expression rate was 93.3% (84/90). Moreover, the Flot2 expression was significantly associated with the histological grade, depth of invasion, lymph node metastasis, and TNM stage. Furthermore, data of survival analysis suggested that Flot2 protein expression was an independent prognostic factor of poor survival. After that, Flot2-specific siRNA was used to decrease the Flot2 expression in gastric cancer AGS and SGC7901 cells. Forced downregulation of Flot2 remarkably inhibited cellular proliferation, migration, and invasion in gastric carcinoma cells. In conclusion, the present study suggests that the Flot2 protein expression is significantly correlated with cancer progression and poor prognosis in gastric carcinomas, probably due to its role in the regulation of cell proliferation, migration, and invasion in gastric carcinoma cells.

MiR-20a inhibits cutaneous squamous cell carcinoma metastasis and proliferation by directly targeting LIMK1

Background MicroRNA-20a (miR-20a) plays a key role in tumorigenesis and progression. But its function is reverse in different kinds of malignant tumor, and its role and mechanism in cutaneous squamous cell carcinoma (CSCC) remains unclear. Object To determine the miR-20a’s roles in CSCC and confirm whether LIMK1 is a direct target gene of miR-20a. Methods First miR-20a and LIMK1 expression levels were detected in six pairs of CSCC tissues and corresponding normal skin by qRT-PCR. Then MTT assays and colony formation assays were performed to evaluate the impact of miR-20a on cell proliferation. In addition, scratch migration assays and transwell invasion assays were performed to check miR-20a’s effect on cell metastasis. Since LIMK1 (LIM kinase-1) was predicted as a target gene of miR-20a, the changes of LIMK1 protein and mRNA were measured by western blot and qRT-RCR methods after miR-20a overexpression. Moreover the dual reporter gene assay was performed to confirm whether LIMK1 is a direct target gene of miR-20a. Finally LIMK1 mRNA and miR-20a in other 30 cases of CSCC pathological specimens were determined and a correlation analysis was evaluated. Results The miR-20a significantly low-expressed in CSCC tissues compared with that in matched normal tissues while LIMK1 has a relative higher expression. MiR-20a inhibited A431 and SCL-1 proliferation and metastasis. Both of LIMK1 protein and mRNA levels were downregulated after miR-20a overexpression. The dual reporter gene assays revealed that LIMK1 is a direct target gene of miR-20a. Furthermore, qRT-PCR results of LIMK1 mRNA and miR-20a in 30 cases of CSCC pathological specimens showed miR-20a is inversely correlated with LIMK1 expression. Conclusion Our study demonstrated that miR-20a is involved in the tumor inhibition of CSCC by directly targeting LIMK1 gene. This finding provides potential novel strategies for therapeutic interventions of CSCC.

MicroRNA-16 sensitizes breast cancer cells to paclitaxel through suppression of IKBKB expression

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treating breast cancer patients. However, chemoresistance is a major obstacle in cancer treatment. Here, we showed that overexpression of miR-16 promoted Taxol-induced cytotoxicity and apoptosis in breast cancer cells. Furthermore, IκB kinase β (IKBKB) was identified as a direct target of miR-16. Up-regulation of IKBKB suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol, and restoring IKBKB expression in miR-16-overexpressing breast cancer cells recovered Taxol resistance. Moreover, miR-16 was highly expressed in Taxol-sensitive breast cancer tissues compared with Taxol-resistant tissues, and there was an inverse correlation between miR-16 expression and IKBKB expression in breast cancer tissues. The expression levels of miR-16 were negatively associated with T stages, whereas the expression of IKBKB was positively correlated with T stages, lymph node metastasis and clinical stages. Taken together, our data demonstrates that miR-16 sensitizes breast cancer cells to Taxol through the suppression of IKBKB expression, and targeting miR-16/IKBKB axis will be a promising strategy for overcoming Taxol resistance in breast cancer.

miR-101 Inhibiting Cell Proliferation, Migration and Invasion in Hepatocellular Carcinoma through Downregulating Girdin

miR-101 is considered to play an important role in hepato-cellular carcinoma (HCC), but the underlying molecular mechanism remains to be elucidated. Here, we aimed to confirm whether Girdin is a target gene of miR-101 and determine the tumor suppressor of miR-101 through Girdin pathway. In our previous studies, we firstly found Girdin protein was overexpressed in HCC tissues, and it closely correlated to tumor size, T stage, TNM stage and Edmondson-Steiner stage of HCC patients. After specific small interfering RNA of Girdin was transfected into HepG2 and Huh7.5.1 cells, the proliferation and invasion ability of tumor cells were significantly inhibited. In this study, we further explored the detailed molecular mechanism of Girdin in HCC. Interestingly, we found that miR-101 significantly low-expressed in HCC tissues compared with that in matched normal tissues while Girdin had a relative higher expression, and miR-101 was inversely correlated with Girdin expression. In addition, after miR-101 transfection, the proliferation, migration and invasion abilities of HepG2 cells were weakened. Furthermore, we confirmed that Girdin is a direct target gene of miR-101. Finally we confirmed Talen-mediated Girdin knockout markedly suppressed cell proliferation, migration and invasion in HCC while down-regulation of miR-101 significantly restored the inhibitory effect. Our findings suggested that miR-101/Girdin axis could be a potential application of HCC treatment.

Inhibition of REDD1 sensitizes bladder urothelial carcinoma to paclitaxel by inhibiting autophagy

Purpose: Regulated in development and DNA damage response-1 (REDD1) is a stress-related protein and is involved in the progression of cancer. The role and regulatory mechanism of REDD1 in bladder urothelial carcinoma (BUC), however, is yet unidentified. Experimental Design: The expression of REDD1 in BUC was detected by Western blot analysis and immunohistochemistry (IHC). The correlation between REDD1 expression and clinical features in patients with BUC were assessed. The effects of REDD1 on cellular proliferation, apoptosis, autophagy, and paclitaxel sensitivity were determined both in vitro and in vivo. Then the targeted-regulating mechanism of REDD1 by miRNAs was explored. Results: Here the significant increase of REDD1 expression is detected in BUC tissue, and REDD1 is first reported as an independent prognostic factor in patients with BUC. Silencing REDD1 expression in T24 and EJ cells decreased cell proliferation, increased apoptosis, and decreased autophagy, whereas the ectopic expression of REDD1 in RT4 and BIU87 cells had the opposite effect. In addition, the REDD1-mediated proliferation, apoptosis, and autophagy are found to be negatively regulated by miR-22 in vitro, which intensify the paclitaxel sensitivity via inhibition of the well-acknowledged REDD1–EEF2K–autophagy axis. AKT/mTOR signaling initially activated or inhibited in response to silencing or enhancing REDD1 expression and then recovered rapidly. Finally, the inhibited REDD1 expression by either RNAi or miR-22 sensitizes BUC tumor cells to paclitaxel in a subcutaneous transplant carcinoma model in vivo. Conclusions: REDD1 is confirmed as an oncogene in BUC, and antagonizing REDD1 could be a potential therapeutic strategy to sensitize BUC cells to paclitaxel. Clin Cancer Res; 24(2); 445–59. ©2017 AACR.

MiR-1204 sensitizes nasopharyngeal carcinoma cells to paclitaxel both in vitro and in vivo

Nasopharyngeal carcinoma (NPC) is an endemic tumor with a relatively high incidence in Southern China and Southeast Asia. Paclitaxel combination chemotherapy has been used for treatment of advanced NPC. However, treatment failure often occurs due to development of acquired paclitaxel resistance. In this study, we first established a paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cell sublines by treating the parental CNE-1, HNE-2 and 5–8F cells with increasing doses of paclitaxel for about 5 months, respectively. Then, microRNA arrays were used to screen differentially expressed miRNAs between the CNE-1/Taxol cells and the parental CNE-1 cells. We found 13 differentially expressed miRNAs, of which miR-1204 was significantly downregulated in the paclitaxel-resistant CNE-1/Taxol cells. We restored miR-1204 expression in the CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells and found that restoration of miR-1204 re-sensitized the paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells to paclitaxel both in vitro. Finally, we demonstrated that restoration of miR-1204 in significantly inhibits tumor growth in vivo. Thus, our study provides important information for the development of targeted gene therapy for reversing paclitaxel resistance in NPC.

SPAG5 promotes proliferation and suppresses apoptosis in bladder urothelial carcinoma by upregulating Wnt3 via activating the AKT/mTOR pathway and predicts poorer survival

Sperm-associated antigen 5 (SPAG5) is involved in various biological processes. However, the roles of SPAG5 in bladder urothelial carcinoma (BUC) are unknown. This study showed that upregulation of SPAG5 was detected frequently in primary BUC tissues, and was associated with significantly worse survival among the 112 patients that underwent radical cystectomy (RC). Up and downregulating the expression of SPAG5 enhanced or inhibited, respectively, the proliferation of BUC cells in vitro and in vivo, and suppressed or enhanced, respectively, apoptosis in vitro and in vivo. Moreover, SPAG5 increased the resistance of BUC cells to chemotherapy-induced apoptosis. Mechanistic investigations showed that SPAG5 promotes proliferation and suppresses apoptosis in BUC at least partially via upregulating Wnt3 through activating the AKT/mTOR signaling pathway. The importance of the SPAG5/AKT-mTOR/Wnt3 axis identified in BUC cell models was confirmed via immunohistochemical analysis of a cohort of human BUC specimens that underwent RC. Collectively, our data suggested that in patients with BUC who underwent RC, high SPAG5 expression is associated with poor survival. In addition, targeting SPAG5 might represent a novel therapeutic strategy to improve the survival of patients with BUC.

Silencing the Girdin gene enhances radio-sensitivity of hepatocellular carcinoma via suppression of glycolytic metabolism

BackgroundRadiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear.MethodsA short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo.ResultsshRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis.ConclusionGirdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.