MiR‐139‐5p inhibits the tumorigenesis and progression of oral squamous carcinoma cells by targeting HOXA9

Our study sought to clarify the effects of microRNA‐139‐5p (miR‐139‐5p) in the tumorigenesis and progression of oral squamous cell carcinoma (OSCC) by regulating HOXA9. MiR‐139‐5p and HOXA9 expression in OSCC tissues, tumour adjacent tissues, OSCC cells and normal cells were tested by qRT‐PCR. SAS and CAL‐27 cell lines were selected in among four OSCC cell lines and then transfected with miR‐139‐5p mimics, pEGFP‐HOXA9 and cotransfected with miR‐139‐5p mimics + pEGFP‐HOXA9. We used MTT, colony formation, transwell and wound healing assays to analyse cell viability, proliferation, invasion and migration. The target relationship between miR‐139‐5p and HOXA9 was verified by luciferase reporter assay and Western blot, respectively. MiR‐139‐5p was down‐regulated, whereas HOXA9 was up‐regulated in OSCC tissues and cells. The proliferation, invasion and migration ability of SAS and CAL‐27 cells in miR‐139‐5p mimics group were significantly weaker than those in the control group and the miR‐NC group (P < 0.01). MiR‐139‐5p can negatively regulate HOXA9. The proliferation, invasion and migration of SAS and CAL‐27 cells in the miR‐139‐5p mimics + pEGFP‐HOXA9 group were not significantly different from those in the blank control and negative control groups (P > 0.05). Our results indicated that miR‐139‐5p could directly inhibit HOXA9, which might be a potential mechanism in inhibiting the proliferation, invasiveness and migration of OSCC cells.     

CCN5 inhibits proliferation and promotes apoptosis of oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) is a common cancer with poor prognosis and high mortality. The role of CCN5 has attracted a great focus on the regulation of cancer progression. However, the biological function and mechanism of CCN5 in OSCC are still not well elucidated. The current study was designed to determine the effects of CCN5 on OSCC cell proliferation and apoptosis using two OSCC cell lines. Further, LY294002, a PI3K/AKT antagonist, was employed to explore the mechanism underlying the effects of CCN5 in the regulation of OSCC. The results showed that overexpression of CCN5 in TSCCa cells significantly reduced viable cell number, arrested cell cycle, and suppressed cell‐cycle regulators (cyclin D1, cyclin E, and CDK2). CCN5 overexpression increased the apoptotic ratio and Hoechst‐positive cell number, and altered the apoptotic‐related proteins (caspase‐3/9, Bax, and Bcl‐2). However, CCN5 silencing induced opposite effects on cell proliferation and apoptosis in Tca‐8113 cells. In addition, we observed that CCN5 knockdown increased the expression levels of PI3K (p85α and p110α) and phosphorylated AKT at serine 473 (p‐AKT Ser473) in Tca‐8113 cells. Inhibiting PI3K/AKT signaling with LY294002 rescued the apoptotic process in CCN5‐silenced OSCC cells. Finally, xenograft analysis showed that CCN5 represses tumorigenesis of OSCC cells. These findings together suggest that CCN5 functions as a tumor suppressor for OSCC cell development through inactivation of PI3K/AKT signaling pathway, providing a potential candidate for OSCC therapy.     

miR-204-5p regulates cell proliferation,invasion, and apoptosis by targeting IL-11 in esophageal squamous cell carcinoma

Esophageal cancer (EC) is the world's eighth most common malignant neoplasm and is ranked as the sixth leading cause of death related to cancer. Aberrant microRNA (miRNA) expression has been reported to be associated with esophageal squamous cell carcinoma. However, the molecular mechanism of miR-204-5p in esophageal squamous cell carcinoma (ESCC) is not clear. Therefore, the aim of this study was to investigate the potential role of miR-204-5p in ESCC. In the present study, we found that miR-204-5p could affect ESCC proliferation, invasion, apoptosis, and cell cycle in cell and mouse models. A dual-luciferase reporter assay showed that miR-204-5p expression was negatively correlated with interleukin-11 (IL-11) expression. IL-11 overexpression reversed the suppressive effects of miR-204-5p in the cell lines. These results indicated that miR-204-5p functions as a tumor suppressor by directly targeting IL-11 in ESCC.     

CircTIMELESS regulates the proliferation and invasion of lung squamous cell carcinoma cells via the miR-136-5p/ROCK1 axis

Numerous studies demonstrate that circular RNAs (circRNAs) are critical regulators of the occurrence and progression of tumors. However, research on the involvement of circRNAs in lung squamous cell carcinoma (LUSC) is limited. In our study, circTIMELESS (also named hsa_circ_0000408 in the Human circRNA Database) was upregulated in both LUSC tissues and LUSC cells, and circTIMELESS expression was positively associated with the TNM stage. Moreover, circTIMELESS silencing markedly suppressed invasion in vitro and disrupted proliferation in vitro as well as in vivo. Additional investigations have shown that circTIMELESS functions as a miR-136-5p “sponge” and regulates miR-136-5p expression. Furthermore, the impact of miR-136-5p upregulation was consistent with the results of circTIMELESS silencing, both of which inhibited the proliferation and invasion of LUSC cells. Additional results showed that Rho-associated coiled-coil containing protein kinase 1 (ROCK1) is targeted by miR-136-5p. The results of recovery experiments showed that ROCK1 overexpression partly rescued the impact of circTIMELESS silencing and miR-136-5p upregulation on proliferation and invasion. Consequently, our findings confirmed that circTIMELESS exists in LUSC and acts as a tumor promoter through the miR-136-5p/ROCK1 axis. Based on these findings, circTIMELESS may be potentially utilized as a therapeutic target for LUSC.     

Urinary exosome miR‐30c‐5p as a biomarker of clear cell renal cell carcinoma that inhibits progression by targeting HSPA5

Exosome‐derived miRNAs are regarded as biomarkers for the diagnosis and prognosis of many human cancers. However, its function in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, differentially expressed miRNAs from urinal exosomes were identified using next‐generation sequencing (NGS) and verified using urine samples of ccRCC patients and healthy donors. Then, the exosomes were analysed in early‐stage ccRCC patients, healthy individuals and patients suffering from other urinary system cancers. Thereafter, the target gene of the miRNA was detected. Its biological function was investigated in vitro and in vivo. The results showed that miR‐30c‐5p could be amplified in a stable manner. Its expression pattern was significantly different only between ccRCC patients and healthy control individuals, but not compared with that of other urinary system cancers, which indicated its specificity for ccRCC. Additionally, the overexpression of miR‐30c‐5p inhibited ccRCC progression in vitro and in vivo. Heat‐shock protein 5 (HSPA5) was found to be a direct target gene of miR‐30c‐5p. The depletion of HSPA5 caused by miR‐30c‐5p inhibition reversed the promoting effect of ccRCC growth. In conclusion, urinary exosomal miR‐30c‐5p acts as a potential diagnostic biomarker of early‐stage ccRCC and may be able to modulate the expression of HSPA5, which is correlated with the progression of ccRCC.     

Up‐regulation of miR‐10b‐3p promotes the progression of hepatocellular carcinoma cells via targeting CMTM5

In this study, we investigated how miR‐10b‐3p regulated the proliferation, migration, invasion in hepatocellular carcinoma (HCC) at both in vitro and in vivo levels. CMTM5 was among the differentially expressed genes (data from TCGA). The expression of miR‐10b‐3p and CMTM5 was detected by qRT‐PCR and Western blot (WB). TargetScan was used to acquire the binding sites. Dual‐luciferase reporter gene assay was used to verify the direct target relationship between miR‐10b‐3p and CMTM5. WB analysis proved that miR‐10b‐3p suppressed CMTM5 expression. Furthermore, proliferation, invasion and migration of HCC cells were measured by MTT assay, colony formation assay, transwell assay and wound‐healing assay, respectively. Kaplan‐Meier plotter valued the overall survival of CMTM5. Finally, xenograft assay was also conducted to verify the effects of miR‐10b‐3p/CMTM5 axis in vivo. Up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were detected in HCC tissues and cell lines. CMTM5 was verified as a target gene of miR‐10b‐3p. The overexpression of CMTM5 contributed to the suppression of the proliferative, migratory and invasive abilities of HCC cells. Moreover, the up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were observed to be associated with worse overall survival. Lastly, we have confirmed the carcinogenesis‐related roles of miR‐10b‐3p and CMTM5 in vivo. We concluded that the up‐regulation of miR‐10b‐3p promoted the progression of HCC cells via targeting CMTM5.     

Linc-ROR regulates apoptosis in esophageal squamous cell carcinoma via modulation of p53 ubiquitination by targeting miR-204-5p/MDM2

The long intergenic noncoding RNA, regulator of reprogramming (linc-ROR) has been reported to participate in tumorigenesis, while its functions and fundamental mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, gain-of-function assays showed that linc-ROR upregulation enhanced cell viability, promoted cell proliferation, and inhibited apoptosis. Mechanistically, the regulatory network of linc-ROR/miR-204-5p/MDM2 was established with bioinformatics analysis and online databases, then validated via dual-luciferase reporter assays, RNA immunoprecipitation assays in ESCC cells. Linc-ROR positively regulates the expression of MDM2 as a molecular sponge of miR-204-5p. Moreover, results of western blot and coimmunoprecipitation indicated that linc-ROR overexpression enhanced the ubiquitination level of p53, and its downstream apoptosis-related genes have showed higher bcl-2 expression, lower bax, and cleaved caspase-3 expressions, while miR-204-5p could counteract with this effect. Finally, small interfering RNAs tailored to linc-ROR were established to further evaluate its effects on ESCC comprehensively. In summary, this study revealed that linc-ROR modulated cell apoptosis and regulated p53 ubiquitination via targeting miR-204-5p/MDM2 axis, which provides a novel therapeutic insight into treatments for ESCC.     

LncRNA WDFY3‐AS2 suppresses proliferation and invasion in oesophageal squamous cell carcinoma by regulating miR‐2355‐5p/SOCS2 axis

Long non‐coding RNAs (lncRNAs) widely participate in ESCC development and progression; however, the prognostic factors and therapeutic strategies implicated in ESCC development and progression remain to be under investigation. The purpose of the current study was to explore whether WDFY3‐AS2 may be a potential prognostic factor and investigate its biological functions in ESCC. Here, WDFY3‐AS2 was frequently down‐regulated in ESCC tissues and cells, and its expression was correlated with TNM stage, lymph node metastasis and poor prognosis of ESCC patients. Moreover, WDFY3‐AS2 down‐regulation significantly promoted cell proliferation and invasion, whereas WDFY3‐AS2 up‐regulation markedly suppressed cell proliferation and invasion in ESCC EC9706 and TE1 cells, coupled with EMT phenotype alterations. WDFY3‐AS2 functioned as a competing endogenous RNA (ceRNA) for sponging miR‐2355‐5p, further resulted in the up‐regulation of its target gene SOCS2, followed by suppression of JAK2/Stat5 signalling pathway, to suppress ESCC cell proliferation and invasion in EC9706 and TE1 cells. These findings suggest that WDFY3‐AS2 may participate in ESCC development and progression, and may be a novel prognostic factor for ESCC patients, and thus targeting WDFY3‐AS2/miR‐2355‐5p/SOCS2 signalling axis may be a novel therapeutic strategy for ESCC patients.     

LIN28B regulates androgen receptor in human trophoblast cells through Let‐7c

LIN28B is an RNA‐binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let‐7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let‐7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first‐trimester trophoblast cell line, ACH‐3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral‐based vectors. LIN28B knockdown in ACH‐3P cells resulted in significantly decreased levels of AR and increased levels of Let‐7 miRNAs. Moreover, treatment of ACH‐3P cells with Let‐7c mimic, but not Let‐7e or Let‐7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin‐induced syncytialization and Let‐7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW‐1 and a significant decrease in AR in ACH‐3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let‐7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.     

Curcumin inhibits oral squamous cell carcinoma SCC-9 cells proliferation by regulating miR-9 expression

Curcumin, a phytochemical derived from the rhizome of Curcuma longa, has shown anticancer effects against a variety of tumors. In the present study, we investigated the effects of curcumin on the miR-9 expression in oral squamous cell carcinoma (OSCC) and explored the potential relationships between miR-9 and Wnt/β-catenin pathway in curcumin-mediated OSCC inhibition in vitro. As the results shown, the expression levels of miR-9 were significantly lower in clinical OSCC specimens than those in the adjacent non-tumor tissues. Furthermore, our results indicated that curcumin inhibited OSCC cells (SCC-9 cells) proliferation through up-regulating miR-9 expression, and suppressing Wnt/β-catenin signaling by increasing the expression levels of the GSK-3β, phosphorylated GSK-3β and β-catenin, and decreasing the cyclin D1 level. Additionally, the up-regulation of miR-9 by curcumin in SCC-9 cells was significantly inhibited by delivering anti-miR-9 but not control oligonucleotides. Downregulation of miR-9 by anti-miR-9 not only attenuated the growth-suppressive effects of curcumin on SCC-9 cells, but also re-activated Wnt/β-catenin signaling that was inhibited by curcumin. Therefore, our findings would provide a new insight into the use of curcumin against OSCC in future.     

LINC01783 accelerated tongue squamous cell carcinoma progression via inhibiting miR-199b-5p

Growing studies illustrated that lncRNAs exert critical roles in development and occurrence of tumours including TSCC. In this research, we indicated that LINC01783 was up-regulated in TSCC cells (SCC1, Cal27, UM1 and SCC4) when compared to NHOK cell. RT-qPCR analysis indicated that LINC01783 was overexpressed in 22 TSCC cases (73.3%, 22/30) compared with no-tumour specimens. LINC01783 level was up-regulated in TSCC specimens when compared to no-tumour specimens. Ectopic expression of LINC01783 promoted TSCC cell cycle and growth and EMT progression in both TSCC cell SCC1 and Cal27. Overexpression of LINC01783 sponged miR-199b-5p in TSCC cell and elevated expression of LINC01783 inhibited miR-199b-5p expression. Moreover, we illustrated that miR-199b-5p was down-regulated in TSCC cells and specimen and LINC01783 level was up-regulated in TSCC specimens when compared to no-tumour specimens. Elevated expression of LINC01783 promoted TSCC cell growth, cycle and EMT progression by sponging miR-199b-5p. These data suggested that LINC01783 functioned as one oncogene and might be one treatment target for TSCC.